Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

Energy Technology Data Exchange (ETDEWEB)

Morton, M.J., E-mail: michael.morton@astrazeneca.com [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Armstrong, D.; Abi Gerges, N. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Bridgland-Taylor, M. [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Pollard, C.E.; Bowes, J.; Valentin, J.-P. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom)

2014-09-01

Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility.

Elaboration of a radioligand receptor assay for TSH and thyroid-stimulating immunoglobulins

International Nuclear Information System (INIS)

Wille, A.

1980-01-01

125 J-TSH is bound by membrane preparations from human thyroid glands. The removal of the radioactive hormone from the bond, interpreted by means of a standard curve, is an indicator of the unknown quantity of TSH or TSI. The specific binding of the TSH to the membrane proceeds as a function of hydrogen ion concentration, temperature, and incubation time. Since all globulins exhibit an unspecific binding to the membranes, it is necessary to separate the gamma globulin fraction from the serum in order to detect the TSI. The separation is achieved by QAE Sephadex A-50 columns. The displacement characteristics of the gamma globulin fractions are determined in the radioligand receptor assay. The classification into normal and pathological findings is done in accordance with the TSI index of Smith and Hall. The poor detection of TSI in the sera studied is attributed to the fact that the group of patients of this study had already been treated at the time the blood sera were taken. The TSH content of homogenates from human, postmortally taken pituitary glands is determined by the RRA and compared with the TSH values of the RIA. The comparison shows a positive correlation, with the TSH data of the RRA being above those of the radioimmunoassay. (orig./MG) [de

A kinetic analysis of kappa-opioid agonist binding using the selective radioligand (/sup 3/H)U69593

Energy Technology Data Exchange (ETDEWEB)

Smith, J.A.; Hunter, J.C.; Hill, R.G.; Hughes, J.

1989-07-01

The interaction of the nonselective opioid ligand (3H)bremazocine and of the kappa-opioid (3H)U69593 with the kappa-receptor was investigated in guinea-pig cortical membranes. Each radioligand bound to a single population of high-affinity sites, although (3H)U69593 apparently recognised only 70% of those sites labelled by (3H)bremazocine. Naloxone and the kappa-selective ligands U69593 and PD117302 exhibited full inhibition of the binding of both radioligands. Kinetic analysis demonstrated biphasic rates of association and dissociation for both (3H)bremazocine and (3H)U69593. Detailed analysis of the binding of (3H)U69593 revealed that the fast rate of association was dependent on radioligand concentration, in contrast to the slow rate, which was independent of ligand concentration. Guanylyl-5'-imidodiphosphate (GppNHp) inhibited binding of (3H)U69593; saturation analysis demonstrated that the inhibitory effects of GppNHp resulted in a decrease in affinity without any significant change in binding capacity. GppNHp attenuated the formation of the slow component of (3H)U69593 binding, while accelerating the fast component. The data are consistent with the formation of a high-affinity complex between the kappa-receptor and a guanine nucleotide binding protein. Guanine nucleotides promote the dissociation of this ternary complex and the stabilisation of a lower-affinity state of the receptor.

Effects of dopaminergic drug treatments on in vivo radioligand binding to brain vesicular monoamine transporters

Energy Technology Data Exchange (ETDEWEB)

Kilbourn, Michael R; Frey, Kirk A; Vander Borght, Thierry; Sherman, Phillip S

1996-05-01

The effects of various dopaminergic drug treatments on the in vivo regional brain distribution of high-affinity radioligands ([{sup 11}C]dihydrotetrabenazine and [{sup 11}C]methoxytetrabenazine) for the rat brain vesicular monoamine transporter (VMAT2) were determined. Acute treatments with reserpine (2 mg/kg i.p.), tetrabenazine (10 mg/kg i.v.) or related benzoisoquinolines significantly reduced radiotracer binding in vivo. In contrast, radiotracer distributions remained unchanged after treatments with other dopaminergic drugs, whether given by single injection (haloperidol, 1 mg/kg i.p., pargyline 80 mg/kg), repeatedly (pargyline, 80 mg/kg s.c., 14 days), or by continuous infusion (deprenyl, 10 mg/kg/day, 5 days; L-DOPA methyl ester 100 mg/kg/day, 5 days). Repeated injections of tetrabenazine (5 mg/kg i.p., twice daily, 3 days) did not alter in vivo radioligand binding measured after allowing drug washout from the brain. These studies support the proposal that in vivo PET imaging of VMAT2 radioligands in patients with extrapyramidal movement disorders will not be affected by concurrent use of L-DOPA or deprenyl.

Human CRF{sub 2} {alpha} and {beta} splice variants: pharmacological characterization using radioligand binding and a luciferase gene expression assay

Energy Technology Data Exchange (ETDEWEB)

Ardati, A. [Rhone-Poulenc Rorer, Cardiovascular Biology, NW4, 500 Arcola Road, Collegeville, PA (United States); Goetschy, V.; Gottowick, J.; Henriot, S.; Deuschle, U.; Kilpatrick, G.J. [Central Nervous System, Pharma Division, F. Hoffmann-La Roche AG, CH-4070 Basel (Switzerland); Valdenaire, O. [Cardiovascular Research, Pharma Division, F. Hoffmann-La Roche AG, CH-4070 Basel (Switzerland)

1999-03-14

Corticotropin releasing factor (CRF) receptors belong to the super-family of G protein-coupled receptors. These receptors are classified into two subtypes (CRF{sub 1} and CRF{sub 2}). Both receptors are positively coupled to adenylyl cyclase but they have a distinct pharmacology and distribution in brain. Two isoforms belonging to the CRF{sub 2} subtype receptors, CRF{sub 2{alpha}} and CRF{sub 2{beta}}, have been identified in rat and man. The neuropeptides CRF and urocortin mediate their actions through this CRF G protein-coupled receptor family. In this report, we describe the pharmacological characterization of the recently identified hCRF{sub 2{beta}} receptor. We have used radioligand binding with [{sup 125}I]-tyr{sup 0}-sauvagine and a gene expression assay in which the firefly luciferase gene expression is under the control of cAMP responsive elements. Association kinetics of [{sup 125}I]-tyr{sup 0}-sauvagine binding to the hCRF{sub 2{beta}} receptor were monophasic while dissociation kinetics were biphasic, in agreement with the kinetics results obtained with the hCRF{sub 2{alpha}} receptor. Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically and with parameters obtained with the hCRF{sub 2{alpha}} receptor. A non-hydrolysable GTP analog, Gpp(NH)p, reduced the high affinity binding of [{sup 125}I]-tyr{sup 0}-sauvagine to both hCRF{sub 2} receptor isoforms in a similar manner. The rank order of potency of CRF agonist peptides in competition experiments was identical for both hCRF{sub 2}{alpha}-helical CRF{sub (9-41)}oCRF). Similarly, agonist potency was similar for the two isoforms when studied using the luciferase gene reporter system. The peptide antagonist {alpha}-helical CRF{sub (9-41)} exhibited a non-competitive antagonism of urocortin-stimulated luciferase expression with both hCRF{sub 2} receptor isoforms. Taken together, these results indicate that the

Improved receptor analysis in PET using a priori information from in vitro binding assays

Energy Technology Data Exchange (ETDEWEB)

Litton, J.-E.; Hall, H.; Blomqvist, G. [Department of Clinical Neuroscience, Karolinska Hospital, S-171 76 Stockholm (Sweden)

1997-08-01

An accurate determination of non-specific binding is required for the analysis of in vitro and in vivo receptor binding data. For some radioligands the non-specific binding is of the same magnitude as the specific binding. Furthermore, in vitro measurements have shown that the non-specific binding can be different in different brain regions. If this is the case in a PET study for determining B{sub max} and K{sub d}, a correction for the non-specific binding has to be applied. The aim of the present communication is to present a means for determining corrected B{sub max} and K{sub d} with Scatchard analysis using in vitro binding studies. The influence of non-specific binding on the free and specifically bound radioligand is expressed with the aid of a correction factor, which can be calculated from measurable quantities. Introduction of the corrected free and specifically bound radioligand should give binding parameters closer to reality than previously obtained results. (author)

Brain radioligands. State of the art and new trends

International Nuclear Information System (INIS)

Halldin, C.; Gulyas, B.; Langer, O.; Farde, L.

2001-01-01

Non-invasive radioligand imaging methods for brain receptor studies use either short-lived positron-emitting radionuclides such as 11 C and 18 F for positron emission tomography (PET) or single photon-emitting radionuclides such as 123 I for single photon emission computed tomography (SPECT). PET and SPECT use radioligands which are injected intravenously into experimental animals, human volunteers or patients. The main applications of radioligands in brain research concern human neuro psychopharmacology and the discovery and development of novel drugs to be used in the therapy of neurological and psychiatric disorders. A basic problem in PET and SPECT brain receptor studies is the lack of useful radioligands with appropriate binding characteristics. Prerequisite criteria need to be satisfied for a radioligand to reveal target binding sites in vivo. This section will discuss these important criteria and also review recent examples in neuro receptor radioligand development such as selective radioligands for brain monoamine transporters

Measuring specific receptor binding of a PET radioligand in human brain without pharmacological blockade: The genomic plot.

Science.gov (United States)

Veronese, Mattia; Zanotti-Fregonara, Paolo; Rizzo, Gaia; Bertoldo, Alessandra; Innis, Robert B; Turkheimer, Federico E

2016-04-15

PET studies allow in vivo imaging of the density of brain receptor species. The PET signal, however, is the sum of the fraction of radioligand that is specifically bound to the target receptor and the non-displaceable fraction (i.e. the non-specifically bound radioligand plus the free ligand in tissue). Therefore, measuring the non-displaceable fraction, which is generally assumed to be constant across the brain, is a necessary step to obtain regional estimates of the specific fractions. The nondisplaceable binding can be directly measured if a reference region, i.e. a region devoid of any specific binding, is available. Many receptors are however widely expressed across the brain, and a true reference region is rarely available. In these cases, the nonspecific binding can be obtained after competitive pharmacological blockade, which is often contraindicated in humans. In this work we introduce the genomic plot for estimating the nondisplaceable fraction using baseline scans only. The genomic plot is a transformation of the Lassen graphical method in which the brain maps of mRNA transcripts of the target receptor obtained from the Allen brain atlas are used as a surrogate measure of the specific binding. Thus, the genomic plot allows the calculation of the specific and nondisplaceable components of radioligand uptake without the need of pharmacological blockade. We first assessed the statistical properties of the method with computer simulations. Then we sought ground-truth validation using human PET datasets of seven different neuroreceptor radioligands, where nonspecific fractions were either obtained separately using drug displacement or available from a true reference region. The population nondisplaceable fractions estimated by the genomic plot were very close to those measured by actual human blocking studies (mean relative difference between 2% and 7%). However, these estimates were valid only when mRNA expressions were predictive of protein levels (i

Preparation of steroid radioligands for ultrafiltration assays by a solid-phase transport globulin method using concanavalin A-Sepharose

International Nuclear Information System (INIS)

Hiramatsu, R.; Nisula, B.C.

1985-01-01

Concanavalin A-Sepharose (Con A-Sepharose) was applied to separate non-protein-bound and albumin-bound radioactive impurities from steroid radioligands. Con A-Sepharose gel, plasma, and steroid radioligand were mixed, incubated, and then washed with buffer. This method was compared with an affinity diafiltration method which separates non-protein-bound radioactive impurities with a filter membrane. 3 H-Water and 3 H-estrone sulfate, chosen to serve as molecules representative of non-protein-bound and albumin-bound impurities, were removed quite effectively by the Con A-Sepharose method, while 85% of 3 H-estrone sulfate could not be removed by the diafiltration method. Plasma unbound cortisol (F) and testosterone (T) values determined by ultrafiltration using 3 H-F and 3 H-T prepared by the Con A-Sepharose method were significantly lower than those determined using the radioligands unprocessed or prepared by the diafiltration method. The whole procedure of the Con A-Sepharose method takes only 3-4 hours. This method is a simple, rapid, and effective technique for preparation of steroid radioligands for plasma protein binding studies

A novel radioligand for glycine transporter 1: characterization and use in autoradiographic and in vivo brain occupancy studies

Energy Technology Data Exchange (ETDEWEB)

Zeng Zhizhen [Imaging, Merck Research Laboratories, West Point, PA 19486 (United States)], E-mail: zhizhen_zeng@merck.com; O' Brien, Julie A. [Sleep and Psychiatric Disorders, Merck Research Laboratories, West Point, PA 19486 (United States); Lemaire, Wei [Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486 (United States); O' Malley, Stacey S.; Miller, Patricia J. [Imaging, Merck Research Laboratories, West Point, PA 19486 (United States); Zhao Zhijian [Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486 (United States); Wallace, Michael A. [Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065 (United States); Raab, Conrad [Drug Metabolism, Merck Research Laboratories, West Point, PA 19486 (United States); Lindsley, Craig W. [Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486 (United States); Departments of Pharmacology and Chemistry, Vanderbilt University, Nashville, TN 37232 (United States); Sur, Cyrille; Williams, David L. [Imaging, Merck Research Laboratories, West Point, PA 19486 (United States)

2008-04-15

Introduction: In an effort to develop agents to test the NMDA hypofunction hypothesis of schizophrenia, benchmark compounds from a program to discover potent, selective, competitive glycine transporter 1 (GlyT1) inhibitors were radiolabeled in order to further study the detailed pharmacology of these inhibitors and the distribution of GlyT1 in brain. We here report the in vitro characterization of [{sup 35}S](S)-2-amino-4-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl) ethyl)benzamide ([{sup 35}S]ACPPB), a radiotracer developed from a potent and selective non-sarcosine-derived GlyT1 inhibitor, its use in autoradiographic studies to localize (S)-2-amino-6-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl) benzamide (ACPPB) binding sites in rat and rhesus brain and for in vivo occupancy assays of competitive GlyT1 inhibitors. Methods: Functional potencies of unlabeled compounds were characterized by [{sup 14}C]glycine uptake into JAR (human placental choriocarcinoma) cells and synaptosomes. Radioligand binding studies were performed with tissue homogenates. Autoradiographic studies were performed on tissue slices. Results: ACPPB is a potent (K{sub d}=1.9 nM), selective, GlyT1 inhibitor that, when radiolabeled with [{sup 35}S], is a well-behaved radioligand with low nondisplaceable binding. Autoradiographic studies of rat and rhesus brain slices with this ligand showed that specific binding sites were plentiful and nonhomogeneously distributed, with high levels of binding in the brainstem, cerebellar white matter, thalamus, cortical white matter and spinal cord gray matter. In vivo studies demonstrate displaceable binding of [{sup 35}S]ACPPB in rat brain tissues following iv administration of this radioligand. Conclusions: This is the first report of detailed anatomical localization of GlyT1 using direct radioligand binding, and the first demonstration that an in vivo occupancy assay is feasible, suggesting that it may also be feasible to develop

A novel radioligand for glycine transporter 1: characterization and use in autoradiographic and in vivo brain occupancy studies

International Nuclear Information System (INIS)

Zeng Zhizhen; O'Brien, Julie A.; Lemaire, Wei; O'Malley, Stacey S.; Miller, Patricia J.; Zhao Zhijian; Wallace, Michael A.; Raab, Conrad; Lindsley, Craig W.; Sur, Cyrille; Williams, David L.

2008-01-01

Introduction: In an effort to develop agents to test the NMDA hypofunction hypothesis of schizophrenia, benchmark compounds from a program to discover potent, selective, competitive glycine transporter 1 (GlyT1) inhibitors were radiolabeled in order to further study the detailed pharmacology of these inhibitors and the distribution of GlyT1 in brain. We here report the in vitro characterization of [ 35 S](S)-2-amino-4-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl) ethyl)benzamide ([ 35 S]ACPPB), a radiotracer developed from a potent and selective non-sarcosine-derived GlyT1 inhibitor, its use in autoradiographic studies to localize (S)-2-amino-6-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl) benzamide (ACPPB) binding sites in rat and rhesus brain and for in vivo occupancy assays of competitive GlyT1 inhibitors. Methods: Functional potencies of unlabeled compounds were characterized by [ 14 C]glycine uptake into JAR (human placental choriocarcinoma) cells and synaptosomes. Radioligand binding studies were performed with tissue homogenates. Autoradiographic studies were performed on tissue slices. Results: ACPPB is a potent (K d =1.9 nM), selective, GlyT1 inhibitor that, when radiolabeled with [ 35 S], is a well-behaved radioligand with low nondisplaceable binding. Autoradiographic studies of rat and rhesus brain slices with this ligand showed that specific binding sites were plentiful and nonhomogeneously distributed, with high levels of binding in the brainstem, cerebellar white matter, thalamus, cortical white matter and spinal cord gray matter. In vivo studies demonstrate displaceable binding of [ 35 S]ACPPB in rat brain tissues following iv administration of this radioligand. Conclusions: This is the first report of detailed anatomical localization of GlyT1 using direct radioligand binding, and the first demonstration that an in vivo occupancy assay is feasible, suggesting that it may also be feasible to develop positron emission

A strategy for increasing the brain uptake of a radioligand in animals: use of a drug that inhibits plasma protein binding

International Nuclear Information System (INIS)

Haradahira, Terushi; Zhang, Ming-Rong; Maeda, Jun; Okauchi, Takashi; Kawabe, Kouichi; Kida, Takayo; Suzuki, Kazutoshi; Suhara, Tetsuya

2000-01-01

A positron-emitter labeled radioligand for the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor, [ 11 C]L-703,717, was examined for its ability to penetrate the brain in animals by simultaneous use with drugs having high-affinity separate binding sites on human serum albumin. [ 11 C]L-703,717 has poor blood-brain barrier (BBB) permeability because it binds tightly to plasma proteins. Co-injection of warfarin (50-200 mg/kg), a drug that binds to albumin and resembles L-703,717 in structure, dose-dependently enhanced the penetration by [ 11 C]L-703,717 in mice, resulting in a five-fold increase in the brain radioactivity at 1 min after the injection. Drugs structurally unrelated to L-703,717, salicylate, phenol red, and L-tryptophan, were less effective or ineffective in increasing the uptake of [ 11 C]L-703,717. These results suggest that the simultaneous use of a drug that inhibits the binding of a radioligand to plasma proteins is a useful way to overcome the poor BBB permeability of the radioligand triggered by its tight binding to plasma proteins. In brain distribution studies in rodents, it was found that, after the increase in brain uptake with warfarin, much of the glycine site antagonist accumulates in the cerebellum but its pharmacological specificity did not match the glycine site of NMDA receptors

( sup 125 I)Ifenprodil: a convenient radioligand for binding and autoradiographic studies of the polyamine-sensitive site of the NMDA receptor

Energy Technology Data Exchange (ETDEWEB)

Beart, P M; Mercer, L D; Jarrott, B [University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospital, Heidelberg, Vic (Australia)

1991-04-01

Iodination of ifenprodil, a non-competitive NMDA antagonist, with Na{sup 125}I/Chloramin-T gave a radioligand which bound rapidly and saturably to a single population of sites (dissociation constant 145 nM) in membranes of rat cerebral cortex. In competition studies, specific binding of ({sup 125}I)-ifenprodil was inhibited by analogues of ifenprodil, as well as by spermine and spermidine. Binding was sensitive to Ca{sup 2+}, Mg{sup 2+} and Zn{sup 2+}. ({sup 125}I)-Ifenprodil labelled a population of binding sites, which was topographically distributed in rat forebrain, as shown by autoradiography. ({sup 125}I)-Ifenprodil is a useful radioligand for the investigation of the polyamine site of the N-methyl-D-aspartate (NMDA) receptor-complex. (author).

A strategy for increasing the brain uptake of a radioligand in animals: use of a drug that inhibits plasma protein binding

Energy Technology Data Exchange (ETDEWEB)

Haradahira, Terushi E-mail: terushi@nirs.go.jp; Zhang, Ming-Rong; Maeda, Jun; Okauchi, Takashi; Kawabe, Kouichi; Kida, Takayo; Suzuki, Kazutoshi; Suhara, Tetsuya

2000-05-01

A positron-emitter labeled radioligand for the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor, [{sup 11}C]L-703,717, was examined for its ability to penetrate the brain in animals by simultaneous use with drugs having high-affinity separate binding sites on human serum albumin. [{sup 11}C]L-703,717 has poor blood-brain barrier (BBB) permeability because it binds tightly to plasma proteins. Co-injection of warfarin (50-200 mg/kg), a drug that binds to albumin and resembles L-703,717 in structure, dose-dependently enhanced the penetration by [{sup 11}C]L-703,717 in mice, resulting in a five-fold increase in the brain radioactivity at 1 min after the injection. Drugs structurally unrelated to L-703,717, salicylate, phenol red, and L-tryptophan, were less effective or ineffective in increasing the uptake of [{sup 11}C]L-703,717. These results suggest that the simultaneous use of a drug that inhibits the binding of a radioligand to plasma proteins is a useful way to overcome the poor BBB permeability of the radioligand triggered by its tight binding to plasma proteins. In brain distribution studies in rodents, it was found that, after the increase in brain uptake with warfarin, much of the glycine site antagonist accumulates in the cerebellum but its pharmacological specificity did not match the glycine site of NMDA receptors.

Photolabeling and radioligand binding of human erythrocyte NaK-ATPase with 125I-derivatives of cymarin and digitoxigenin

International Nuclear Information System (INIS)

Lowndes, J.M.

1988-01-01

NaK-ATPase is an enzyme which maintains Na + and K + gradients across the plasma membrane of eukaryotic cells, and is specifically inhibited by cardiac glycosides. The cardiac glycoside binding site is located primarily on the catalytic α subunit but the glycoprotein β and proteolipid-γ subunits may also contribute to the structure of the site. In order to label the cardiac glycoside binding site of human erythrocytes, four photoaffinity ligands with very high specific radioactivity were synthesized. The compounds, which are abbreviated [ 125 I]AISC, [ 125 I]AIPP-GluD, [ 125 I]AIPP-GalD and [ 125 I]IA-GalD, were all effective photolabels for NaK-ATPase as shown by ouabain-protectable, covalent labeling of the α, β, and proteolipid-γ subunits. In order to study the possible existence of a very high affinity binding site in erythrocyte NaK-ATPase, a carrier-free radioligand, [ 125 I]I-TASC, was synthesized; this compound had the same structure as [ 125 I]AISC except that a light-sensitive azide group was replaced with a hydroxyl group. Competitive binding assays with cymarin against 0.2 nM [ 125 I]I-TASC suggested two classes of erythrocyte binding sites. Scatchard analysis of direct [ 125 I]I-TASC binding indicated that the very high affinity, low capacity class of erythrocyte bindings sites had a K D of 54 pM and a B max of 23 fmol/mg protein

Assays for calcitonin receptors

International Nuclear Information System (INIS)

Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

1985-01-01

The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is 125 I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed

New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics.

Science.gov (United States)

Zeilinger, Markus; Pichler, Florian; Nics, Lukas; Wadsak, Wolfgang; Spreitzer, Helmut; Hacker, Marcus; Mitterhauser, Markus

2017-12-01

Resolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A 3 R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the K i of eight different A 3 R antagonists, using CHO-K1 cells stably expressing the hA 3 R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants k on and k off , as well as the dedicated K d of the A 3 R agonist [ 125 I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds. Two novel real-time cell-binding approaches were successfully developed and established. Both experimental procedures were found to visualize the kinetic binding characteristics with high spatial and temporal resolution, resulting in reliable affinity values, which are in good agreement with values previously reported with traditional methods. Taking into account the lipophilicity of the A 3 R antagonists, no influences on the experimental performance and the resulting affinity were investigated. Both kinetic binding approaches comprise tracer administration and subsequent binding to living cells, expressing the dedicated target protein. Therefore, the experiments resemble better the true in vivo physiological conditions and provide important markers of cellular feedback and biological response.

A comparative study in three commercial kits of radioligand assay for GAD-Ab

International Nuclear Information System (INIS)

Yang Yehua; Zhou Zhiguang; Huang Gan; Yang Feike; Li Zhangwei

2006-01-01

Objective: To provide data for perfect selection of glutamic acid decarboxylase antibodies (GAD-Ab) assay commercial kits. Methods: The sera of 88 titer-graded samples by radioligand assay (RLA) were measured with two types of enzyme-linked immunosorbent assay (ELISA) kits and one type of immunoradiometric assay (IRMA) kit for GAD-Ab in order to test their consistency with RLA. Another 140 diabetic sera were measured with RLA and screened by a type of suitable kit to search the cutoff point for discriminating patients who needed rechecking. Results: Compared with RLA, concordance ratio of 3 kits: Medizym kit(75%) > RSR kit(73.9%) > Biomerica kit(62.5%); Kappa value: Medizym kit (0.408, common) > RSR kit (0.405, common) > Biomerica kit (0.185, failing); correlation to RLA index: RSR kit (r= 0.992) > Medizym kit(r= 0. 791 ) > Biomerica kit (r = -0. 055); area under the receiver operating characteristic (ROC) curve: Medizym kit (0.812) > RSR kit (0. 727 ) > Biomerica kit (0.666). Conclusions: The efficiencies of RSR kit and Medizym kit are good. Serum concentration between 0.25-0.99 U/ml measured by RSR kit is suggested to further recheck by RLA. (authors)

Synthesis and evaluation of translocator 18 kDa protein (TSPO) positron emission tomography (PET) radioligands with low binding sensitivity to human single nucleotide polymorphism rs6971.

Science.gov (United States)

Zanotti-Fregonara, Paolo; Zhang, Yi; Jenko, Kimberly J; Gladding, Robert L; Zoghbi, Sami S; Fujita, Masahiro; Sbardella, Gianluca; Castellano, Sabrina; Taliani, Sabrina; Martini, Claudia; Innis, Robert B; Da Settimo, Federico; Pike, Victor W

2014-10-15

The imaging of translocator 18 kDa protein (TSPO) in living human brain with radioligands by positron emission tomography (PET) has become an important means for the study of neuroinflammatory conditions occurring in several neuropsychiatric disorders. The widely used prototypical PET radioligand [(11)C](R)-PK 11195 ([(11)C](R)-1; [N-methyl-(11)C](R)-N-sec-butyl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide) gives a low PET signal and is difficult to quantify, whereas later generation radioligands have binding sensitivity to a human single nucleotide polymorphism (SNP) rs6971, which imposes limitations on their utility for comparative quantitative PET studies of normal and diseased subjects. Recently, azaisosteres of 1 have been developed with improved drug-like properties, including enhanced TSPO affinity accompanied by moderated lipophilicity. Here we selected three of these new ligands (7-9) for labeling with carbon-11 and for evaluation in monkey as candidate PET radioligands for imaging brain TSPO. Each radioligand was readily prepared by (11)C-methylation of an N-desmethyl precursor and was found to give a high proportion of TSPO-specific binding in monkey brain. One of these radioligands, [(11)C]7, the direct 4-azaisostere of 1, presents many radioligand properties that are superior to those reported for [(11)C]1, including higher affinity, lower lipophilicity, and stable quantifiable PET signal. Importantly, 7 was also found to show very low sensitivity to the human SNP rs6971 in vitro. Therefore, [(11)C]7 now warrants evaluation in human subjects with PET to assess its utility for imaging TSPO in human brain, irrespective of subject genotype.

Long-term reproducibility of in vivo measures of specific binding of radioligands in rat brain

Energy Technology Data Exchange (ETDEWEB)

Kilbourn, Michael R. E-mail: mkilbour@umich.edu

2004-07-01

The long-term reproducibility of measures of in vivo specific binding of radiolabeled forms of (+)-{alpha}-dihydrotetrabenazine (DTBZ) and d-threo-methylphenidate (MPH) in rat brain was examined. All studies were done using a consistent bolus plus infusion protocol and calculation of equilibrium distribution volume ratios (DVR). Over a period of eight years striatal DVR values for DTBZ binding to the vesicular monoamine transporter 2 (VMAT2) in young adult (8-10 wks old) rats showed very good reproducibility (3.62{+-}0.33, N=35). Equivalent values were obtained using either tritiated or carbon-11 labeled DTBZ, and were irrespective of sex of animals. Older animals (78 wks old) showed losses (-45%) of specific binding. Striatal binding of MPH to the dopamine transporter (DAT) showed a similar reproducibility over a five year period (DVR=2.17{+-}0.39, N=52), again irrespective of radionuclide or sex. These studies demonstrate that use of a consistent in vivo technique can provide reliable measures of specific binding of radioligands to high affinity sites in the rat brain.

Characterisation of the binding of [3H]methyllycaconitine: a new radioligand for labelling α7-type neuronal nicotinic acetylcholine receptors

International Nuclear Information System (INIS)

Davies, A.R.L.; Wolstenholme, A.J.; Wonnacott, S.; Hardick, D.J.; Blagbrough, I.S.; Potter, B.V.L.

1999-01-01

Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphinium seeds, is one of the most potent non-proteinacious ligands that is selective for αbungarotoxin-sensitive neuronal nicotinic acetylcholine receptors (nAChR). [ 3 H]MLA bound to rat brain membranes with high affinity (K d =1.86±0.31 nM) with a good ratio of specific to non-specific binding. The binding of [ 3 H]MLA was characterised by rapid association (t 1/2 =2.3 min) and dissociation (t 1/2 =12.6 min) kinetics. The radioligand binding displayed nicotinic pharmacology, consistent with an interaction with αbungarotoxin-sensitive nAChR. The snake α-toxins, αbungarotoxin and αcobratoxin, displaced [ 3 H]MLA with high affinity (K i =1.8±0.5 and 5.5±0.9 nM, respectively), whereas nicotine was less potent (K i =6.1±1.1 μM). The distribution of [ 3 H]MLA binding sites in crudely dissected rat brain regions was identical to that of [ 125 I]αbungarotoxin binding sites, with a high binding site density in hippocampus and hypothalamus, but low density in striatum and cerebellum. [ 3 H]MLA also labelled a sub-population of binding sites which are not sensitive to the snake αtoxins, but which did not differ significantly from the major population with respect to their other pharmacological properties or regional distribution. [ 3 H]MLA, therefore, is a novel radiolabel for characterising α7-type nAChR. A good signal to noise ratio and rapid binding kinetics provide advantages over the use of radiolabelled αbungarotoxin for rapid and accurate equilibrium binding assays. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

Competitive receptor binding radioassay for β-1 and β-2 adrenergic agents

International Nuclear Information System (INIS)

Hussain, M.N.; Culbreth, W.; Dalrymple, R.; Fung, C.; Ricks, C.

1987-01-01

A rapid and sensitive competitive receptor bonding assay for β-1 and β-2 adrenergic binding for adrenergic agents has been developed. The steps that are critical for the success of the assay are given in detail so that the assay can be set up in any routine laboratory with relative ease. The rationale behind the use of specific reagents is discussed. The assay requires microgram quantities of test compound, a radiolabeled specific β adrenergic antagonist [ 3 H]dihydroalprenolol (DHA), and turkey erythrocyte β-1 and rat erythrocyte β-2 receptor membranes. Serial dilutions of sample are incubated with appropriate receptor membranes and DHA for 1 hr at room temperature. After equilibrium is attained, the bound radioligand is separated by rapid filtration under vacuum through Whatman GF/B filters. The amount of bound DHA trapped on the filter is inversely proportional to the degree of β-1 and β-2 adrenergic binding of the sample. Separation of bound from free radioligand by filtration permits rapid determination of a large number of samples. This assay quantitates and differentiates β-1 and β-2 adrenergic binding of synthetic adrenergic agents

Modeling of the Renal Kinetics of the AT1 Receptor Specific PET Radioligand [11C]KR31173

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Nedim C. M. Gulaldi

2013-01-01

Full Text Available Purpose. The radioligand [11C]KR31173 has been introduced for PET imaging of the angiotensin II subtype 1 receptor (AT1R. The purpose of the present project was to employ and validate a compartmental model for quantification of the kinetics of this radioligand in a porcine model of renal ischemia followed by reperfusion (IR. Procedures. Ten domestic pigs were included in the study: five controls and five experimental animals with IR of the left kidney. To achieve IR, acute ischemia was created with a balloon inserted into the left renal artery and inflated for 60 minutes. Reperfusion was achieved by deflation and removal of the balloon. Blood chemistries, urine specific gravity and PH values, and circulating hormones of the renin angiotensin system were measured and PET imaging was performed one week after IR. Cortical time-activity curves obtained from a 90 min [11C]KR31173 dynamic PET study were processed with a compartmental model that included two tissue compartments connected in parallel. Radioligand binding quantified by radioligand retention (80 min value to maximum value ratio was compared to the binding parameters derived from the compartmental model. A binding ratio was calculated as DVR=DVS/DVNS, where DVS and DVNS represented the distribution volumes of specific binding and nonspecific binding. Receptor binding was also determined by autoradiography in vitro. Results. Correlations between rate constants and binding parameters derived by the convolution and deconvolution curve fittings were significant (r>0.9. Also significant was the correlation between the retention parameter derived from the tissue activity curve (Yret and the retention parameter derived from the impulse response function (fret. Furthermore, significant correlations were found between these two retention parameters and DVR. Measurements with PET showed no significant changes in the radioligand binding parameters caused by IR, and these in vivo findings were

Alternative Radioligands for Investigating the Molecular Pharmacology of Melatonin Receptors.

Science.gov (United States)

Legros, Céline; Brasseur, Chantal; Delagrange, Philippe; Ducrot, Pierre; Nosjean, Olivier; Boutin, Jean A

2016-03-01

Melatonin exerts a variety of physiologic activities that are mainly relayed through the melatonin receptors MT1 and MT2 Low expressions of these receptors in tissues have led to widespread experimental use of the agonist 2-[(125)I]-iodomelatonin as a substitute for melatonin. We describe three iodinated ligands: 2-(2-[(2-iodo-4,5-dimethoxyphenyl)methyl]-4,5-dimethoxy phenyl) (DIV880) and (2-iodo-N-2-[5-methoxy-2-(naphthalen-1-yl)-1H-pyrrolo[3,2-b]pyridine-3-yl])acetamide (S70254), which are specific ligands at MT2 receptors, and N-[2-(5-methoxy-1H-indol-3-yl)ethyl]iodoacetamide (SD6), an analog of 2-[(125)I]-iodomelatonin with slightly different characteristics. Here, we further characterized these new ligands with regards to their molecular pharmacology. We performed binding experiments, saturation assays, association/dissociation rate measurements, and autoradiography using sheep and rat tissues and recombinant cell lines. Our results showed that [(125)I]-S70254 is receptor, and can be used with both cells and tissue. This radioligand can be used in autoradiography. Similarly, DIV880, a partial agonist [43% of melatonin on guanosine 5'-3-O-(thio)triphosphate binding assay], selective for MT2, can be used as a tool to selectively describe the pharmacology of this receptor in tissue samples. The molecular pharmacology of both human melatonin receptors MT1 and MT2, using a series of 24 ligands at these receptors and the new radioligands, did not lead to noticeable variations in the profiles. For the first time, we described radiolabeled tools that are specific for one of the melatonin receptors (MT2). These tools are amenable to binding experiments and to autoradiography using sheep or rat tissues. These specific tools will permit better understanding of the role and implication in physiopathologic processes of the melatonin receptors. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Synthesis and pharmacological characterization of the selective GluK1 radioligand (S)-2-amino-3-(6-[3H]-2,4-dioxo-3,4-dihydrothieno.3,2-d] pyrimidin1(2H)- yl) propanoic acid ([3H]-NF608)

DEFF Research Database (Denmark)

Alcaide, Anna; Marconi, Laura; Marek, Ales

2016-01-01

The kainic acid receptors belong to the class of ionotropic glutamate receptors and comprise five subunits named GluK1-5. Radioligands are essential tools for use in binding assays aimed at ligand-receptor structure-activity-relationship studies. Previous work has led to the synthesis of GluK1...... radioligands [H-3]SYM2081, [H-3]-UBP310 and [H-3]-ATPA, however all strategies were work-intensive and thus not attractive. Herein, we report the synthesis of [H-3]-NF608 and subsequent pharmacological evaluation at homomeric recombinant rat GluK1 receptors. Binding affinities of a series of standard GluK1...... ligands were shown to be in line with previously reported affinities obtained by use of already reported radioligands....

Studies of food folates and folic acid deficiency by radioligand competitive binding assay techniques. Part of a coordinated programme on in vitro assay techniques

International Nuclear Information System (INIS)

Hettiarachchy, N.S.

1981-01-01

Conjugese extracted from winged bean or sweet potato leaves was used to release folate from Sri Lankan foodstuffs. Total folate was then estimated by competitive binding assay using goat milk as binding agent. Of 33 foodstuffs investigated, green gram, cow pea, and red gram among the pulses and mukunuvenna, amaranth and centella among the leafy vegetables were shown to be rich sources of folate. Between 20 and 60% of total folate was lost when such foodstuffs were boiled for 60 minutes. It is thus advisable that pulses and leafy vegetables be boiled only for the minimum time necessary for tenderization before consumption

Characterisation of the binding of [{sup 3}H]methyllycaconitine: a new radioligand for labelling {alpha}7-type neuronal nicotinic acetylcholine receptors

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Davies, A.R.L.; Wolstenholme, A.J.; Wonnacott, S. [Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY (United Kingdom); Hardick, D.J.; Blagbrough, I.S.; Potter, B.V.L. [Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY (United Kingdom)

1999-05-15

Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphinium seeds, is one of the most potent non-proteinacious ligands that is selective for {alpha}bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors (nAChR). [{sup 3}H]MLA bound to rat brain membranes with high affinity (K{sub d}=1.86{+-}0.31 nM) with a good ratio of specific to non-specific binding. The binding of [{sup 3}H]MLA was characterised by rapid association (t{sub 1/2}=2.3 min) and dissociation (t{sub 1/2}=12.6 min) kinetics. The radioligand binding displayed nicotinic pharmacology, consistent with an interaction with {alpha}bungarotoxin-sensitive nAChR. The snake {alpha}-toxins, {alpha}bungarotoxin and {alpha}cobratoxin, displaced [{sup 3}H]MLA with high affinity (K{sub i}=1.8{+-}0.5 and 5.5{+-}0.9 nM, respectively), whereas nicotine was less potent (K{sub i}=6.1{+-}1.1 {mu}M). The distribution of [{sup 3}H]MLA binding sites in crudely dissected rat brain regions was identical to that of [{sup 125}I]{alpha}bungarotoxin binding sites, with a high binding site density in hippocampus and hypothalamus, but low density in striatum and cerebellum. [{sup 3}H]MLA also labelled a sub-population of binding sites which are not sensitive to the snake {alpha}toxins, but which did not differ significantly from the major population with respect to their other pharmacological properties or regional distribution. [{sup 3}H]MLA, therefore, is a novel radiolabel for characterising {alpha}7-type nAChR. A good signal to noise ratio and rapid binding kinetics provide advantages over the use of radiolabelled {alpha}bungarotoxin for rapid and accurate equilibrium binding assays. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

Science.gov (United States)

Müller, C E; Maurinsh, J; Sauer, R

2000-01-01

The present study describes the preparation and binding properties of a new, potent, and selective A(2A) adenosine receptor (AR) antagonist radioligand, [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargy lxanth ine ([3H]MSX-2). [3H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [3H]MSX-2 labeled a single class of binding sites with high affinity (K(d)=8.0 nM) and limited capacity (B(max)=1.16 fmol.mg(-1) of protein). The presence of 100 microM GTP, or 10 mM magnesium chloride, respectively, had no effect on [3H]MSX-2 binding. AR agonists competed with the binding of 1 nM [3H]MSX-2 with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxami doaden osine (CGS-21680)>2-chloroadenosine (2-CADO)>N(6)-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX)>1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5, 6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2, 3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K(i) values for antagonists were in accordance with data from binding studies with the agonist radioligand [3H]CGS21680, while agonist affinities were 3-7-fold lower. [3H]MSX-2 is a highly selective A(2A) AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20-30%, at 1 nM.

Competitive protein binding assay

International Nuclear Information System (INIS)

Kaneko, Toshio; Oka, Hiroshi

1975-01-01

The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

Characterization of the binding of [3H]-(+/-)-L-364,718: a new potent, nonpeptide cholecystokinin antagonist radioligand selective for peripheral receptors

International Nuclear Information System (INIS)

Chang, R.S.; Lotti, V.J.; Chen, T.B.; Kunkel, K.A.

1986-01-01

[3H]-(+/-)-L-364,718 a new, potent and selective nonpeptide peripheral cholecystokinin (CCK) antagonist bound saturably and reversibly to rat pancreatic membranes. The radioligand recognized a single class of binding sites with a high affinity (Kd = 0.23 nM). The binding of [ 3 H]-(+/-)-L-364,718 was stereospecific in that the more biologically active (-)-enantiomer demonstrated greater potency than the (+)-enantiomer. The rank order of potency of various CCK agonists and antagonists in displacing [ 3 H]-(+/-)-L-364,718 correlated with their ability to displace [ 125 I]CCK-8 and their known pharmacological activities in peripheral tissues. However, the absolute potencies of agonists were greater in displacing [ 125 I]CCK-8 than [ 3 H]-(+/-)-L-364,718. As described for other physiologically relevant receptor systems, the potency for displacement of [ 3 H]-(+/-)-L-364,718 binding by CCK agonists, but not antagonists, was reduced by guanosine 5'-(beta, gamma-imido)triphosphate and NaCl and enhanced by MgCl 2 . [ 3 H]-(+/-)-L-364,718 also demonstrated specific binding to bovine gall bladder tissue but not guinea pig brain or gastric glands, consistent with its selectivity as a peripheral CCK antagonist. [ 3 H]-(+/-)-L-364,718 binding to pancreatic membranes was not affected by various pharmacological agents known to interact with other common peptide and nonpeptide receptor systems. These data indicate that [ 3 H]-(+/-)-L-364,718 represents a new potent nonpeptide antagonist radioligand for the study of peripheral CCK receptors which may allow differentiation of agonist and antagonist interactions

Interference of anaesthetics with radioligand binding in neuroreceptor studies

International Nuclear Information System (INIS)

Elfving, Betina; Knudsen, Gitte Moos; Bjoernholm, Berith

2003-01-01

Evaluations of new emission tomography ligands are usually carried out in animals. In order to keep the animals in a restricted position during the scan session, anaesthesia is almost inevitable. In ex vivo rat studies we investigated the interference of ketamine/xylazine, zoletile mixture, isoflurane and halothane with the serotonin re-uptake site, the serotonin 2A receptor and the dopamine re-uptake site by use of [ 3 H]-(S)-citalopram, [ 18 F]altanserin and [ 125 I]PE2I, respectively. Ketamine/xylazine decreased the target-to-background ratio (mean ± SD) of [ 3 H]-(S)-citalopram from 1.5±0.19 to 0.81±0.19 (P 18 F]altanserin. The [ 125 I]PE2I target-to-background ratio decreased with both ketamine/xylazine (from 12.4±0.81 to 10.1±1.4, P<0.05) and isoflurane (from 12.4±0.81 to 9.5±1.1, P<0.05) treated rats, whereas treatment with zoletile mixture and halothane left the ratio unaltered. It is concluded that prior to performance of neuroreceptor radioligand studies, the possible interaction between radioligands and anaesthetics should be carefully evaluated. (orig.)

Radioreceptor assay: theory and applications to pharmacology

International Nuclear Information System (INIS)

Perret, G.; Simon, P.

1984-01-01

The aim of the first part of this work is to present the theory of the radioreceptor assay and to compare it to the other techniques of radioanalysis (radioimmunoassay, competitive protein binding assays). The technology of the radioreceptor assay is then presented and its components (preparation of the receptors, radioligand, incubation medium) are described. The analytical characteristics of the radioreceptor assay (specificity, sensitivity, reproductibility, accuracy) and the pharmacological significance of the results are discussed. The second part is devoted to the description of the radioreceptor assays of some pharmacological classes (neuroleptics, tricyclic antidepressants, benzodiazepines, β-blockers, anticholinergic drugs) and to their use in therapeutic drug monitoring. In conclusion, by their nature, radioreceptor assays are highly sensitive, reliable, precise, accurate and simple to perform. Their chief disadvantage relates to specificity, since any substance having an appreciable affinity to the receptor site will displace the specifically bound radioligand. Paradoxically in some cases, this lack of specificity may be advantageous in that it allows for the detection of not only the apparent compound but of active metabolites and endogenous receptor agonists as well and in that radioreceptors assays can be devised for a whole pharmacological class and not only for one drug as it is the case for classical physico-chemical techniques. For all these reasons future of radioreceptor assay in pharmacology appears promising [fr

Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

International Nuclear Information System (INIS)

Liang, B.T.

1989-01-01

Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand [3H]-8-cyclopentyl-1,3-diproylxanthine ([3H]CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or the maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that [3H] CPX is an antagonist radioligand. Competition curves for [3H] CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific [3H]CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid)

Determination of luteinizing hormone in bovine blood by radioligand receptor assay and comparison with radioimmunological evaluation

International Nuclear Information System (INIS)

Schams, D.; Menzer, C.

1978-01-01

A sensitive and specific radioligand receptor assay (RRA) using rat testis homogenate as the receptor source is described for measurement of luteinizing hormone (LH) in bovine blood. Interfering and nonspecific substances in blood were removed by means of ion-exchange chromatography on CM-Sephadex C-50. Criteria of validation such as recovery of added LH to plasma or serum, reproducibility, and specificity gave good results. Inhibition curves obtained with bovine plasma and serum were parallel to those obtained with the bovine standard preparation. The range of the dose-response curve was between 0.5-20 ng of bovine LH. The pattern of LH concentrations in purified serum samples under different physiological conditions such as during the oestrous cycle and after administration of GnRH showed a very close correlattion whether measured by means of radioimmunoassay (RIA) or receptor assay. Values of RRA-LH were consistently higher than those of RIA-LH. Thus the lower the RIA-LH levels, the more pronounced were the discrepancies between results of both assay systems. The mean ratio of RRA-LH/RIA-LH for basal levels (less than 1 ng RIA-LH/ml plasma) was 17.8 as compared to a mean ratio for higher peak values (more than 20 ng RIA-LH/ml plasma) of only 1.2. (author)

Neurokinin-3 Receptor Binding in Guinea Pig, Monkey, and Human Brain: In Vitro and in Vivo Imaging Using the Novel Radioligand, [18F]Lu AF10628.

Science.gov (United States)

Varnäs, Katarina; Finnema, Sjoerd J; Stepanov, Vladimir; Takano, Akihiro; Tóth, Miklós; Svedberg, Marie; Møller Nielsen, Søren; Khanzhin, Nikolay A; Juhl, Karsten; Bang-Andersen, Benny; Halldin, Christer; Farde, Lars

2016-08-01

Previous autoradiography studies have suggested a marked interspecies variation in the neuroanatomical localization and expression levels of the neurokinin 3 receptor, with high density in the brain of rat, gerbil, and guinea pig, but at the time offered no conclusive evidence for its presence in the human brain. Hitherto available radioligands have displayed low affinity for the human neurokinin 3 receptor relative to the rodent homologue and may thus not be optimal for cross-species analyses of the expression of this protein. A novel neurokinin 3 receptor radioligand, [(18)F]Lu AF10628 ((S)-N-(cyclobutyl(3-fluorophenyl)methyl)-8-fluoro-2-((3-[(18)F]-fluoropropyl)amino)-3-methyl-1-oxo-1,2-dihydroisoquinoline-4-carboxamide), was synthesized and used for autoradiography studies in cryosections from guinea pig, monkey, and human brain as well as for positron emission tomography studies in guinea pig and monkey. The results confirmed previous observations of interspecies variation in the neurokinin 3 receptor brain localization with more extensive distribution in guinea pig than in primate brain. In the human brain, specific binding to the neurokinin 3 receptor was highest in the amygdala and in the hypothalamus and very low in other regions examined. Positron emission tomography imaging showed a pattern consistent with that observed using autoradiography. The radioactivity was, however, found to accumulate in skull bone, which limits the use of this radioligand for in vivo quantification of neurokinin 3 receptor binding. Species differences in the brain distribution of neurokinin 3 receptors should be considered when using animal models for predicting human neurokinin 3 receptor pharmacology. For positron emission tomography imaging of brain neurokinin 3 receptors, additional work is required to develop a radioligand with more favorable in vivo properties. © The Author 2016. Published by Oxford University Press on behalf of CINP.

Difference in brain distributions of carbon 11-labeled 4-hydroxy-2(1H)-quinolones as PET radioligands for the glycine-binding site of the NMDA ion channel

Energy Technology Data Exchange (ETDEWEB)

Fuchigami, Takeshi [Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582 (Japan); Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Haradahira, Terushi [Molecular Imaging Center, National Institute of Radiological Sciences, Inage-ku, Chiba 263-8555 (Japan)], E-mail: terushi@niu.ac.jp; Fujimoto, Noriko [Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582 (Japan); Okauchi, Takashi; Maeda, Jun; Suzuki, Kazutoshi; Suhara, Tetsuya [Molecular Imaging Center, National Institute of Radiological Sciences, Inage-ku, Chiba 263-8555 (Japan); Yamamoto, Fumihiko; Sasaki, Shigeki; Mukai, Takahiro [Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582 (Japan); Yamaguchi, Hiroshi [Molecular Imaging Frontier Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Ogawa, Mikako [Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Magata, Yasuhiro [Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Molecular Imaging Frontier Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Maeda, Minoru [Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582 (Japan)

2008-02-15

High-affinity iodine- and ethyl-C-5 substituted analogs of 4-hydroxy-3-(3-[{sup 11}C]methoxyphenyl)-2(1H)-quinolone ([{sup 11}C]4HQ) were synthesized as new positron emission tomography radioligands for the glycine-binding sites of the N-methyl-D-aspartate (NMDA) ion channel. Although both radioligands showed high in vitro specific binding to rat brain slices, their binding characteristics were quite different from each other. 5-Ethyl-[{sup 11}C]4HQ (5Et-[{sup 11}C]4HQ) showed higher in vitro binding in the forebrain regions than in the cerebellum, bindings that were strongly inhibited by both glycine-site agonists and antagonists. In contrast, 5-iodo-[{sup 11}C]4HQ (5I-[{sup 11}C]4HQ) showed a homogeneous in vitro binding throughout the brain, which was inhibited by antagonists but not by agonists. This difference in in vitro binding between 5Et-[{sup 11}C]4HQ and 5I-[{sup 11}C]4HQ was quite similar to that previously observed between [{sup 11}C]L-703,717 and [{sup 11}C]4HQ, both glycine-site antagonists. In vivo brain uptakes of these {sup 11}C-labeled 4-hydroxyquinolones were examined in mice. Initial brain uptakes of 5Et- and 5I-[{sup 11}C]4HQ at 1 min after intravenous injections were comparable to that of [{sup 11}C]4HQ, but they were 1.3-2.1 times higher than that of [{sup 11}C]L-703,717. The treatment with an anticoagulant, warfarin, only slightly increased the initial uptakes of [{sup 11}C]4HQ and 5Et-[{sup 11}C]4HQ in contrast to [{sup 11}C]L-703,717. The in vivo regional brain distributions were slightly different between the two radioligands. Pretreatment with nonradioactive ligand (2 mg/kg) slightly inhibited the binding of 5Et-[{sup 11}C]4HQ (16-36% inhibition) but not that of 5I-[{sup 11}C]4HQ. In this study, it was found that a small structural change in [{sup 11}C]4HQ resulted in a major change in binding characteristics and distributions, suggesting the existence of two binding sites for [{sup 11}C]4-hydroxyquinolones on the NMDA ion channel

Radiosynthesis and in vitro validation of 3H-NS14492 as a novel high affinity alpha7 nicotinic receptor radioligand

DEFF Research Database (Denmark)

Magnussen, Janus H.; Ettrup, Anders; Donat, Cornelius K.

2015-01-01

The neuronal alpha 7 nicotinic acetylcholine receptor is a homo-pentameric ligand-gated ion channel that is a promising drug target for cognitive deficits in Alzheimer's disease and schizophrenia. We have previously described 11C-NS14492 as a suitable agonist radioligand for in vivo positron...... emission tomography (PET) occupancy studies of the alpha 7 nicotinic receptor in the pig brain. In order to investigate the utility of the same compound for in vitro studies, 3H-NS14492 was synthesized and its binding properties were characterized using in vitro autoradiography and homogenate binding...... assays in pig frontal cortex. 3H-NS14492 showed specific binding to alpha 7 nicotinic receptors in autoradiography, revealing a dissociation constant (Kd) of 2.1 ± 0.7 nM and a maximum number of binding sites (Bmax) of 15.7±2.0 fmol/mg tissue equivalent. Binding distribution was similar...

Use of [125I]-iodohistamine-labelled steroid derivatives as radioligands for radioimmunoassay of natural and synthetic steroids

International Nuclear Information System (INIS)

Stanczyk, F.Z.; Goebelsmann, U.

1981-01-01

[ 125 I]-Iodohistamine-labelled steroid derivatives were prepared and utilized as radioligands in radioimmunoassays of progesterone, testosterone, estradiol, estriol, estriol-16α-glucuronide, levonorgestrel, norethindrone and medroxyprogesterone acetate. The binding of these iodinated radioligands was compared to that of the corresponding tritiated steroids and their effect on the sensitivity and slope of standard curves was examined. The results demonstrate that much higher antibody dilutions could be used with iodinated than with tritiated radioligands. In general, standard curves obtained with iodinated radioligands were more sensitive than those obtained with tritiated steroids, but standard curves had steeper slopes when tritiated rather than iodinated radioligands were used. The data, summarizing our 5-year experience with steroid-[ 125 I]-iodohistamine derivatives, indicate that these tracers play an important role in radioimmunoassay systems for both natural and synthetic steroids. (author)

A consistent and efficient graphical analysis method to improve the quantification of reversible tracer binding in radioligand receptor dynamic PET studies

OpenAIRE

Zhou, Yun; Ye, Weiguo; Brašić, James R.; Crabb, Andrew H.; Hilton, John; Wong, Dean F.

2008-01-01

The widely used Logan plot in radioligand receptor dynamic PET studies produces marked noise-induced negative biases in the estimates of total distribution volume (DVT) and binding potential (BP). To avoid the inconsistencies in the estimates from the Logan plot, a new graphical analysis method was proposed and characterized in this study. The new plot with plasma input and with reference tissue input was first derived to estimate DVT and BP. A condition was provided to ensure that the estima...

Interference of anaesthetics with radioligand binding in neuroreceptor studies

Energy Technology Data Exchange (ETDEWEB)

Elfving, Betina; Knudsen, Gitte Moos [Neurobiology Research Unit N9201, University hospital Rigshospitalet, 9 Blegdamsvej, 2100, Copenhagen (Denmark); Bjoernholm, Berith [Department of Computational Chemistry, H. Lundbeck A/S, Copenhagen-Valby (Denmark)

2003-06-01

Evaluations of new emission tomography ligands are usually carried out in animals. In order to keep the animals in a restricted position during the scan session, anaesthesia is almost inevitable. In ex vivo rat studies we investigated the interference of ketamine/xylazine, zoletile mixture, isoflurane and halothane with the serotonin re-uptake site, the serotonin{sub 2A} receptor and the dopamine re-uptake site by use of [{sup 3}H]-(S)-citalopram, [{sup 18}F]altanserin and [{sup 125}I]PE2I, respectively. Ketamine/xylazine decreased the target-to-background ratio (mean {+-} SD) of [{sup 3}H]-(S)-citalopram from 1.5{+-}0.19 to 0.81{+-}0.19 (P<0.05), whereas isoflurane and halothane increased the ratio from 1.5{+-}0.19 to 1.9{+-}0.24 and 2.1{+-}0.13 (P<0.05), respectively. Only with the zoletile mixture did the ratio remain unaltered. None of the tested anaesthetics affected the target-to-background ratio of [{sup 18}F]altanserin. The [{sup 125}I]PE2I target-to-background ratio decreased with both ketamine/xylazine (from 12.4{+-}0.81 to 10.1{+-}1.4, P<0.05) and isoflurane (from 12.4{+-}0.81 to 9.5{+-}1.1, P<0.05) treated rats, whereas treatment with zoletile mixture and halothane left the ratio unaltered. It is concluded that prior to performance of neuroreceptor radioligand studies, the possible interaction between radioligands and anaesthetics should be carefully evaluated. (orig.)

Histamine type I (H1) receptor radioligand binding studies on normal T cell subsets, B cells, and monocytes

International Nuclear Information System (INIS)

Cameron, W.; Doyle, K.; Rocklin, R.E.

1986-01-01

A single, specific binding site for [ 3 H]pyrilamine on normal human T helper, T suppressor, B cells, and monocytes was documented. The binding of the radioligand to its receptor is reversible with cold H 1 antagonist, saturates at 40 to 60 nM, and binding equilibrium is achieved in 2 to 4 min. Using a computer program (Ligand), the authors calculated the dissociation constants, binding capacities, and numbers of receptors per cell for each of the different cell types. Monocytes were found to have the highest affinity for [ 3 H]pyrilamine, followed by T helper cells, B cells and T suppressor cells (K/sub D/ = 44.6 +/- 49.4 nM). T suppressor cells were found to express the higher number of H 1 receptors per cell followed by B cells, T helper cells, and monocytes. The binding affinity for [ 3 H]pyrilamine increased over a 48-hr period, whereas the number of receptors per T cell was essentially unchanged. In contrast, T cells stimulated with Con A or PHA were shown to have a greater than fourfold increase in the number of receptors per cell, whereas the binding affinity for [ 3 H]pyrilamine decreased over the 48-hr period. Although the function of H 1 receptors on T cells, B cells, and monocytes has not been completely defined, this receptor has the potential of playing an important role in the modulating the immune response

Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

Directory of Open Access Journals (Sweden)

Meyyammai Swaminathan

2014-06-01

Full Text Available Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM, comparable to that of acetylcholine (Ki = 59 µM. Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

Radiotracers for per studies of neurotransmitter binding sites: Design considerations

International Nuclear Information System (INIS)

Kilbourn, M.R.

1991-01-01

Neurotransmitter binding sites, such as receptors, neuronal uptake systems, and vesicular uptake systems, are important targets for new radiopharmaceutical design. Selection of potential radioligands can be guided by in vitro laboratory data including such characteristics as selectivity and affinity for specific binding sites. However, development of PET radiotracers for use in vivo must include considerations of in vivo pharmacokinetics and metabolism. Introduction of potential radioligands is further narrowed by the demands of the radiochemical synthesis, which must produce radioligands of high chemical and radiochemical purity and of high specific activity. This paper will review examples of previous and current attempts by radiopharmaceutical chemists to meet these demands for new positron emitter-labeled radioligands for PET studies of a wide array of neurotransmitter binding sites

Cerebellar heterogeneity and its impact on PET data quantification of 5-HT receptor radioligands

DEFF Research Database (Denmark)

Ganz, Melanie; Feng, Ling; Hansen, Hanne Demant

2017-01-01

standardized uptake values (SUV) and nondisplaceable neocortical binding potential (BPND). Statistical difference was assessed with paired nonparametric two-sided Wilcoxon signed-rank tests and multiple comparison corrected via false discovery rate. We demonstrate significant radioligand specific regional...

125I-labeled 8-phenylxanthine derivatives: antagonist radioligands for adenosine A1 receptors

International Nuclear Information System (INIS)

Linden, J.; Patel, A.; Earl, C.Q.; Craig, R.H.; Daluge, S.M.

1988-01-01

A series of 8-phenylxanthine derivatives has been synthesized with oxyacetic acid on the para phenyl position to increase aqueous solubility and minimize nonspecific binding and iodinatable groups on the 1- or 3-position of the xanthine ring. The structure-activity relationship for binding of these compounds to A1 adenosine receptors of bovine and rat brain and A2 receptors of human platelets was examined. The addition of arylamine or photosensitive aryl azide groups to the 3-position of xanthine had little effect on A1 binding affinity with or without iodination, whereas substitutions at the 1-position caused greatly reduced A1 binding affinity. The addition of an aminobenzyl group to the 3-position of the xanthine had little effect on A2 binding affinity, but 3-aminophenethyl substitution decreased A2 binding affinity. Two acidic 3-(arylamino)-8-phenylxanthine derivatives were labeled with 125 I and evaluated as A1 receptor radioligands. The new radioligands bound to A1 receptors with KD values of 1-1.25 nM. Specific binding represented over 80% of total binding. High concentrations of NaCl or other salts increased the binding affinity of acidic but not neutral antagonists, suggesting that interactions between ionized xanthines and receptors may be affected significantly by changes in ionic strength. On the basis of binding studies with these antagonists and isotope dilution with the agonist [ 125 I]N6-(4-amino-3-iodobenzyl)adenosine, multiple agonist affinity states of A1 receptors have been identified

Differential binding of 125I-IGF-I preparations to human fibroblast monolayers

International Nuclear Information System (INIS)

Conover, C.A.; Misra, P.; Hintz, R.L.; Rosenfeld, R.G.

1988-01-01

Specific, high affinity binding of 125 I-IGF-I to the type IIGF receptor on human fibroblast monolyaers was not altered by varying feeding schedules, serum lots, washing procedures, or incubation times and temperatures. However, markedly different competitive binding curves were obtained when different iodinated IGF-I preparations were used. Five of six radioligands bound preferentially to the type IIGF receptor on human fibroblast monolayers, with 50% displacement at 4-8 μg/l unlabelled IGF-I; with one radioligand a paradoxical 20-200% increase in 125 I-IGF-I binding was observed at low concentrations of unlabelled IGF-I, while concentrations as high as 100 μg/l IGF-I failed to displace this radioligand. The latter binding pattern cannot be accounted for by 125 -I-IGF-I binding to the type II IGF receptor. These data indicate that various radioligands may have preferential affinities for different IGF-I binding sites on human fibroblast monolayers. (author)

A study of 19-0-carboxymethyl ether and 19-hemisuccinate derivatives of testosterone: their immunogenicity and use as iodinated radioligands for radioimmunoassay of testosterone

International Nuclear Information System (INIS)

White, A.; Crosby, S.R.; Ratcliffe, W.A.; Smith, G.N.

1985-01-01

Testosterone 19-0-carboxymethyl ether (T19C) and 19-hemisuccinate (T19H) derivatives were synthesised and coupled to bovine serum albumin (BSA) or porcine thyroglobulin (PT) for immunogens or to iodohistamine for radioligands. The immunogenicity of these conjugates in mice was compared with those of testosterone 3-0-carboxymethyloxime and 15β-thioethyl conjugates. Of 10 immunogens studied, those linked to PT gave the highest antiserum titres and more sensitive standard curves. Cross-reactivity with 5α-dihydrotestosterone (DHT) was in the range 22-100%, for the 19-linked immunogens. Antisera to T19C and T19H conjugated to PT were then raised in rabbits and characterised with 4 radioligands. Homologous assay systems in which the chemical bridge was identical in immunogen and 125 I-radioligand gave the highest antiserum titres but the poorest assay sensitivity while those heterologous with respect to bridge or site gave the most sensitive standard curves. Rabbit antisera to both T19C and T19H immunogens showed good specificities with respect to DHT androstenedione (AN) and progesterone (PO) with all radioligands. The best assay system employed an antiserum to the T19H-PT immunogen with heterologous radioligand [ 125 I]T19C. It had a detection limit of 15pg/tube and low cross-reactivity with DHT and AN. (author)

Rapid, radiochemical-ligand binding assay for methotrexate

International Nuclear Information System (INIS)

Caston, J.D.

1976-01-01

A radiochemical ligand binding assay for methotrexate is provided. A binder factor comprising a partially purified dihydrofolic acid reductase preparation is employed. The binder factor is conveniently prepared by homogenizing a factor containing animal organ such as liver, and extracting with isotonic saline and ammonium sulfate. A binder cofactor, NADPH 2 , is also employed in the binding reaction. The procedure contemplates both direct and sequential assay techniques, and it is not interfered with by vast excesses of many natural folate derivatives. 12 claims, 6 drawing figures

A New Positron Emission Tomography (PET) Radioligand for Imaging Sigma-1 Receptors in Living Subjects

DEFF Research Database (Denmark)

James, Michelle L; Shen, Bin; Zavaleta, Cristina L

2012-01-01

of the radioligand in S1R rich regions of the brain. Pre-treatment with 1 mg/kg haloperidol (2), non-radioactive 13, or BD1047 (18) reduced the binding of [(18)F]13 in the brain at 60 min by 80%, 82% and 81% respectively, suggesting that [(18)F]13 accumulation in mouse brain represents specific binding to S1Rs...

Improved assay for measuring heparin binding to bull sperm

International Nuclear Information System (INIS)

Miller, D.J.; Ax, R.L.

1988-01-01

The binding of heparin to sperm has been used to study capacitation and to rank relative fertility of bulls. Previous binding assays were laborious, used 10 7 sperm per assay point, and required large amounts of radiolabeled heparin. A modified heparin-binding assay is described that used only 5 x 10 4 cells per incubation well and required reduced amounts of [ 3 H] heparin. The assay was performed in 96-well Millititer plates, enabling easy incubation and filtering. Dissociation constants and concentrations of binding sites did not differ if analyzed by Scatchard plots, Woolf plots, or by log-logit transformed weighted nonlinear least squares regression, except in the case of outliers. In such cases, Scatchard analysis was more sensitive to outliers. Nonspecific binding was insignificant using nonlinear logistic fit regression and a proportion graph. The effects were tested of multiple free-thawing of sperm in either a commercial egg yolk extender, 40 mM Tris buffer with 8% glycerol, or 40 mM Tris buffer without glycerol. Freeze-thawing in extender did not affect the dissociation constant or the concentration of binding sites. However, freeze-thawing three times in 40 mM Tris reduced the concentration of binding sites and lowered the dissociation constant (raised the affinity). The inclusion of glycerol in the 40 mM Tris did not significantly affect the estimated dissociation constant or the concentration of binding sites as compared to 40 mM Tris without glycerol

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Modelling the interdependence between the stoichiometry of receptor oligomerization and ligand binding for a coexisting dimer/tetramer receptor system.

Science.gov (United States)

Rovira, X; Vivó, M; Serra, J; Roche, D; Strange, P G; Giraldo, J

2009-01-01

Many G protein-coupled receptors have been shown to exist as oligomers, but the oligomerization state and the effects of this on receptor function are unclear. For some G protein-coupled receptors, in ligand binding assays, different radioligands provide different maximal binding capacities. Here we have developed mathematical models for co-expressed dimeric and tetrameric species of receptors. We have considered models where the dimers and tetramers are in equilibrium and where they do not interconvert and we have also considered the potential influence of the ligands on the degree of oligomerization. By analogy with agonist efficacy, we have considered ligands that promote, inhibit or have no effect on oligomerization. Cell surface receptor expression and the intrinsic capacity of receptors to oligomerize are quantitative parameters of the equations. The models can account for differences in the maximal binding capacities of radioligands in different preparations of receptors and provide a conceptual framework for simulation and data fitting in complex oligomeric receptor situations.

High non-specific binding of the {beta}{sub 1}-selective radioligand 2-{sup 125}I-ICI-H

Energy Technology Data Exchange (ETDEWEB)

Riemann, B. [Muenster Univ. (Germany). Department of Nuclear Medicine; Law, M.P. [Muenster Univ. (Germany). Department of Nuclear Medicine; Hammersmith Hospital, London (United Kingdom). MRC Clinical Sciences Centre; Kopka, K. [Muenster Univ. (DE). Department of Nuclear Medicine] [and others

2003-08-01

Aim: As results of cardiac biopsies suggest, myocardial {beta}{sub 1}-adrenoceptor density is reduced in patients with chronic heart failure. However, changes in cardiac {beta}{sub 2}-adrenoceptors vary. With suitable radiopharmaceuticals single photon emission computed tomography (SPECT) and positron emission tomography (PET) offer the opportunity to assess {beta}-adrenoceptors non-invasively. Among the novel racemic analogues of the established {beta}{sub 1}-selective adrenoceptor antagonist ICI 89.406 the iodinated 2-I-ICI-H showed high affinity and selectivity to {beta}{sub 1}-adrenoceptors in murine ventricular membranes. The aim of this study was its evaluation as a putative subtype selective {beta}{sub 1}-adrenergic radioligand in cardiac imaging. Methods: Competition studies in vitro and in vivo were used to investigate the kinetics of 2-I-ICI-H binding to cardiac {beta}-adrenoceptors in mice and rats. In addition, the radiosynthesis of 2-{sup 125}I-ICI-H from the silylated precursor 2-SiMe{sub 3}-ICI-H was established. The specific activity was 80 GBq/{mu}mol, the radiochemical yield ranged from 70 to 80%. Results: The unlabelled compound 2-I-ICI-H showed high {beta}{sub 1}-selectivity and -affinity in the in vitro competition studies. In vivo biodistribution studies apparently showed low affinity to cardiac {beta}-adrenoceptors. The radiolabelled counterpart 2-{sup 125}I-ICI-H showed a high degree of non-specific binding in vitro and no specific binding to cardiac {beta}{sub 1}-adrenoceptors in vivo. Conclusion: Because of its high non-specific binding 2-{sup 125}I-ICI-H is no suitable radiotracer for imaging in vivo. (orig.)

In Vitro Binding of [³H]PSB-0413 to P2Y₁₂ Receptors.

Science.gov (United States)

Dupuis, Arnaud; Heim, Véronique; Ohlmann, Philippe; Gachet, Christian

2015-12-08

The P2Y₁₂/ADP receptor plays a central role in platelet activation. Characterization of this receptor is mandatory for studying disorders associated with a P2Y₁₂ receptor defect and for evaluating P2Y₁₂ receptor agonists and antagonists. In the absence of suitable anti-P2Y₁₂ antibodies, radioligand binding assays are the only way to conduct such studies. While various radioligands were employed in the past for this purpose, none were found to be suitable for routine use. Described in this unit are protocols for quantitatively and qualitatively assessing P2Y₁₂ receptors with [³H]PSB-0413, a selective antagonist for this site. The saturation and competition assays described herein make possible the determination of P2Y₁₂ receptor density on cells, as well as the potencies and affinities of test agents at this site. Copyright © 2015 John Wiley & Sons, Inc.

Radiosynthesis and evaluation of new α1-adrenoceptor antagonists as PET radioligands for brain imaging

International Nuclear Information System (INIS)

Airaksinen, Anu J.; Finnema, Sjoerd J.; Balle, Thomas; Varnäs, Katarina; Bang-Andersen, Benny; Gulyás, Balázs; Farde, Lars; Halldin, Christer

2013-01-01

Introduction: Evaluation of the α 1 -adrenoceptors in relation to brain pathophysiology and drug treatment has been hindered by lack of α 1 -adrenoceptor specific radioligands with sufficient brain exposure. Our aim was to develop an α 1 -adrenoceptor specific PET radioligand for brain imaging. Methods: Two sertindole analogues 1-(4-fluorophenyl)-5-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(1-[ 11 C] methylpiperidin-4-yl)-1H-indole [ 11 C]3 and 1-(4-fluorophenyl)-3-(1-[ 11 C]methylpiperidin-4-yl)-5-(pyrimidin-5-yl) -1H-indole ([ 11 C]Lu AA27122) [ 11 C]4 were synthesized and evaluated as α 1 -adrenoceptor PET radioligands in cynomolgus monkey. Compounds 3 and 4 were selected due to their promising in vitro preclinical profile; high affinity and selectivity for the α 1 -adrenoceptor, favourable blood brain barrier permeability rates in Caco-2 monolayers and promising brain tissue/plasma ratio, assessed by equilibrium dialysis of free fraction in plasma and brain homogenate. Results: Compounds [ 11 C]3 and [ 11 C]4 were synthesized from their desmethyl piperidine precursors with high specific radioactivity (> 370 GBq/μmol) using [ 11 C]methyl iodide. The 1,2,4-triazole analogue [ 11 C]3 exhibited poor brain uptake, but the corresponding pyrimidyl analogue [ 11 C]4 exhibited high brain exposure and binding in α 1 -adrenoceptor rich brain regions. However, the binding could not be inhibited by pretreatment with prazosin (0.1 mg/kg and 0.3 mg/kg). The results were extended by autoradiography of [ 11 C]4 binding in human brain sections and competition with antagonists from different structural families, revealing that only a minor portion of the observed binding of [ 11 C]4 in brain was α 1 -adrenoceptor specific. Conclusion: Though [ 11 C]3 and [ 11 C]4 proved not suitable as PET radioligands, the study provided further understanding of structural features influencing brain exposure of the chemical class of compounds related to the antipsychotic drug sertindole. It

Competitive binding thyroid assay with improved bound-free separation step

International Nuclear Information System (INIS)

1979-01-01

A competitive binding assay is described for serum thyroid hormone using 125 I-labelled thyroid hormone and exogenous thyroid hormone binding protein. The unbound thyroid hormone is separated from thyroid hormone bound to thyroid hormone binding protein using an intermediate base anion exchange resin. This resin comprises tertiary and quaternary amine groups on a polyalkyleneamine lattice and is compressed with microcrystalline cellulose in a tablet form. The assay technique of the present invention employing an intermediate base anion resin was found to give superior results compared with alternative assay techniques used in serum thyroid hormone estimation. (UK)

Competitive binding assay for fructose 2,6-bisphosphate

International Nuclear Information System (INIS)

Thomas, H.; Uyeda, K.

1986-01-01

A new direct assay method for fructose 2,6-bisphosphate has been developed based on competitive binding of labeled and unlabeled fructose 2,6-P 2 to phosphofructokinase. Phosphofructokinase (0.5-1.3 pmol promoter) is incubated with saturating concentrations (5.0-5.5 pmol) of fructose 2,6[2- 32 P]P 2 and samples containing varying concentrations of fructose 2,6-P 2 . The resulting stable binary complex is retained on nitrocellulose filters with a binding efficiency of up to 70%. Standard curves obtained with this assay show strict linearity with varying fructose 2,6-P 2 in the range of 0.5 to 45 pmol, which exceeds the sensitivity of most of the previously described assay methods. Fructose 2,6-P 2 , ATP, and high concentrations of phosphate interfere with this assay. However, the extent of this inhibition is negligible since their tissue contents are one-half to one-tenth that examined. The new assay is simple, direct, rapid, and does not require pretreatment

Muscarinic and alpha 1-adrenergic receptor binding characteristics of saw palmetto extract in rat lower urinary tract.

Science.gov (United States)

Suzuki, Mayumi; Oki, Tomomi; Sugiyama, Tomomi; Umegaki, Keizo; Uchida, Shinya; Yamada, Shizuo

2007-06-01

To elucidate the in vitro and ex vivo effects of saw palmetto extract (SPE) on autonomic receptors in the rat lower urinary tract. The in vitro binding affinities for alpha 1-adrenergic, muscarinic, and purinergic receptors in the rat prostate and bladder were measured by radioligand binding assays. Rats received vehicle or SPE (0.6 to 60 mg/kg/day) orally for 4 weeks, and alpha 1-adrenergic and muscarinic receptor binding in tissues of these rats were measured. Saw palmetto extract inhibited specific binding of [3H]prazosin and [N-methyl-3H]scopolamine methyl chloride (NMS) but not alpha, beta-methylene adenosine triphosphate [2,8-(3)H]tetrasodium salt in the rat prostate and bladder. The binding activity of SPE for muscarinic receptors was four times greater than that for alpha 1-adrenergic receptors. Scatchard analysis revealed that SPE significantly reduced the maximal number of binding sites (Bmax) for each radioligand in the prostate and bladder under in vitro condition. Repeated oral administration of SPE to rats brought about significant alteration in Bmax for prostatic [3H]prazosin binding and for bladder [3H]NMS binding. Such alteration by SPE was selective to the receptors in the lower urinary tract. Saw palmetto extract exerts significant binding activity on autonomic receptors in the lower urinary tract under in vitro and in vivo conditions.

Investigation and characterization of receptors for pituitary adenylate cyclase-activating polypeptide in human brain by radioligand binding and chemical cross-linking

International Nuclear Information System (INIS)

Suda, K.; Smith, D.M.; Ghatei, M.A.; Murphy, J.K.; Bloom, S.R.

1991-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel peptide of hypothalamic origin which increases adenylate cyclase activity in rat anterior pituitary cell cultures. The 38-amino acid peptide shows a close sequence homology to vasoactive intestinal peptide (VIP). Binding sites for PACAP in membranes from postmortem human brain tissue were studied using [ 125 I]PACAP27 as the radioligand. High specific binding sites (amount of specific binding measured at 0.25 nM [ 125 I]PACAP27 in femtomoles per mg protein +/- SEM; n = 4) were present in hypothalamus (344.5 +/- 13.0), brain stem (343.0 +/- 29.3), cerebellum (292.0 +/- 21.1), cortex (259.6 +/- 19.8), and basal ganglia (259.2 +/- 50.3). Specific binding sites in pituitary, although present, were less abundant (35.0 +/- 8.9). Binding of [ 125 I]PACAP27 was reversible and time, pH, and temperature dependent. Despite the homology with VIP, VIP was a poor inhibitor of [ 125 I]PACAP27 binding (IC50, greater than 1 microM) compared with PACAP27 (IC50, 0.5-1.3 nM) and PACAP38 (IC50, 0.2-1.3 nM). Scatchard plots of [ 125 I]PACAP27 binding showed the presence of both high and lower affinity sites. Chemical cross-linking of PACAP-binding sites revealed that [ 125 I]PACAP27 was bound to polypeptide chains of 67,000 and 48,000 mol wt. Thus, we have demonstrated the presence of PACAP-specific receptors in human brain which are not VIP receptors. This opens the possibility of PACAP functioning as a novel neurotransmitter/neuromodulator in human brain

Evaluation of [O-methyl-11C]RS-15385-197 as a positron emission tomography radioligand for central α2-adrenoceptors

International Nuclear Information System (INIS)

Hume, S.P.; Hirani, E.; Opacka-Juffry, J.; Osman, S.; Myers, R.; Gunn, R.N.; McCarron, J.A.; Pike, V.W.; Clark, R.D.; Melichar, J.; Nutt, D.J.

2000-01-01

Carbon-11 labelled RS-15385-197 and its ethylsulphonyl analogue, RS-79948-197, were evaluated in rats as potential radioligands to image central α 2 -adrenoceptors in vivo. The biodistributions of both compounds were comparable with that obtained in an earlier study using tritiated RS-79948-197 and were consistent with the known localisation of α 2 -adrenoceptors. The maximal signals (total to non-specific binding) were, however, reduced, in the order [ 11 C]RS-79948-197 11 C]RS-15385-197 3 H]RS-79948-197, primarily due to the difference in radiolabel position (O-methyl for carbon-11 compared with S-ethyl for tritium). This resulted in the in-growth of radiolabelled metabolites in plasma, which, in turn, contributed to the non-specific component of brain radioactivity. Nonetheless, the signal ratio of ∝5 for a receptor-dense tissue compared with the receptor-sparse cerebellum, at 90-120 min after radioligand injection, encouraged the development of [O-methyl- 11 C]RS-15385-197 for human positron emission tomography (PET). Unfortunately, in two human PET scans (each of 90 min), brain extraction of the radioligand was minimal, with volumes of distribution more than an order of magnitude lower than that measured in rats. Following intravenous injection, radioactivity was retained in plasma and metabolism of the radiolabelled compound was very low. Retrospective measurements of in vitro plasma protein binding and in vivo brain uptake index (BUI) in rats demonstrated a higher protein binding of the radioligand in human compared with rat plasma and a lower BUI in the presence of human plasma. It is feasible that a higher affinity of RS-15385-197 for human plasma protein compared with receptor limited the transport of the radioligand. Although one of the PET scans showed a slight heterogeneity in biodistribution of radioactivity which was consistent with the known localisation of α 2 -adrenoceptors in human brain, it was concluded that [O-methyl- 11 C]RS-15385

Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. 1

Science.gov (United States)

Meyer, Christiane; Feyerabend, Martin; Weiler, Elmar W.

1989-01-01

Using the novel radioligand, [3H]-9′-nor-fusicoccin-8′-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K+, Mg2+-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [3H]-9′-nor-fusicoccin-8′-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (KD), KD1 = 1.5 nanomolar and KD2 = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 ± 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin. PMID:16666603

Evaluation of radioiodinated (2S,{alpha}S)-2-({alpha}-(2-iodophenoxy)benzyl)morpholine as a radioligand for imaging of norepinephrine transporter in the heart

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Kiyono, Yasushi [Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193 (Japan); Radioisotopes Research Laboratory, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606-8507 (Japan)], E-mail: ykiyono@u-fukui.ac.jp; Sugita, Taku [Department of Pathofunctional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501 (Japan); Ueda, Masashi [Radioisotopes Research Laboratory, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Kawashima, Hidekazu [Department of Nuclear Medicine and Diagnostic Imaging, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Kanegawa, Naoki; Kuge, Yuji [Department of Pathofunctional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501 (Japan); Fujibayashi, Yasuhisa [Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193 (Japan); Saji, Hideo [Department of Pathofunctional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501 (Japan)

2008-02-15

Introduction: The norepinephrine transporter (NET) is located presynaptically on noradrenergic nerve terminals and plays a critical role in the regulation of the synaptic norepinephrine (NE) concentration via the reuptake of NE. Changes in NET have been recently reported in several cardiac failures. Therefore, a NET-specific radioligand is useful for in vivo assessment of changes in NET density in various cardiac disorders. Recently, we developed a radioiodinated reboxetine analogue, (2S,{alpha}S)-2-({alpha}-(2-iodophenoxy)benzyl)morpholine ((S,S)-IPBM), for NET imaging. In the current study, we assessed the applicability of radioiodinated (S,S)-IPBM to NET imaging in the heart. Methods: The NET affinity and selectivity were measured from the ability to displace specific [{sup 3}H]nisoxetine and (S,S)-[{sup 125}I]IPBM binding to rat heart membrane, respectively. To evaluate the distribution of (S,S)-[{sup 125}I]IPBM in vivo, biodistribution experiment was performed in rats. With the use of several monoamine transporter binding agents, pharmacological blocking experiments were performed in rats. Results: In vitro binding assays showed that the affinity of (S,S)-IPBM to NET was similar to those of the well-known NET-specific binding agents, nisoxetine and desipramine. Furthermore, (S,S)-[{sup 125}I]IPBM binding was inhibited by nisoxetine and desipramine, but not by dopamine or serotonin transporter binding agents. These data indicated that (S,S)-IPBM had high affinity and selectivity for NET in vitro. Biodistribution studies in rats showed rapid and high uptake of (S,S)-[{sup 125}I]IPBM by the heart and rapid clearance from the blood. The heart-to-blood ratio was 31.9 at 180 min after the injection. The administration of nisoxetine and desipramine decreased (S,S)-[{sup 125}I]IPBM accumulation in the heart, but injection of fluoxetine and GBR12909 had little influence. Conclusions: Radioiodinated (S,S)-IPBM is a potential radioligand for NET imaging in the heart.

A radioreceptor assay for measurement of plasma glucocorticoid binding activity

International Nuclear Information System (INIS)

Fan Jie

1990-01-01

A radioreceptor assay (RRA) for plasma glucocorticoid binding activity (GCBA) has been developed using glucocorticoid receptor in rat thymocytes. Unlike other assays for natural and certain synthetic corticosteroids, RRA measures the GCBA of all natural and synthetic GC in plasma. The range of standard curve was 0 ∼ 1.00 mg/L. The sensitivity was 0.01 mg/l. The recovery rate was 92.1%, and the intra and inter assay CV was 0.7% (n = 3) and 4.4% (n = 3) respectively. The level of corticosterone in 9 rat plasma samples was determined by RRA and CBG-isotope binding assay. There was a general correlation over a wide range between the values determined by the two assays (r = 0.95; P < 0.001). The measuring condition was described in detail

Fenfluramine Reduces [11C]Cimbi-36 Binding to the 5-HT2A Receptor in the Nonhuman Primate Brain

DEFF Research Database (Denmark)

Yang, Kai-Chun; Stepanov, Vladimir; Martinsson, Stefan

2017-01-01

Background: [11C]Cimbi-36 is a serotonin 2A receptor agonist positron emission tomography radioligand that has recently been examined in humans. The binding of agonist radioligand is expected to be more sensitive to endogenous neurotransmitter concentrations than antagonist radioligands. In the c...... sensitive radioligands. [11C]Cimbi-36 is a promising radioligand to examine serotonin release in the primate brain....

Specific in vivo binding in the rat brain of [18F]RP 62203: A selective 5-HT2A receptor radioligand for positron emission tomography

International Nuclear Information System (INIS)

Besret, Laurent; Dauphin, Francois; Huard, Cecile; Lasne, Marie-Claire; Vivet, Richard; Mickala, Patrick; Barbelivien, Alexandra; Baron, Jean-Claude

1996-01-01

In vivo pharmacokinetic and brain binding characteristics of [ 18 F]RP 62203, a selective high-affinity serotonergic 5-HT 2A receptor antagonist, were assessed in the rat following intravenous injection of trace amount of the radioligand. The radioactive distribution profile observed in the brain 60 min after injection was characterized by greater than fourfold higher uptake in neocortex as compared to cerebellum (0.38 ± 0.07% injected dose/g, % ID/g and 0.08 ± 0.01 ID/g, respectively), consistent with in vivo specific binding to the 5-HT 2A receptor. Furthermore, specific [ 18 F]RP 62203 binding significantly correlated with the reported in vitro distribution of 5-HT 2A receptors, but not with known concentration profiles of dopaminergic D 2 or adrenergic α 1 receptors. Finally, detectable specific binding was abolished by pretreatment with large doses of ritanserin, a selective 5-HT 2A antagonist, which resulted in uniform uptakes across cortical, striatal and cerebellar tissues. Thus, [ 18 F]RP 62203 appears to be a promising selective tool to visualize and quantify 5-HT 2A brain receptors in vivo with positron emission tomography

Lectin binding assays for in-process monitoring of sialylation in protein production.

Science.gov (United States)

Xu, Weiduan; Chen, Jianmin; Yamasaki, Glenn; Murphy, John E; Mei, Baisong

2010-07-01

Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galbeta1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(beta1-4) exposure) is inversely proportional to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII producing cells. To examine the Galbeta1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment. To establish the correlation between Galbeta1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be rapidly performed, thereby making them effective for in-process monitoring of protein sialylation.

Synthesis of 125I Labeled Estradiol-17-Hemisuccinate and Its Binding Study to Estrogen Receptors Using Scintillation Proximity Assay Method

Directory of Open Access Journals (Sweden)

Y. Susilo

2012-12-01

Full Text Available Research was carried out to obtain a selective ligand which strongly bind to estrogen receptors through determination of binding affinity of estradiol-17β-hemisuccinate. Selectivity of these compounds for estrogen receptor was studied using Scintillation Proximity Assay (SPA method. Primary reagents required in the SPA method including radioligand and receptor, the former was obtained by labeling of estradiol-17β-hemisuccinate with 125I, while MCF7 was used as the receptor. The labeling process was performed by indirect method via two-stage reaction. In this procedure, first step was activation of estradiol-17β-hemisuccinate using isobutylchloroformate and tributylamine as a catalist, while labeling of histamine with 125I was carried out using chloramin-T method to produce 125I-histamine. The second stage was conjugation of activated estradiol-17β-hemisuccinate with 125I-histamine. The product of estradiol-17β-hemisuccinate labeled 125I was extracted using toluene. Furtherly, the organic layer was purified by TLC system. Characterization of estradiol-17β-hemisuccinate labeled 125I from this solvent extraction was carried out by determining its radiochemical purity and the result was obtained using paper electrophoresis and TLC were 79.8% and 84.4% respectively. Radiochemical purity could be increased when purification step was repeated using TLC system, the result showed up to 97.8%. Determination of binding affinity by the SPA method was carried out using MCF7 cell lines which express estrogen receptors showed the value of Kd at 7.192 x 10-3 nM and maximum binding at 336.1 nM. This low value of Kd indicated that binding affinity of estradiol-17β-hemisuccinate was high or strongly binds to estrogen recepto

Development of a PET radioligand for the central 5-HT{sub 1B} receptor: radiosynthesis and characterization in cynomolgus monkeys of eight radiolabeled compounds

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Andersson, Jan D., E-mail: j.d.andersson@ki.s [Psychiatry Section, Department of Clinical Neuroscience, Karolinska Institutet, Karolinska University Hospital, SE-17176 Stockholm (Sweden); Pierson, M. Edward [AstraZeneca Pharmaceuticals, CNS Discovery, Wilmington, DE 19850 (United States); Finnema, Sjoerd J.; Gulyas, Balazs [Psychiatry Section, Department of Clinical Neuroscience, Karolinska Institutet, Karolinska University Hospital, SE-17176 Stockholm (Sweden); Heys, Richard; Elmore, Charles S. [AstraZeneca Pharmaceuticals, CNS Discovery, Wilmington, DE 19850 (United States); Farde, Lars [AstraZeneca Pharmaceuticals, Neuroscience Clinical, SE-15185 Soedertaelje (Sweden); Halldin, Christer [Psychiatry Section, Department of Clinical Neuroscience, Karolinska Institutet, Karolinska University Hospital, SE-17176 Stockholm (Sweden)

2011-02-15

Introduction: The serotonin 1B (5-HT{sub 1B}) receptor has been implicated in several psychiatric disorders and is a potential pharmacological target in the treatment of depression. The aim of this study was to develop a radioligand for positron emission tomography (PET) imaging of the 5-HT{sub 1B} receptor in the primate brain in vivo. Methods: Eight carboxamide radioligands (1-8) from three different core structures were radiolabeled with carbon-11 employing N-methylation with [{sup 11}C]methyl triflate on the piperazine structural moiety. In vivo PET evaluation of each radioligand was performed in cynomolgus monkeys and included analysis of radioactive metabolites measured in plasma using high-performance liquid chromatography. Results: In a total of 12 radiosynthesis of the eight radioligands, the mean decay corrected yield was 11%, and the mean specific radioactivity was 299 GBq/{mu}mol (8075 Ci/mmol) at time of administration. Of the eight tested candidates, [{sup 11}C]6 demonstrated the most promising in vivo characteristics, showing high binding in 5-HT{sub 1B} receptor-rich regions and low binding in the cerebellum. When inspecting data from all eight compounds, lipophilicity appeared as a physicochemical property that could be related to favorable in vivo imaging characteristics. Conclusion: Candidate [{sup 11}C]6, i.e., [{sup 11}C]AZ10419369, exhibited high binding potentials in regions known to contain 5-HT{sub 1B} receptors and was nominated for further preclinical characterization and PET examination in human subjects.

Time-dependent recovery of in vivo binding sites after drug dosing: A method for radiotracer evaluation

International Nuclear Information System (INIS)

Kilbourn, Michael R.

1997-01-01

The recovery of in vivo binding sites for (±)-α-[ 11 C]methoxytetrabenazine, a radioligand for the monoamine vesicular transporter (VMAT2), was determined in mouse brain at various times following a pharmacological dose of tetrabenazine. Concentrations of in vivo radioligand binding sites progressively increased and had reached control values by 8.5 h, and this recovery was consistent with the pharmacokinetics of the competing drug tetrabenazine and its active metabolite, dihydrotetrabenazine. This study demonstrates a simple experimental protocol of using a single dose of a reversible competing drug and time-dependent measurements of in vivo binding of a radioligand. This protocol is suitable for testing the sensitivity of an in vivo radiotracer for measurement of varying concentrations of in vivo binding sites

5-tert-Butyl-2-(4'-[{sup 18}F]fluoropropynylphenyl)-1,3-dithiane oxides: potential new GABA{sub A} receptor radioligands

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Li Xuehe; Jung, Yong-Woon; Snyder, Scott E.; Blair, Joseph; Sherman, Philip S.; Desmond, Timothy; Frey, Kirk A. [Department of Radiology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States); Kilbourn, Michael R. [Department of Radiology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States)], E-mail: mkilbour@umich.edu

2008-07-15

As potential new ligands targeting the binding site of {gamma}-aminobutyric acid (GABA) receptor ionophore, trans-5-tert-butyl-2-(4'-fluoropropynylphenyl)-2-methyl-1,1-dioxo-1, 3-dithiane (1) and cis/trans-5-tert-butyl-2-(4'-fluoropropynylphenyl)-2-methyl-1,1,3, 3-tetroxo-1,3-dithiane (2) were selected for radiolabeling and initial evaluation as in vivo imaging agents for positron emission tomography (PET). Both compounds exhibited identical high in vitro binding affinities (K{sub i}=6.5 nM). Appropriate tosylate-substituted ethynyl precursors were prepared by multistep syntheses involving stepwise sulfur oxidation and chromatographic isolation of desired trans isomers. Radiolabeling was accomplished in one step using nucleophilic [{sup 18}F]fluorination. In vivo biodistribution studies with trans-[{sup 18}F]1 and trans-[{sup 18}F]2 showed significant initial uptake into mouse brain and gradual washout, with heterogeneous regional brain distributions and higher retention in the cerebral cortex and cerebellum and lower retention in the striatum and pons-medulla. These regional distributions of the new radioligands correlated with in vitro and ex vivo measures of standard radioligands binding to the ionophore- and benzodiazepine-binding sites of GABA{sub A} receptor in rodent brain. A comparison of these results with previously prepared radiotracers for other neurochemical targets, including successes and failures as in vivo radioligands, suggests that higher-affinity compounds with increased retention in target brain tissues will likely be needed before a successful radiopharmaceutical for human PET imaging can be identified.

Guanidine-acylguanidine bioisosteric approach in the design of radioligands: synthesis of a tritium-labeled N(G)-propionylargininamide ([3H]-UR-MK114) as a highly potent and selective neuropeptide Y Y1 receptor antagonist.

Science.gov (United States)

Keller, Max; Pop, Nathalie; Hutzler, Christoph; Beck-Sickinger, Annette G; Bernhardt, Günther; Buschauer, Armin

2008-12-25

Synthesis and characterization of (R)-N(alpha)-(2,2-diphenylacetyl)-N-(4-hydroxybenzyl)-N(omega)-([2,3-(3)H]-propanoyl)argininamide ([(3)H]-UR-MK114), an easily accessible tritium-labeled NPY Y(1) receptor (Y(1)R) antagonist (K(B): 0.8 nM, calcium assay, HEL cells) derived from the (R)-argininamide BIBP 3226, is reported. The radioligand binds with high affinity (K(D), saturation: 1.2 nM, kinetic experiments: 1.1 nM, SK-N-MC cells) and selectivity for Y(1)R over Y(2), Y(4), and Y(5) receptors. The title compound is a useful pharmacological tool for the determination of Y(1)R ligand affinities, quantification of Y(1)R binding sites, and autoradiography.

Radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

International Nuclear Information System (INIS)

Pierce, E.A.; Dame, M.C.; DeLuca, H.F.

1986-01-01

A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approx. 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D 3 and should be useful for the detection of antibodies to ligand-binding proteins in general

Radioligands for brain 5-HT2 receptor imaging in vivo: why do we need them?

International Nuclear Information System (INIS)

Busatto, G.F.

1996-01-01

Recently, PET and SPET radiotracers with high specificity for 5-HT 2 receptors have been developed. These have been studied in baboons and humans with promising results, displaying a binding profile compatible with the brain distribution of 5-HT 2 receptors. It is predicted that studies with the newly developed 5-HT radioligands will substantially increase knowledge about the pharmacology of brain disorders. (orig./MG)

Specific in vivo binding in the rat brain of [{sup 18}F]RP 62203: A selective 5-HT{sub 2A} receptor radioligand for positron emission tomography

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Besret, Laurent; Dauphin, Francois; Huard, Cecile; Lasne, Marie-Claire; Vivet, Richard; Mickala, Patrick; Barbelivien, Alexandra; Baron, Jean-Claude

1996-02-01

In vivo pharmacokinetic and brain binding characteristics of [{sup 18}F]RP 62203, a selective high-affinity serotonergic 5-HT{sub 2A} receptor antagonist, were assessed in the rat following intravenous injection of trace amount of the radioligand. The radioactive distribution profile observed in the brain 60 min after injection was characterized by greater than fourfold higher uptake in neocortex as compared to cerebellum (0.38 {+-} 0.07% injected dose/g, % ID/g and 0.08 {+-} 0.01 ID/g, respectively), consistent with in vivo specific binding to the 5-HT{sub 2A} receptor. Furthermore, specific [{sup 18}F]RP 62203 binding significantly correlated with the reported in vitro distribution of 5-HT{sub 2A} receptors, but not with known concentration profiles of dopaminergic D{sub 2} or adrenergic {alpha}{sub 1} receptors. Finally, detectable specific binding was abolished by pretreatment with large doses of ritanserin, a selective 5-HT{sub 2A} antagonist, which resulted in uniform uptakes across cortical, striatal and cerebellar tissues. Thus, [{sup 18}F]RP 62203 appears to be a promising selective tool to visualize and quantify 5-HT{sub 2A} brain receptors in vivo with positron emission tomography.

Radiochemical synthesis and biological evaluation of 3-[4-(4-[{sup 18}F]fluorobenzyl)piperazin-1-ylmethyl]pyrazolo[1,5-a]pyridine as dopamine D{sub 4} receptor radioligand

Energy Technology Data Exchange (ETDEWEB)

Li, Gu-Cai; Zhang, Ru; Jiang, Kai-Jun; Chen, Bo [Hunan Institute of Engineering, Xiangtan (China). College of Chemistry and Chemical Engineering

2014-09-01

A potential dopamine D{sub 4} receptor radioligand, 3-[4-(4-[{sup 18}F]fluorobenzyl)piperazin-1-ylmethyl]pyrazolo[1,5-a]pyridine was synthesized through a one-pot two-step procedure with total yield 18.5% (decay corrected). The molar radioactivity was 115 GBq/μmol and the radiochemical purity was greater than 95.5%. Its affinity and selectivity for dopamine D{sub 2}-like receptors were measured through in vitro receptor binding experiments and the K{sub i} for D{sub 4} receptor was determined to be 17 ± 0.5 nM. The partition coefficient (Log P) of it was determined to be 2.80 ± 0.10 through octanol experiment. The in vivo biodistribution of it in rat brain exposed that the radioligand penetrates through blood-brain- barrier (BBB) and may specifically bind to dopamine D{sub 4} receptor. The results indicated that the radioligand shows promise for the in vivo study of dopamine D{sub 4} receptor. (orig.)

Test-retest reliability of the novel 5-HT1B receptor PET radioligand [11C]P943

International Nuclear Information System (INIS)

Saricicek, Aybala; Chen, Jason; Ruf, Barbara; Planeta, Beata; Labaree, David; Gallezot, Jean-Dominique; Huang, Yiyun; Subramanyam, Kalyani; Maloney, Kathleen; Matuskey, David; Deserno, Lorenz; Neumeister, Alexander; Krystal, John H.; Carson, Richard E.; Bhagwagar, Zubin

2015-01-01

[ 11 C]P943 is a novel, highly selective 5-HT 1B PET radioligand. The aim of this study was to determine the test-retest reliability of [ 11 C]P943 using two different modeling methods and to perform a power analysis with each quantification technique. Seven healthy volunteers underwent two PET scans on the same day. Regions of interest (ROIs) were the amygdala, hippocampus, pallidum, putamen, insula, frontal, anterior cingulate, parietal, temporal and occipital cortices, and cerebellum. Two multilinear radioligand quantification techniques were used to estimate binding potential: MA1, using arterial input function data, and the second version of the multilinear reference tissue model analysis (MRTM2), using the cerebellum as the reference region. Between-scan percent variability and intraclass correlation coefficients (ICC) were used to assess test-retest reliability. We also performed power analyses to determine the method that would allow the least number of subjects using within-subject or between-subject study designs. A voxel-wise ICC analysis for MRTM2 BP ND was performed for the whole brain and all the ROIs studied. Mean percent variability between two scans across regions ranged between 0.4 % and 12.4 % for MA1 BP ND , 0.5 % and 11.5 % for MA1 BP P , 16.7 % and 28.3 % for MA1 BP F , and between 0.2 % and 5.4 % for MRTM2 BP ND . The power analyses showed a greater number of subjects were required using MA1 BP F compared with other outcome measures for both within-subject and between-subject study designs. ICC values were the highest using MRTM2 BP ND and the lowest with MA1 BP F in ten ROIs. Small regions and regions with low binding had lower ICC values than large regions and regions with high binding. Reliable measures of 5-HT 1B receptor binding can be obtained using the novel PET radioligand [ 11 C]P943. Quantification of 5-HT 1B receptor binding with MRTM2 BP ND and with MA1 BP P provided the least variability and optimal power for within-subject and

In vivo imaging of neuroinflammation in the rodent brain with [{sup 11}C]SSR180575, a novel indoleacetamide radioligand of the translocator protein (18 kDa)

Energy Technology Data Exchange (ETDEWEB)

Chauveau, Fabien [CEA, DSV, IBM, Service Hospitalier Frederic Joliot, Orsay (France); Universite Paris Sud, INSERM U1023, Orsay (France); Universite Lyon 1, Creatis, CNRS UMR 5220, INSERM U630, INSA Lyon, Lyon (France); Boutin, Herve [CEA, DSV, IBM, Service Hospitalier Frederic Joliot, Orsay (France); Universite Paris Sud, INSERM U1023, Orsay (France); University of Manchester, Faculty of Life Sciences, Manchester (United Kingdom); Camp, Nadja van; Tavitian, Bertrand [CEA, DSV, IBM, Service Hospitalier Frederic Joliot, Orsay (France); Universite Paris Sud, INSERM U1023, Orsay (France); Thominiaux, Cyrille; Dolle, Frederic [CEA, DSV, IBM, Service Hospitalier Frederic Joliot, Orsay (France); Hantraye, Philippe [CEA, DSV, IBM, MIRCEN, Fontenay-aux-Roses (France); Rivron, Luc [Sanofi-Aventis, GMPK-Global Isotope Chemistry and Metabolite Synthesis Department (ICMS), Paris (France); Marguet, Frank; Castel, Marie-Noelle; Rooney, Thomas; Benavides, Jesus [Sanofi-Aventis, CNS Department, Paris (France)

2011-03-15

Neuroinflammation is involved in neurological disorders through the activation of microglial cells. Imaging of neuroinflammation with radioligands for the translocator protein (18 kDa) (TSPO) could prove to be an attractive biomarker for disease diagnosis and therapeutic evaluation. The indoleacetamide-derived 7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide, SSR180575, is a selective high-affinity TSPO ligand in human and rodents with neuroprotective effects. Here we report the radiolabelling of SSR180575 with {sup 11}C and in vitro and in vivo imaging in an acute model of neuroinflammation in rats. The image contrast and the binding of [{sup 11}C]SSR180575 are higher than that obtained with the isoquinoline-based TSPO radioligand, [{sup 11}C]PK11195. Competition studies demonstrate that [{sup 11}C]SSR180575 has high specific binding for the TSPO. [{sup 11}C]SSR180575 is the first PET radioligand for the TSPO based on an indoleacetamide scaffold designed for imaging neuroinflammation in animal models and in the clinic. (orig.)

Receptors for vasoactive intestinal peptide in rat anterior pituitary glands: Localization of binding to lactotropes

International Nuclear Information System (INIS)

Wanke, I.E.; Rorstad, O.P.

1990-01-01

Vasoactive intestinal peptide (VIP) has been implicated as a physiological PRL-releasing factor; however, characterization of VIP receptors on normal pituitaries using radioligand-binding methods has been problematic. In this study we demonstrated specific receptors for VIP in anterior pituitary glands of female rats using HPLC-purified monoiodinated [Tyr(125I)10]VIP. Binding of VIP was reversible, saturable to receptor and radioligand, regulated by guanine nucleotides, and dependent on time and temperature. Scatchard analysis of competitive binding studies indicated high and low affinity binding sites, with equilibrium dissociation constants (Kd) of 0.19 +/- 0.03 and 28 +/- 16 nM, respectively. The corresponding maximum numbers of binding sites were 158 +/- 34 fmol/mg and 11.7 +/- 6.9 pmol/mg. Binding was specific, as peptides with structural homology to VIP were less than 100th as potent as VIP. The rank order of potency of the peptides tested was VIP greater than rat (r) peptide histidine isoleucine = human (h) PHI greater than rGRF greater than bovine GRF = porcine PHI = VIP-(10-28) greater than hGRF greater than secretin greater than apamin greater than glucagon. Radioligand binding was associated primarily with lactotrope-enriched fractions prepared by unit gravity sedimentation of dispersed anterior pituitary cells. VIP stimulated PRL release from cultured rat anterior pituitary cells, with an ED50 of 1 nM. These results, comprising the first identification of specific VIP receptors in normal rat anterior pituitary tissue using radioligand-binding methods, provide additional support for a biological role of VIP in lactotrope function

Endogenous opioid peptides as neurotransmitters in the rat hippocampus

International Nuclear Information System (INIS)

Neumaier, J.F.

1989-01-01

The role of endogenous opioid peptides as neurotransmitters in the rat hippocampus was investigated by using extracellular recording and radioligand binding techniques in the hippocampal slice preparation. Synaptic conductances from endogenously released opioid peptides have been difficult to detect. This problem was approach by designing a novel assay of opioid peptide release, in which release was detected by measuring binding competition between endogenous opioids and added radioligand. Membrane depolarization displaced [ 3 H]-diprenorphine binding in a transient, calcium-dependent, and peptidase-sensitive manner. Autoradiographic localization of the sites of [ 3 H]-diprenorphine binding displacement showed that significant opioid peptide release and receptor occupancy occurred in each major subregion of the hippocampal slices. This assay method can not be used to define optimal electrical stimulation conditions for releasing endogenous opioids. The binding displacement method was extended to the study of the sigma receptor. Depolarization of hippocampal slices was found to reduce the binding of the sigma-selective radioligand [ 3 H]-ditolylguanidine in a transient and calcium-dependent manner with no apparent direct effects on sigma receptor affinity

Radioligands for brain 5-HT{sub 2} receptor imaging in vivo: why do we need them?

Energy Technology Data Exchange (ETDEWEB)

Busatto, G.F. [Section of Clinical Neuropharmacology, Dept. of Psychological Medicine, Inst. of Psychiatry, London (United Kingdom)

1996-08-01

Recently, PET and SPET radiotracers with high specificity for 5-HT{sub 2} receptors have been developed. These have been studied in baboons and humans with promising results, displaying a binding profile compatible with the brain distribution of 5-HT{sub 2} receptors. It is predicted that studies with the newly developed 5-HT radioligands will substantially increase knowledge about the pharmacology of brain disorders. (orig./MG)

Assessment of a recombinant androgen receptor binding assay: initial steps towards validation.

Science.gov (United States)

Freyberger, Alexius; Weimer, Marc; Tran, Hoai-Son; Ahr, Hans-Jürgen

2010-08-01

Despite more than a decade of research in the field of endocrine active compounds with affinity for the androgen receptor (AR), still no validated recombinant AR binding assay is available, although recombinant AR can be obtained from several sources. With funding from the European Union (EU)-sponsored 6th framework project, ReProTect, we developed a model protocol for such an assay based on a simple AR binding assay recently developed at our institution. Important features of the protocol were the use of a rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain (LBD) of the rat AR (which is identical to the human AR-LBD) and performance in a 96-well plate format. Besides two reference compounds [dihydrotestosterone (DHT), androstenedione] ten test compounds with different affinities for the AR [levonorgestrel, progesterone, prochloraz, 17alpha-methyltestosterone, flutamide, norethynodrel, o,p'-DDT, dibutylphthalate, vinclozolin, linuron] were used to explore the performance of the assay. At least three independent experiments per compound were performed. The AR binding properties of reference and test compounds were well detected, in terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using recombinant AR preparations. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.6. Our data demonstrate that the assay reliably ranked compounds with strong, weak, and no/marginal affinity for the AR with high accuracy. It avoids the manipulation and use of animals, as a recombinant protein is used and thus contributes to the 3R concept. On the whole, this assay is a promising candidate for further validation. Copyright 2009 Elsevier Inc. All rights reserved.

C5a binding to human polymorphonuclear leukocyte plasma membrane (PMNLM) receptors

International Nuclear Information System (INIS)

Conway, R.G.; Mollison, K.W.; Carter, G.W.; Lane, B.

1986-01-01

Previous investigations of the C5a receptor have been performed using intact human PMNL. To circumvent some of the potential problems with such whole cell assays (e.g. internalization or metabolism of radioligand) the authors have developed a PMNLM binding assay. Human PMNLM were prepared by nitrogen cavitation and Percoll gradient centrifugation. Specific binding of [ 125 I]C5a to PMNLM was: high affinity, K/sub D/ = 0.6 nM; saturable, B/sub max/ = 8.7 pmol/mg protein; and reversible. Kinetic measurements agree with the K/sub D/ value obtained by Scatchard analysis. Furthermore, the binding activity of C5a correlates with biological activity as measured by myeloperoxidase release from human PMNL. Human serum C5a and recombinant C5a bind with similar affinities when measured by competition or direct binding and label the same number of sites in human PMNLM. The nonhydrolyzable GTP analog, GppNHp, induces a low affinity state of the C5a receptor (4-6 fold shift in K/sub D/) with little effect on B/sub max/. In summary, the criteria have been satisfied for identification of a biologically relevant C5a binding site in human PMNLM. Regulation of the C5a receptor and its membrane transduction mechanism(s) appears to involve guanyl nucleotides, as has been found for other chemoattractant receptors

Novel Photochrome Aptamer Switch Assay (PHASA) for adaptive binding to aptamers.

Science.gov (United States)

Papper, Vladislav; Pokholenko, Oleksandr; Wu, Yuanyuan; Zhou, Yubin; Jianfeng, Ping; Steele, Terry W J; Marks, Robert S

2014-11-01

A novel Photochrome-Aptamer Switch Assay (PHASA) for the detection and quantification of small environmentally important molecules such as toxins, explosives, drugs and pollutants, which are difficult to detect using antibodies-based assays with high sensitivity and specificity, has been developed. The assay is based on the conjugation of a particular stilbene-analyte derivative to any aptamer of interest. A unique feature of the stilbene molecule is its reporting power via trans-cis photoisomerisation (from fluorescent trans-isomer to non-fluorescent cis-isomer) upon irradiation with the excitation light. The resulting fluorescence decay rate for the trans-isomer of the stilbene-analyte depends on viscosity and spatial freedom to rotate in the surrounding medium and can be used to indicate the presence of the analyte. Quantification of the assay is achieved by calibration of the fluorescence decay rate for the amount of the tested analyte. Two different formats of PHASA have been recently developed: direct conjugation and adaptive binding. New stilbene-maleimide derivatives used in the adaptive binding format have been prepared and characterised. They demonstrate effective binding to the model thiol compound and to the thiolated Malachite Green aptamer.

Peptide Binding to HLA Class I Molecules: Homogenous, High-Throughput Screening, and Affinity Assays

DEFF Research Database (Denmark)

Harndahl, Mikkel; Justesen, Sune Frederik Lamdahl; Lamberth, Kasper

2009-01-01

, better signal-to-background ratios, and a higher capacity. They also describe an efficient approach to screen peptides for binding to HLA molecules. For the occasional user, this will serve as a robust, simple peptide-HLA binding assay. For the more dedicated user, it can easily be performed in a high-throughput...... the luminescent oxygen channeling immunoassay technology (abbreviated LOCI and commercialized as AlphaScreen (TM)). Compared with an enzyme-linked immunosorbent assay-based peptide-HLA class I binding assay, the LOCI assay yields virtually identical affinity measurements, although having a broader dynamic range...... screening mode using standard liquid handling robotics and 384-well plates. We have successfully applied this assay to more than 60 different HLA molecules, leading to more than 2 million measurements. (Journal of Biomolecular Screening 2009: 173-180)...

Radiosynthesis and autoradiographic evaluation of [11C]NAD-299, a radioligand for visualization of the 5-HT1A receptor

International Nuclear Information System (INIS)

Sandell, Johan; Halldin, Christer; Hall, Haakan; Thorberg, Seth-Olov; Werner, Tom; Sohn, Daniel; Sedvall, Goeran; Farde, Lars

1999-01-01

The selective 5-HT 1A receptor antagonist NAD-299 ([R]-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran- 5-carboxamide) was labeled with the positron emitting radionucldie carbon-11. The radioligand was synthesized from NAD-195 ([R]-3-N,N-dicyclobutylamino-8-fluoro-5-trifluoromethylsulfonyloxy-3, 4-dihydro-2H-1-benzopyran) in two radiochemical steps. A palladium-catalyzed reaction of NAD-195 and [ 11 C]cyanide was followed by hydrolysis of the carbon-11-labeled nitrile intermediate with basic hydrogen peroxide. The total radiochemical yield, based on [ 11 C]CO 2 and corrected for decay, was 20-40%. The specific radioactivity was 24 GBq/μmol (900 Ci/mmol) at end of synthesis, with a radiochemical purity better than 99% and a total synthesis time of 40-45 min. Autoradiographic examination of [ 11 C]NAD-299 binding in human brain postmortem demonstrated high binding in hippocampus, raphe nuclei, and neocortex. The binding in the hippocampus was higher than in the neocortex. Within the hippocampus, the densest binding was observed in the CA1 region. [ 11 C]NAD-299 binding was inhibited by addition of the 5-HT 1A receptor ligands WAY-100635, pindolol, (±)-8-OH-DPAT, 5-HT, and buspirone, leaving a low background of nonspecific binding. The results indicate that [ 11 C]NAD-299 binds specifically to 5-HT 1A receptors in the human brain in vitro and is a potential radioligand for positron emission tomography (PET) examination of 5-HT 1A receptors in vivo

Characterization of [(11)C]Cimbi-36 as an agonist PET radioligand for the 5-HT(2A) and 5-HT(2C) receptors in the nonhuman primate brain

DEFF Research Database (Denmark)

Finnema, Sjoerd J; Stepanov, Vladimir; Ettrup, Anders

2014-01-01

a more meaningful assessment of available receptors than antagonist radioligands. In the current study we characterized [(11)C]Cimbi-36 receptor binding in the primate brain. On five experimental days, a total of 14 PET measurements were conducted in three female rhesus monkeys. On each day, PET...... agonist radioligand suitable for examination of 5-HT2A receptors in the cortical regions and of 5-HT2C receptors in the choroid plexus of the primate brain....

A simple ligand-binding assay for thyroxine-binding globulin on reusable Sephadex columns

International Nuclear Information System (INIS)

Bastomsky, C.H.; Kalloo, H.; Frenkel-Leith, D.B.; McGill Univ., Montreal, Quebec

1977-01-01

A method for the assay of thyroxine-binding globulin on reusable Sephadex G-25 columns is described. It depends upon elution by diluted iodothyronine-free serum of protein-bound [ 125 I]thyroxine from the columns under conditions where binding to thyroxine-binding prealbumin and albumin are abolished. It is simple, rapid and precise, and permits determinations inlarge numbers of samples. Values (mg/l; mean +- S.D.) were: normals 31.6+-5.4, hyperthyroid 28.3+-4.8, hypothyroid 40.6+-7.5, oral contraceptives 40.1+-6.8, pregnant 50.3+-5.4, cirrhotics 20.7+-4.3. Concentrations were reduced in serum heated at 56degC, while the uptake of [ 125 I]triiodothyronine was increased. There was a significant negative correlation between thyroxine-binding globulin concentration and triiodothyronine uptake in the heated serum samples and in euthyroid subjects

Comparison of acid ethanol extraction and acid gel filtration prior to IGF-I and IGF-II radioimmunoassays; Improvement of determinations in acid ethanol extracts by the use of truncated IGF-I as radioligand

Energy Technology Data Exchange (ETDEWEB)

Bang, P; Eriksson, U; Wivall, I -L; Hall, K [Department of Endocrinology, Karolinska Institute, Stockholm (Sweden); Sara, V [Department of Pathology, Karolinska Institute, Stockholm (Sweden)

1991-01-01

Insulin-like growth factor binding proteins interfere in the IGF-I and -II radioimmunoassays. In an attempt to overcome this problem, we have compared the use of truncated IGF-I, with reduced IGFBP affinity, and IGF-I as radioligands for IGF-I RIA measurements in serum separated by acid gel filtration or acid ethanol extraction followed by cryo-precipitation. With truncated IGF-I as radioligand the IGF-I measurements in acid gel filtrates and acid ethanol extracts were significantly correlated in healthy subjects (N=42, r=0.91, p<0.001) and in patients with acromegaly (N=10, r=0.85, p<0.01), GH deficiency (N=10, r=0.88, p<0.001) or Type I diabetes mellitus (N=10, r=0.90, p<0.001). In contrast, the IGF-I concentrations in acid ethanol extracts determined with IGF-I as radioligand did not correlate with those in acid gel filtrates using truncated IGF-I radioligand in patients with acromegaly (r=0.61, NS) or GH deficiency (r=0.46, NS). In the latter group the mean IGF-I concentrations measured in acid ethanol extracts were erroneously elevated by 112%. Low-affinity antibodies used for IGF-II RIA determinations failed to give reliable results in acid ethanol extracts from patients with Type I diabetes mellitus or GH deficiency. In conclusion, erroneously high IGF-I concentrations owing to binding of the radioligand to IGFBPs not completely removed by acid ethanol extraction can be avoided by the use of truncated IGF-I as radioligand. (author).

Comparison of the binding characteristics of [18F]THK-523 and other amyloid imaging tracers to Alzheimer's disease pathology

International Nuclear Information System (INIS)

Harada, Ryuichi; Okamura, Nobuyuki; Yoshikawa, Takeo; Yanai, Kazuhiko; Furumoto, Shozo; Tago, Tetsuro; Iwata, Ren; Maruyama, Masahiro; Higuchi, Makoto; Arai, Hiroyuki; Kudo, Yukitsuka

2013-01-01

Extensive deposition of senile plaques and neurofibrillary tangles in the brain is a pathological hallmark of Alzheimer's disease (AD). Although several PET imaging agents have been developed for in vivo detection of senile plaques, no PET probe is currently available for selective detection of neurofibrillary tangles in the living human brain. Recently, [ 18 F]THK-523 was developed as a potential in vivo imaging probe for tau pathology. The purpose of this study was to compare the binding properties of [ 18 F]THK-523 and other amyloid imaging agents, including PiB, BF-227 and FDDNP, to synthetic protein fibrils and human brain tissue. In vitro radioligand binding assays were conducted using synthetic amyloid β 42 and K18ΔK280-tau fibrils. Nonspecific binding was determined by the addition of unlabelled compounds at a concentration of 2 μM. To examine radioligand binding to neuropathological lesions, in vitro autoradiography was conducted using sections of AD brain. [ 18 F]THK-523 showed higher affinity for tau fibrils than for Aβ fibrils, whereas the other probes showed a higher affinity for Aβ fibrils. The autoradiographic analysis indicated that [ 18 F]THK-523 accumulated in the regions containing a high density of tau protein deposits. Conversely, PiB and BF-227 accumulated in the regions containing a high density of Aβ plaques. These findings suggest that the unique binding profile of [ 18 F]THK-523 can be used to identify tau deposits in AD brain. (orig.)

Specific binding assay technique; standardization of reagent

International Nuclear Information System (INIS)

Huggins, K.G.; Roitt, I.M.

1979-01-01

The standardization of a labelled constituent, such as anti-IgE, for use in a specific binding assay method is disclosed. A labelled ligand, such as IgE, is standardized against a ligand reference substance, such as WHO standard IgE, to determine the weight of IgE protein represented by the labelled ligand. Anti-light chain antibodies are contacted with varying concentrations of the labelled ligand. The ligand is then contacted with the labelled constituent which is then quantitated in relation to the amount of ligand protein present. The preparation of 131 I-labelled IgE is described. Also disclosed is an improved specific binding assay test method for determining the potency of an allergen extract in serum from an allergic individual. The improvement involved using a parallel model system of a second complex which consisted of anti-light chain antibodies, labelled ligand and the standardized labelled constituent (anti-IgE). The amount of standardized labelled constituent bound to the ligand in the first complex was determined, as described above, and the weight of ligand inhibited by addition of soluble allergen was then used as a measure of the potency of the allergen extract. (author)

Test-retest reliability of the novel 5-HT{sub 1B} receptor PET radioligand [{sup 11}C]P943

Energy Technology Data Exchange (ETDEWEB)

Saricicek, Aybala [Yale University, Department of Psychiatry, New Haven, CT (United States); Connecticut Mental Health Center, Abraham Ribicoff Research Facilities, New Haven, CT (United States); Izmir Katip Celebi University, Department of Psychiatry, Izmir (Turkey); Chen, Jason; Ruf, Barbara [Yale University, Department of Psychiatry, New Haven, CT (United States); Planeta, Beata; Labaree, David; Gallezot, Jean-Dominique; Huang, Yiyun [Yale University, PET Center, Department of Diagnostic Radiology, New Haven, CT (United States); Subramanyam, Kalyani; Maloney, Kathleen [Yale University, Department of Psychiatry, New Haven, CT (United States); Connecticut Mental Health Center, Abraham Ribicoff Research Facilities, New Haven, CT (United States); Matuskey, David [Yale University, Department of Psychiatry, New Haven, CT (United States); Connecticut Mental Health Center, Abraham Ribicoff Research Facilities, New Haven, CT (United States); Yale University, PET Center, Department of Diagnostic Radiology, New Haven, CT (United States); Deserno, Lorenz [Charite - Universitaetsmedizin Berlin, Department of Psychiatry and Psychotherapy, Campus Charite Mitte, Berlin (Germany); Max-Planck-Institute for Human Cognitive and Brain Sciences, Leipzig, Berlin (Germany); Neumeister, Alexander [Yale University, Department of Psychiatry, New Haven, CT (United States); Mount Sinai School of Medicine, Department of Psychiatry, New York, NY (United States); VA Connecticut Healthcare System, Clinical Neuroscience Division, VA National Center for PTSD, West Haven, CT (United States); Krystal, John H. [Yale University, Department of Psychiatry, New Haven, CT (United States); Connecticut Mental Health Center, Abraham Ribicoff Research Facilities, New Haven, CT (United States); VA Connecticut Healthcare System, Clinical Neuroscience Division, VA National Center for PTSD, West Haven, CT (United States); Carson, Richard E. [Connecticut Mental Health Center, Abraham Ribicoff Research Facilities, New Haven, CT (United States); Bhagwagar, Zubin [Yale University, Department of Psychiatry, New Haven, CT (United States); Connecticut Mental Health Center, Abraham Ribicoff Research Facilities, New Haven, CT (United States); Bristol-Myers Squibb, Wallingford, CT (United States)

2014-11-27

[{sup 11}C]P943 is a novel, highly selective 5-HT{sub 1B} PET radioligand. The aim of this study was to determine the test-retest reliability of [{sup 11}C]P943 using two different modeling methods and to perform a power analysis with each quantification technique. Seven healthy volunteers underwent two PET scans on the same day. Regions of interest (ROIs) were the amygdala, hippocampus, pallidum, putamen, insula, frontal, anterior cingulate, parietal, temporal and occipital cortices, and cerebellum. Two multilinear radioligand quantification techniques were used to estimate binding potential: MA1, using arterial input function data, and the second version of the multilinear reference tissue model analysis (MRTM2), using the cerebellum as the reference region. Between-scan percent variability and intraclass correlation coefficients (ICC) were used to assess test-retest reliability. We also performed power analyses to determine the method that would allow the least number of subjects using within-subject or between-subject study designs. A voxel-wise ICC analysis for MRTM2 BP{sub ND} was performed for the whole brain and all the ROIs studied. Mean percent variability between two scans across regions ranged between 0.4 % and 12.4 % for MA1 BP{sub ND}, 0.5 % and 11.5 % for MA1 BP{sub P}, 16.7 % and 28.3 % for MA1 BP{sub F}, and between 0.2 % and 5.4 % for MRTM2 BP{sub ND}. The power analyses showed a greater number of subjects were required using MA1 BP{sub F} compared with other outcome measures for both within-subject and between-subject study designs. ICC values were the highest using MRTM2 BP{sub ND} and the lowest with MA1 BP{sub F} in ten ROIs. Small regions and regions with low binding had lower ICC values than large regions and regions with high binding. Reliable measures of 5-HT{sub 1B} receptor binding can be obtained using the novel PET radioligand [{sup 11}C]P943. Quantification of 5-HT{sub 1B} receptor binding with MRTM2 BP{sub ND} and with MA1 BP{sub P

In vivo imaging of neuro-inflammation in the rodent brain with [11C]SSR180575, a novel indoleacetamide radioligand of the translocator protein (18 kDa)

International Nuclear Information System (INIS)

Chauveau, F.; Boutin, H.; Van Camp, N.; Thominiaux, C.; Dolle, F.; Tavitian, B.; Chauveau, F.; Boutin, H.; Van Camp, N.; Tavitian, B.; Hantraye, P.; Rivron, L.; Marguet, F.; Castel, M.N.; Rooney, T.; Benavides, J.; Chauveau, F.; Boutin, H.

2011-01-01

Purpose Neuro-inflammation is involved in neurological disorders through the activation of micro-glial cells. Imaging of neuro-inflammation with radioligands for the translocator protein (18 kDa) (TSPO) could prove to be an attractive bio-marker for disease diagnosis and therapeutic evaluation. The indoleacetamide-derived 7-chloro-N, N, 5- trimethyl-4-oxo-3-phenyl-3, 5-dihydro-4H-pyridazino[4, 5-b] indole-1-acetamide, SSR180575, is a selective high-affinity TSPO ligand in human and rodents with neuro-protective effects. Methods Here we report the radiolabelling of SSR180575 with 11 C and in vitro and in vivo imaging in an acute model of neuro-inflammation in rats. Results The image contrast and the binding of [ 11 C] SSR180575 are higher than that obtained with the isoquinoline- based TSPO radioligand, [ 11 C]PK11195. Competition studies demonstrate that [ 11 C]SSR180575 has high specific binding for the TSPO. Conclusion [ 11 C]SSR180575 is the first PET radioligand for the TSPO based on an indoleacetamide scaffold designed for imaging neuro-inflammation in animal models and in the clinic. (authors)

Flow Cytometry-Based Bead-Binding Assay for Measuring Receptor Ligand Specificity

NARCIS (Netherlands)

Sprokholt, Joris K.; Hertoghs, Nina; Geijtenbeek, Teunis B. H.

2016-01-01

In this chapter we describe a fluorescent bead-binding assay, which is an efficient and feasible method to measure interaction between ligands and receptors on cells. In principle, any ligand can be coated on fluorescent beads either directly or via antibodies. Binding between ligand-coated beads

Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

International Nuclear Information System (INIS)

Mong, S.; Wu, H.L.; Clark, M.A.; Stadel, J.M.; Gleason, J.G.; Crooke, S.T.

1984-01-01

A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [ 3 H]-leukotriene D4 [( 3 H]-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, the authors have identified specific binding sites for [ 3 H]-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for [ 3 H]-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [ 3 H]-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with [ 3 H]-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with [ 3 H]-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung

Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

International Nuclear Information System (INIS)

Barrington, W.W.; Jacobson, K.A.; Hutchison, A.J.; Williams, M.; Stiles, G.L.

1989-01-01

A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-[4-(2-[2-[(4- aminophenyl)methylcarbonylamino]ethylaminocarbonyl]- ethyl)phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-[(R)-1-methyl- 2-phenylethyl]adenosine (R-PIA) greater than (+)-N6-[(S)-1-methyl-2- phenylethyl]adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors and is decreased in the presence of 10(-4) M guanosine 5'-[beta, gamma-imido]triphosphate

Brain α1-adrenergic receptors: suitability of [125I]HEAT as a radioligand for in vitro autoradiography

International Nuclear Information System (INIS)

Jones, L.S.; Gauger, L.L.; Davis, J.N.

1983-01-01

[2-(β-4-Hydroxyphenyl)-ethylaminomethyl)-tetralone] (BE 2254, HEAT) is a new potent α 1 -adrenergic receptor blocker. The iodinated radioligand, [ 125 I]HEAT appears to be even more potent than HEAT (Engel and Hoyer, 1981; Glossman et al., 1981) and has proved useful for the studying of α 1 -adrenergic receptors in membrane preparations of rat brain. The authors report the suitability of [ 125 I]HEAT for α 1 -adrenergic binding site autoradiography and a degree of localization of α 1 -adrenergic receptor binding sites that has not been possible with [ 3 H]WB 4101 and [ 3 H]prazosin autoradiography. (Auth.)

In-Solution SH2 Domain Binding Assay Based on Proximity Ligation.

Science.gov (United States)

Machida, Kazuya

2017-01-01

Protein-protein interactions mediated by SH2 domains confer specificity in tyrosine kinase pathways. Traditional assays for assessing interactions between an SH2 domain and its interacting protein such as far-Western and pull-down are inherently low throughput. We developed SH2-PLA, an in-solution SH2 domain binding assay, that takes advantage of the speed and sensitivity of proximity ligation and real-time PCR. SH2-PLA allows for rapid assessment of SH2 domain binding to a target protein using only a few microliters of cell lysate, thereby making it an attractive new tool to study tyrosine kinase signaling.

Specific binding-adsorbent assay method and test means

International Nuclear Information System (INIS)

1981-01-01

A description is given of an improved specific binding assay method and test means employing a nonspecific adsorbent for the substance to be determined, particularly hepatitis B surface (HBsub(s)) antigen, in its free state or additionally in the form of its immune complex. The invention is illustrated by 1) the radioimmunoadsorbent assay for HBsub(s) antigen, 2) the radioimmunoadsorbent assay for HBsub(s) antigen in the form of immune complex with antibody, 3) a study of adsorption characteristics of various anion exchange materials for HBsub(s) antigen, 4) the use of hydrophobic adsorbents in a radioimmunoadsorbent assay for HBsub(s) antigen and 5) the radioimmunoadsorbent assay for antibody to HBsub(s) antigen. The advantages of the present method for detecting HBsub(s) antigen compared to previous methods include the manufacturing advantages of eliminating the need for insolubilised anti-HBsub(s) and the advantages of a single incubation step, fewer manipulations, storability of adsorbent materials, increased sensitivity and versatility of detecting HBsub(s) antigen in the form of its immune complex if desired. (U.K.)

Establishment of a novel radioligand assay using eukaryotically expressed cytochrome P4502D6 for the measurement of liver kidney microsomal type 1 antibody in patients with autoimmune hepatitis and hepatitis C virus infection.

Science.gov (United States)

Ma, Y; Gregorio, G; Gäken, J; Muratori, L; Bianchi, F B; Mieli-Vergani, G; Vergani, D

1997-06-01

Liver kidney microsomal type 1 antibody (LKM1) is the diagnostic marker of autoimmune hepatitis (AIH) type 2 and is also found in patients with hepatitis C virus (HCV) infection. Cytochrome P4502D6 (CYP2D6) is the documented target antigen of LKM1 in AIH, but not in HCV infection. To compare the reactivity in the two conditions, we established a radioligand assay using eukaryotically expressed CYP2D6 as target. A 1.2-kb human CYP2D6 cDNA was isolated from a human liver cDNA library and subcloned into an in vitro transcription vector pSP64 Poly(A). Recombinant CYP2D6 was then produced by in vitro transcription/translation, metabolically labelled with 35S methionine and used in the immunoprecipitation assay. Antibodies that bound radiolabelled CYP2D6 were immunoprecipitated and their levels assessed as cpm. Sera from 50 LKM1-positive patients (26 with AIH; 24 with HCV infection), 128 LKM1-negative patients and 57 normal controls were tested. Reactivity to 35S labelled CYP2D6 was observed in all LKM1-positive sera from patients with AIH and HCV infection, but in none of the controls. The cpm in both conditions were significantly higher than in normal controls (pLKM1 (r 0.87, p<0.001 and r=0.64, p<0.001 for AIH and HCV infection, respectively). Reactivity to 35S labelled CYP2D6 was inhibited by addition of an excess of eukaryotically expressed CYP2D6. CYP2D6 is a major target antigen of both AIH and HCV infection. The novel radioligand assay is highly sensitive and specific.

Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

Science.gov (United States)

Hardison, D Ransom; Holland, William C; McCall, Jennifer R; Bourdelais, Andrea J; Baden, Daniel G; Darius, H Taiana; Chinain, Mireille; Tester, Patricia A; Shea, Damian; Quintana, Harold A Flores; Morris, James A; Litaker, R Wayne

2016-01-01

Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

Directory of Open Access Journals (Sweden)

D Ransom Hardison

Full Text Available Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs. One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R. However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R in certain labs. A fluorescence based receptor binding assay (RBA(F was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2 for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1. Fish (N = 61 of six different species were screened using the RBA(F. Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a correlated well (R2 = 0.71 with those of the RBA(F, given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F, which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F advantages include the long-term (> 5 years stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R. The RBA(F is cost-effective, allows high sample

Measurement of biologically active interleukin-1 by a soluble receptor binding assay

International Nuclear Information System (INIS)

Riske, F.; Chizzonite, R.; Nunes, P.; Stern, A.S.

1990-01-01

A soluble receptor binding assay has been developed for measuring human interleukin-1 alpha (IL-1 alpha), human IL-1 beta, and mouse IL-1 alpha. The assay is based on a competition between unlabeled IL-1 and 125I-labeled mouse recombinant IL-1 alpha for binding to soluble IL-1 receptor prepared from mouse EL-4 cells. The assay measures only biologically active IL-1 folded in its native conformation. The ratio of human IL-1 alpha to human IL-1 beta can be measured in the same sample by a pretreatment step which removes human IL-1 beta from samples prior to assay. This technique has been used to monitor the purification of recombinant IL-1, and may be utilized to specifically and accurately measure bioactive IL-1 in human serum and cell culture supernatants

Key structural features of nonsteroidal ligands for binding and activation of the androgen receptor.

Science.gov (United States)

Yin, Donghua; He, Yali; Perera, Minoli A; Hong, Seoung Soo; Marhefka, Craig; Stourman, Nina; Kirkovsky, Leonid; Miller, Duane D; Dalton, James T

2003-01-01

The purposes of the present studies were to examine the androgen receptor (AR) binding ability and in vitro functional activity of multiple series of nonsteroidal compounds derived from known antiandrogen pharmacophores and to investigate the structure-activity relationships (SARs) of these nonsteroidal compounds. The AR binding properties of sixty-five nonsteroidal compounds were assessed by a radioligand competitive binding assay with the use of cytosolic AR prepared from rat prostates. The AR agonist and antagonist activities of high-affinity ligands were determined by the ability of the ligand to regulate AR-mediated transcriptional activation in cultured CV-1 cells, using a cotransfection assay. Nonsteroidal compounds with diverse structural features demonstrated a wide range of binding affinity for the AR. Ten compounds, mainly from the bicalutamide-related series, showed a binding affinity superior to the structural pharmacophore from which they were derived. Several SARs regarding nonsteroidal AR binding were revealed from the binding data, including stereoisomeric conformation, steric effect, and electronic effect. The functional activity of high-affinity ligands ranged from antagonist to full agonist for the AR. Several structural features were found to be determinative of agonist and antagonist activities. The nonsteroidal AR agonists identified from the present studies provided a pool of candidates for further development of selective androgen receptor modulators (SARMs) for androgen therapy. Also, these studies uncovered or confirmed numerous important SARs governing AR binding and functional properties by nonsteroidal molecules, which would be valuable in the future structural optimization of SARMs.

Preliminary studies of 99mTc-PQQ-NMDAR binding and effect of specificity binding by mannitol

International Nuclear Information System (INIS)

Xingqin Zhou; Yanyan Kong; Guoxian Cao; Jiankang Zhang

2013-01-01

Pyrroloquinoline quinone (PQQ) is a powerful neuroprotectant that specifically binds to brain NMDA receptors and inhibits excitotoxicity. Imaging this binding reaction in the brain remains a long sought goal in this field of study, and one of the primary challenges remaining is enabling soluble labeled PQQ to pass the blood-brain barrier (BBB). Previously, our group successfully labeled PQQ with Technetium-99m ( 99m Tc), a metastable nuclear isomer used in radioactive isotope medical tests. In this work, we determined the specific binding of 99m Tc-PQQ and NMDAR by radioligand receptor assay. Ebselen (EB) and MK-801 both effectively inhibited 99m Tc-PQQ binding. We then investigated methods of opening the BBB using mannitol to enable entry to the brain by 99m Tc-PQQ. Our results showed that 7.5 mL/kg of 20 % mannitol effectively opened the BBB and 20 min was the optimum treatment time. Competition studies showed that mannitol did not affect the specific binding between 99m Tc-PQQ and NMDA receptors. Using this method, the amount of 99m Tc-PQQ uptake and retention was increased most significantly in the hippocampus and cortex, and re-opening the BBB did not affect binding. Together, our results demonstrate that the use of mannitol to open the BBB may contribute significantly to improving image quality by increasing the uptake amount of a water-soluble agent in brain. (author)

Radiosynthesis and preliminary evaluation of 5-[123/125I]iodo-3-(2(S)-azetidinylmethoxy)pyridine: a radioligand for nicotinic acetylcholine receptors

International Nuclear Information System (INIS)

Horti, Andrew G.; Koren, Andrei O.; Lee, Kan Sam; Mukhin, Alexey G.; Vaupel, D. Bruce; Kimes, Alane S.; Stratton, Morgan; London, Edythe D.

1999-01-01

The radiochemical syntheses of 5-[ 125 I]iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-[ 125 I]-iodo-A-85380, [ 125 I]1) and 5-[ 123 I]-iodo-A-85380, [ 123 I]1, were accomplished by radioiodination of 5-trimethylstannyl-3-((1-tert-butoxycarbonyl-2(S) -azetidinyl)methoxy)pyridine, 2, followed by acidic deprotection. Average radiochemical yields of [ 125 I]1 and [ 123 I]1 were 40-55%; and the average specific radioactivities were 1,700 and 7,000 mCi/μmol, respectively. Binding affinities of [ 125 I]1 and [ 123 I]1 in vitro (rat brain membranes) were each characterized by a K d value of 11 pM. Preliminary in vivo assay and ex vivo autoradiography of mouse brain indicated that [ 125 I]1 selectively labels nicotinic acetylcholine receptors (nAChRs) with very high affinity and specificity. These studies suggest that [ 123 I]1 may be useful as a radioligand for single photon emission computed tomography (SPECT) imaging of nAChRs

[125Iodo]BE 2254, a new radioligand for alpha 1-adrenoceptors

International Nuclear Information System (INIS)

Engel, G.; Hoyer, D.

1982-01-01

[ 125 cIodo]2-[beta-(14-hydroxyphenyl)-ethyl-aminomethyl]-tetralone([ 125 Iodo]BE 2254 or IBE 2254), a new iodinated radioligand of high specific radioactivity (2175 Ci/mmol), was synthesized and used to characterize alpha 1-adrenoceptors in rat lung and cerebral cortex membranes. The binding constants of IBE 2254, using rat lung and cortex membranes, were Kd . 53 +/- 10 pM, Bmax . 53 +/- 8 fmol/mg; and Kd . 78 +/- 14 pM, Bmas . 210 +/- 26 fmol/mg, respectively (Kd . dissociation constant of IBE 2254 determined in saturation experiments). In equilibrium binding experiments with IBE 2254, at concentrations of the free ligand up to 1.2 nM, only one class of binding sites could be detected. In kinetic experiments, the association and dissociation rate constants were 2.3 X 10(9) M-1 min-1 and 0.10 min-1, respectively. In rat cerebral cortex membranes, alpha-adrenoceptor antagonists competed for IBE 2254 binding in the following order: prazosin greater than BE 2254 greater than WB 4101 greater than phentolamine greater than corynanthine greater than yohimbine greater than rauwolscine, indicating strongly that IBE 2254 binds to alpha 1-adrenoceptors. The calculated affinities of different alpha-adrenoceptor blocking agents from inhibition of IBE 2254 binding were nearly identical in rat lung and cerebral cortex. The low dissociation constant of the ligand together with its high specific radioactivity allows binding studies to be carried out with tissue samples where only small densities of alpha 1-adrenoceptors are present

Radioiodinated ligands for the estrogen receptor: Effect of different 7-cyanoalkyl chains on the binding affinity of novel iodovinyl-6-dehydroestradiols

International Nuclear Information System (INIS)

Neto, Carina; Oliveira, Maria Cristina; Gano, Lurdes; Marques, Fernanda; Santos, Isabel; Morais, Goreti Ribeiro; Yasuda, Takumi; Thiemann, Thies; Botelho, Filomena; Oliveira, Carlos F.

2009-01-01

Three novel 17α-ethynyl-Δ 6,7 -estra-3,17β-diols and their 17α-[ 125 I]-iodovinyl derivatives, containing different C7-cyanoalkyl chains, were studied as potential radioligands for the estrogen receptor. The influence of the chain length on the biological behaviour of the compounds was assessed through in vitro ER binding assays of the ethynyl derivatives and breast cancer cell uptake studies of the 17α-[ 125 I]-iodovinyl-Δ 6,7 -estra-3,17β-diols. A difference in alkyl chain induced a decrease in ER binding affinities of substances, however, the receptor-binding affinities (RBA) of all compounds were lower than that of estradiol itself. In addition, a non-specific cell binding was observed which is in accordance with the encountered ethynyl RBA values suggesting that the uptake is not ER mediated

[(3)H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([(3)H]PSB-11), a novel high-affinity antagonist radioligand for human A(3) adenosine receptors.

Science.gov (United States)

Müller, Christa E; Diekmann, Martina; Thorand, Mark; Ozola, Vita

2002-02-11

This study describes the preparation and binding properties of [(3)H]PSB-11, a novel, potent, and selective antagonist radioligand for human A(3) adenosine receptors (ARs). [(3)H]PSB-11 binding to membranes of Chinese hamster ovary (CHO) cells expressing the human A(3) AR was saturable and reversible. Saturation experiments showed that [(3)H]PSB-11 labeled a single class of binding sites with high affinity (K(D)=4.9 nM) and limited capacity (B(max)=3500 fmol/mg of protein). PSB-11 is highly selective versus the other adenosine receptor subtypes. The new radioligand shows an extraordinarily low degree of non-specific binding rendering it a very useful tool for studying the (patho)physiological roles of A(3 )ARs.

Development of a Surface Plasmon Resonance Assay for the Characterization of Small-Molecule Binding Kinetics and Mechanism of Binding to Kynurenine 3-Monooxygenase.

Science.gov (United States)

Poda, Suresh B; Kobayashi, Masakazu; Nachane, Ruta; Menon, Veena; Gandhi, Adarsh S; Budac, David P; Li, Guiying; Campbell, Brian M; Tagmose, Lena

2015-10-01

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway, was identified as a potential therapeutic target for treating neurodegenerative and psychiatric disorders. In this article, we describe a surface plasmon resonance (SPR) assay that delivers both kinetics and the mechanism of binding (MoB) data, enabling a detailed characterization of KMO inhibitors for the enzyme in real time. SPR assay development included optimization of the protein construct and the buffer conditions. The stability and inhibitor binding activity of the immobilized KMO were significantly improved when the experiments were performed at 10°C using a buffer containing 0.05% n-dodecyl-β-d-maltoside (DDM) as the detergent. The KD values of the known KMO inhibitors (UPF648 and RO61-8048) from the SPR assay were in good accordance with the biochemical LC/MS/MS assay. Also, the SPR assay was able to differentiate the binding kinetics (k(a) and k(d)) of the selected unknown KMO inhibitors. For example, the inhibitors that showed comparable IC50 values in the LC/MS/MS assay displayed differences in their residence time (τ = 1/k(d)) in the SPR assay. To better define the MoB of the inhibitors to KMO, an SPR-based competition assay was developed, which demonstrated that both UPF648 and RO61-8048 bound to the substrate-binding site. These results demonstrate the potential of the SPR assay for characterizing the affinity, the kinetics, and the MoB profiles of the KMO inhibitors.

Murine alpha1-adrenoceptor subtypes. I. Radioligand binding studies

NARCIS (Netherlands)

Yang, M.; Reese, J.; Cotecchia, S.; Michel, M. C.

1998-01-01

Alpha1-adrenoceptors were identified in murine tissues by [3H]prazosin saturation binding studies, with a rank order of cerebral cortex > cerebellum > liver > lung > kidney > heart > spleen, with the spleen not exhibiting detectable expression. Competition binding studies were performed with

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding.

Science.gov (United States)

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-05-01

The P2X(7) receptor exhibits complex pharmacological properties. In this study, binding of a [(3)H]-labelled P2X(7) receptor antagonist to human P2X(7) receptors has been examined to further understand ligand interactions with this receptor. The P2X(7) receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X(7) receptors. Binding of [(3)H]-compound-17 was higher in membranes prepared from cells expressing P2X(7) receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X(7) receptors. Binding was reversible, saturable and modulated by P2X(7) receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. These data demonstrate that human P2X(7) receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X(7) receptor complex enhances subsequent binding to other P2X(7) subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X(7) receptor.

Effects of common anesthetic agents on [(18)F]flumazenil binding to the GABAA receptor

DEFF Research Database (Denmark)

Palner, Mikael; Beinat, Corinne; Banister, Sam

2016-01-01

in preclinical imaging studies and clinical imaging studies involving patient populations that do not tolerate relatively longer scan times. The objective of this study was to examine the effects of anesthesia on the binding of [(18)F]flumazenil to GABAA receptors in mice. METHODS: Brain and whole blood...... mice. CONCLUSIONS: Anesthesia has pronounced effects on the binding and blood-brain distribution of [(18)F]flumazenil. Consequently, considerable caution must be exercised in the interpretation of preclinical and clinical PET studies of GABAA receptors involving the use of anesthesia.......BACKGROUND: The availability of GABAA receptor binding sites in the brain can be assessed by positron emission tomography (PET) using the radioligand, [(18)F]flumazenil. However, the brain uptake and binding of this PET radioligand are influenced by anesthetic drugs, which are typically needed...

Radiosynthesis and autoradiographic evaluation of [{sup 11}C]NAD-299, a radioligand for visualization of the 5-HT{sub 1A} receptor

Energy Technology Data Exchange (ETDEWEB)

Sandell, Johan E-mail: Johan.Sandell@psyk.ks.se; Halldin, Christer; Hall, Haakan; Thorberg, Seth-Olov; Werner, Tom; Sohn, Daniel; Sedvall, Goeran; Farde, Lars

1999-02-01

The selective 5-HT{sub 1A} receptor antagonist NAD-299 ([R]-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran- 5-carboxamide) was labeled with the positron emitting radionucldie carbon-11. The radioligand was synthesized from NAD-195 ([R]-3-N,N-dicyclobutylamino-8-fluoro-5-trifluoromethylsulfonyloxy-3, 4-dihydro-2H-1-benzopyran) in two radiochemical steps. A palladium-catalyzed reaction of NAD-195 and [{sup 11}C]cyanide was followed by hydrolysis of the carbon-11-labeled nitrile intermediate with basic hydrogen peroxide. The total radiochemical yield, based on [{sup 11}C]CO{sub 2} and corrected for decay, was 20-40%. The specific radioactivity was 24 GBq/{mu}mol (900 Ci/mmol) at end of synthesis, with a radiochemical purity better than 99% and a total synthesis time of 40-45 min. Autoradiographic examination of [{sup 11}C]NAD-299 binding in human brain postmortem demonstrated high binding in hippocampus, raphe nuclei, and neocortex. The binding in the hippocampus was higher than in the neocortex. Within the hippocampus, the densest binding was observed in the CA1 region. [{sup 11}C]NAD-299 binding was inhibited by addition of the 5-HT{sub 1A} receptor ligands WAY-100635, pindolol, ({+-})-8-OH-DPAT, 5-HT, and buspirone, leaving a low background of nonspecific binding. The results indicate that [{sup 11}C]NAD-299 binds specifically to 5-HT{sub 1A} receptors in the human brain in vitro and is a potential radioligand for positron emission tomography (PET) examination of 5-HT{sub 1A} receptors in vivo.

Cholecystokinin receptors: Biochemical demonstration and autoradiographical localization in rat brain and pancreas using [3H] cholecystokinin8 as radioligand

International Nuclear Information System (INIS)

Van Dijk, A.; Richards, J.G.; Trzeciak, A.; Gillessen, D.; Moehler, H.

1984-01-01

Since cholecystokinin8 (CCK8) seems to be the physiological ligand of CCK receptors in the brain, it would be the most suitable probe for the characterization of CCK receptors in radioligand binding studies. [ 3 H]CCK8 was synthetized with a specific radioactivity sufficient for the detection of high affinity binding sites. [ 3 H]CCK8 binds saturably and reversibly to distinct sites in rat brain and pancreas with nanomolar affinity. While the C-terminal tetrapeptide of CCK is the minimal structure required for nanomolar affinity in the brain, the entire octapeptide sequence is required for binding affinity in pancreas. Desulfated CCK8 and several gastrin-I peptides, which are likewise unsulfated, show virtually no affinity to the binding sites in pancreas but high affinity in cerebral cortex. The ligand specificity of the CCK peptides corresponds to their electrophysiological potency in the brain and their stimulation of secretion in pancreas, respectively. Autoradiographically, high densities of [ 3 H]CCK8 binding sites were found in cerebral cortex and olfactory bulb, medium levels in nucleus accumbens, hippocampus, dentate gyrus, and striatum with virtually no labeling in cerebellum. This pattern is similar to the distribution of CCK-like immunoreactivity in the brain. In pancreas, equally high levels of [ 3 H]CCK8 labeling were found in the exocrine and endocrine region. [ 3 H]CCK8 binding sites differ from those identified previously with [ 125 I]Bolton-Hunter-CCK33 by their sensitivity to guanyl nucleotides in the brain, their ion dependency in the brain, and pancreas, and their different autoradiographical localization in some parts of the brain. The distribution of CCK binding sites labeled with [ 3 H]CCK8 appears to correlate better with the CCK immunoreactivity than those labeled with [ 125 I]Bolton-Hunter-CCK33. Thus, [ 3 H]CCK8 appears to be the radioligand of choice for the investigation of CCK receptors

Serotonin 2A receptor agonist binding in the human brain with [C]Cimbi-36

DEFF Research Database (Denmark)

Ettrup, A.; da Cunha-Bang, S.; McMahon, Barry P.

2014-01-01

[C]Cimbi-36 was recently developed as a selective serotonin 2A (5-HT) receptor agonist radioligand for positron emission tomography (PET) brain imaging. Such an agonist PET radioligand may provide a novel, and more functional, measure of the serotonergic system and agonist binding is more likely ....... Thus, we here describe [C]Cimbi-36 as the first agonist PET radioligand to successfully image and quantify 5-HT receptors in the human brain.Journal of Cerebral Blood Flow & Metabolism advance online publication, 30 April 2014; doi:10.1038/jcbfm.2014.68....... than antagonist binding to reflect 5-HT levels in vivo. Here, we show data from a first-in-human clinical trial with [C]Cimbi-36. In 29 healthy volunteers, we found high brain uptake and distribution according to 5-HT receptors with [C]Cimbi-36 PET. The two-tissue compartment model using arterial input...

Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography

Energy Technology Data Exchange (ETDEWEB)

Dissanayake, V.U.; Hughes, J.; Hunter, J.C. (Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge (England))

1991-07-01

The specific binding of the selective {mu}-, {delta}-, and {kappa}-opioid ligands (3H)(D-Ala2,MePhe4,Gly-ol5)enkephalin ((3H) DAGOL), (3H)(D-Pen2,D-Pen5)enkephalin ((3H)DPDPE), and (3H)U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of {mu}- and {kappa}-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that (3H)DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the {delta}-opioid binding site. Autoradiography experiments demonstrated that specific (3H)DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the {delta}-opioid binding site in medulla homogenates, using agonist ((3H)DPDPE) and antagonist ((3H)diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the {delta}-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the {delta} binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.

Microbubble Enzyme-Linked Immunosorbent Assay for the Detection of Targeted Microbubbles in in Vitro Static Binding Assays.

Science.gov (United States)

Wischhusen, Jennifer; Padilla, Frederic

2017-07-01

Targeted microbubbles (MBs) are ultrasound contrast agents that are functionalized with a ligand for ultrasound molecular imaging of endothelial markers. Novel targeted MBs are characterized in vitro by incubation in protein-coated wells, followed by binding quantification by microscopy or ultrasound imaging. Both methods provide operator-dependent results: Between 3 and 20 fields of view from a heterogeneous sample are typically selected for analysis by microscopy, and in ultrasound imaging, different acoustic settings affect signal intensities. This study proposes a new method to reproducibly quantify MB binding based on enzyme-linked immunosorbent assay (ELISA), in which bound MBs are revealed with an enzyme-linked antibody. MB-ELISA was adapted to in vitro static binding assays, incubating the MBs in inverted position or by agitation, and compared with microscopy. The specificity and sensitivity of MB-ELISA enable the reliable quantification of MB binding in a rapid, high-throughput and whole-well analysis, facilitating the characterization of new targeted contrast agents. Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Comparison of the binding characteristics of [{sup 18}F]THK-523 and other amyloid imaging tracers to Alzheimer's disease pathology

Energy Technology Data Exchange (ETDEWEB)

Harada, Ryuichi; Okamura, Nobuyuki; Yoshikawa, Takeo; Yanai, Kazuhiko [Tohoku University School of Medicine, Department of Pharmacology, Sendai (Japan); Furumoto, Shozo [Tohoku University School of Medicine, Department of Pharmacology, Sendai (Japan); Tohoku University, Division of Radiopharmaceutical Chemistry, Cyclotron and Radioisotope Center, Sendai (Japan); Tago, Tetsuro; Iwata, Ren [Tohoku University, Division of Radiopharmaceutical Chemistry, Cyclotron and Radioisotope Center, Sendai (Japan); Maruyama, Masahiro; Higuchi, Makoto [Molecular Imaging Center, National Institute of Radiological Sciences, Chiba (Japan); Arai, Hiroyuki [Tohoku University, Department of Geriatrics and Gerontology, Institute of Development, Aging and Cancer, Sendai (Japan); Kudo, Yukitsuka [Tohoku University, Innovation of New Biomedical Engineering Center, Sendai (Japan)

2013-01-15

Extensive deposition of senile plaques and neurofibrillary tangles in the brain is a pathological hallmark of Alzheimer's disease (AD). Although several PET imaging agents have been developed for in vivo detection of senile plaques, no PET probe is currently available for selective detection of neurofibrillary tangles in the living human brain. Recently, [{sup 18}F]THK-523 was developed as a potential in vivo imaging probe for tau pathology. The purpose of this study was to compare the binding properties of [{sup 18}F]THK-523 and other amyloid imaging agents, including PiB, BF-227 and FDDNP, to synthetic protein fibrils and human brain tissue. In vitro radioligand binding assays were conducted using synthetic amyloid {beta}{sub 42} and K18{Delta}K280-tau fibrils. Nonspecific binding was determined by the addition of unlabelled compounds at a concentration of 2 {mu}M. To examine radioligand binding to neuropathological lesions, in vitro autoradiography was conducted using sections of AD brain. [{sup 18}F]THK-523 showed higher affinity for tau fibrils than for A{beta} fibrils, whereas the other probes showed a higher affinity for A{beta} fibrils. The autoradiographic analysis indicated that [{sup 18}F]THK-523 accumulated in the regions containing a high density of tau protein deposits. Conversely, PiB and BF-227 accumulated in the regions containing a high density of A{beta} plaques. These findings suggest that the unique binding profile of [{sup 18}F]THK-523 can be used to identify tau deposits in AD brain. (orig.)

A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

Science.gov (United States)

Wilson, Kris; Mole, Damian J; Homer, Natalie Z M; Iredale, John P; Auer, Manfred; Webster, Scott P

2015-02-01

Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO. © 2014 Society for Laboratory Automation and Screening.

Calibration and validation of the 14C-labelled polyethylene glycol-binding assay for tannins in tropical browse

International Nuclear Information System (INIS)

Mlambo, V.; Makkar, H.P.S.

2005-01-01

This study evaluates the radiolabelled polyethylene glycol (PEG)-binding procedure [Silanikove, N., Shinder, D., Gilboa, N., Eyal, M., Nitsan, Z., 1996. Polyethylene glycol-binding to plant samples as an assay for the biological effects of tannins: predicting the negative effects of tannins in Mediterranean browse on rumen degradation. J. Agric. Food Chem. 44, 3230-3234] for tannin analysis, using 27 tropical browse plants. In this method, the amount of PEG bound to a plant sample is assumed to be a reflection of its tannin content. The method was modified to exclude the use of non-tanniniferous substrate for estimating non-specific binding (NSB) in tannin-containing substrates. Non-specific binding values varied widely (0.4-2.8 mg PEG/100 mg DM tannin-free substrate) when the tannin-free substrate was changed from wheat straw to either rye grass or maize shoots. We therefore propose a modified radiolabelled PEG-binding method to estimate the level of PEG-binding (PEGb) to tannin-containing foliage without using tannin-free substrate to correct for non-specific binding. In this approach, incremental levels of each tanniniferous substrate were used to generate PEGb values. The resultant linear response was analysed and tannin activity was expressed as the slope of the response curve (PEGbSlope) observed for each substrate. The slope takes into account the non-specific binding in each substrate, thus PEGbSlope does not require correction for NSB using tannin-free samples. This approach improved the correlation between PEGb and the 125 I-labelled bovine serum albumin precipitation assay. Relationships between the modified PEG-binding assay and radiolabelled bovine serum albumin assay, in vitro tannin bioassay and colorimetric assays are presented. (author)

A sensitive competitive binding assay for exogenous and endogenous heparins

International Nuclear Information System (INIS)

Dawes, J.; Pepper, D.S.

1982-01-01

A new type of assay for heparins has been devised, in which the test material competes with 125 I-labelled heparin for binding to protamine-Sepharose. The assay is very sensitive and will measure heparin concentrations down to 10 ng ml-1. It responds to both the degree of sulphation and the molecular weight of acidic polysaccharides, but is independent of their biological activities. It can be used to quantitate heparins in biological fluids after pretreatment of the samples with protease. In this way endogenous heparins were measured in normal human serum, plasma and urine. The assay is extremely versatile and has great potential for the investigation of endogenous and exogenous heparins

Development of Plate Reader and On-Line Microfluidic Screening to Identify Ligands of the 5-Hydroxytryptamine Binding Protein in Venoms

Directory of Open Access Journals (Sweden)

Reka A. Otvos

2015-06-01

Full Text Available The 5-HT3 receptor is a ligand-gated ion channel, which is expressed in the nervous system. Its antagonists are used clinically for treatment of postoperative- and radiotherapy-induced emesis and irritable bowel syndrome. In order to better understand the structure and function of the 5-HT3 receptor, and to allow for compound screening at this receptor, recently a serotonin binding protein (5HTBP was engineered with the Acetylcholine Binding Protein as template. In this study, a fluorescence enhancement assay for 5HTBP ligands was developed in plate-reader format and subsequently used in an on-line microfluidic format. Both assay types were validated using an existing radioligand binding assay. The on-line microfluidic assay was coupled to HPLC via a post-column split which allowed parallel coupling to a mass spectrometer to collect MS data. This high-resolution screening (HRS system is well suitable for compound mixture analysis. As a proof of principle, the venoms of Dendroapsis polylepis, Pseudonaja affinis and Pseudonaja inframacula snakes were screened and the accurate masses of the found bioactives were established. To demonstrate the subsequent workflow towards structural identification of bioactive proteins and peptides, the partial amino acid sequence of one of the bioactives from the Pseudonaja affinis venom was determined using a bottom-up proteomics approach.

Considerations for dosimetry calculations with neuroreceptor binding radioligands

International Nuclear Information System (INIS)

Wong, D.F.; Bice, A.N.; Beck, T.; Dannals, R.F.; Links, J.M.; Wagner, H.N. Jr.

1986-01-01

Neuroreceptor binding radiotracers have unique characteristics which must be considered in absorbed dose calculations. In this article the authors outline some of the important issues to be considered such as the high specific binding to various receptor bearing tissue regions, the receptor kinetics, the specific activity of the injected ligand and the presence of competing unlabeled substances. As an example of these considerations they have shown the outline of the measurements for animal and human biodistribution data of the D2 dopamine receptor binding ligand 11 C-3N-methylspiperone (NMSP) and they calculated the absorbed doses for the important body organs. This includes dose estimates using various species including mice, followed by primate and human data. Because of the selective uptake of NMSP to brain regions such as the basal ganglia they calculated values specifically for these areas in the cerebellum. Since kinetic modeling and therapeutic drug monitoring employ competing unlabeled ligands such as haloperidol which alter the NMSP distribution they estimated the dose in both unblocked and cases blocked with haloperidol. In such cases the doses were about 50% lower in the blocked cases for the basal ganglia. Target organs such as the bladder using external probes and a model based upon changing urine volumes suggests a 30% decrease from mouse estimates. 13 references, 4 figures, 7 tables

A Soluble Fluorescent Binding Assay Reveals PIP2 Antagonism of TREK-1 Channels

Directory of Open Access Journals (Sweden)

Cerrone Cabanos

2017-08-01

Full Text Available Lipid regulation of ion channels by low-abundance signaling lipids phosphatidylinositol 4,5-bisphosphate (PIP2 and phosphatidic acid (PA has emerged as a central cellular mechanism for controlling ion channels and the excitability of nerves. A lack of robust assays suitable for facile detection of a lipid bound to a channel has hampered the probing of the lipid binding sites and measuring the pharmacology of putative lipid agonists for ion channels. Here, we show a fluorescent PIP2 competition assay for detergent-purified potassium channels, including TWIK-1-related K+-channel (TREK-1. Anionic lipids PA and phosphatidylglycerol (PG bind dose dependently (9.1 and 96 μM, respectively and agonize the channel. Our assay shows PIP2 binds with high affinity (0.87 μM but surprisingly can directly antagonize TREK-1 in liposomes. We propose a model for TREK-1 lipid regulation where PIP2 can compete with PA and PG agonism based on the affinity of the lipid for a site within the channel.

A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding

Science.gov (United States)

Frauer, Carina; Leonhardt, Heinrich

2009-01-01

We present a simple, non-radioactive assay for DNA methyltransferase activity and DNA binding. As most proteins are studied as GFP fusions in living cells, we used a GFP binding nanobody coupled to agarose beads (GFP nanotrap) for rapid one-step purification. Immobilized GFP fusion proteins were subsequently incubated with different fluorescently labeled DNA substrates. The absolute amounts and molar ratios of GFP fusion proteins and bound DNA substrates were determined by fluorescence spectroscopy. In addition to specific DNA binding of GFP fusion proteins, the enzymatic activity of DNA methyltransferases can also be determined by using suicide DNA substrates. These substrates contain the mechanism-based inhibitor 5-aza-dC and lead to irreversible covalent complex formation. We obtained covalent complexes with mammalian DNA methyltransferase 1 (Dnmt1), which were resistant to competition with non-labeled canonical DNA substrates, allowing differentiation between methyltransferase activity and DNA binding. By comparison, the Dnmt1C1229W catalytic site mutant showed DNA-binding activity, but no irreversible covalent complex formation. With this assay, we could also confirm the preference of Dnmt1 for hemimethylated CpG sequences. The rapid optical read-out in a multi-well format and the possibility to test several different substrates in direct competition allow rapid characterization of sequence-specific binding and enzymatic activity. PMID:19129216

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

Science.gov (United States)

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

2017-08-01

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

Evaluation in vitro and in animals of a new 11C-labeled PET radioligand for metabotropic glutamate receptors 1 in brain

International Nuclear Information System (INIS)

Zanotti-Fregonara, Paolo; Liow, Jeih-San; Zoghbi, Sami S.; Clark, David T.; Morse, Cheryl; Pike, Victor W.; Barth, Vanessa N.; Rhoads, Emily; Siuda, Edward; Heinz, Beverly A.; Nisenbaum, Eric; Dressman, Bruce; Joshi, Elizabeth; Luffer-Atlas, Debra; Fisher, Matthew J.; Masters, John J.; Goebl, Nancy; Kuklish, Steven L.; Tauscher, Johannes; Innis, Robert B.

2013-01-01

Two allosteric modulators of the group I metabotropic glutamate receptors (mGluR1 and mGluR5) were evaluated as positron emission tomography (PET) radioligands for mGluR1. LY2428703, a full mGluR1 antagonist (IC 50 8.9 nM) and partial mGluR5 antagonist (IC 50 118 nM), and LSN2606428, a full mGluR1 and mGluR5 antagonist (IC 50 35.3 nM and 10.2 nM, respectively) were successfully labeled with 11 C and evaluated as radioligands for mGluR1. The pharmacology of LY2428703 was comprehensively assessed in vitro and in vivo, and its biodistribution was investigated by liquid chromatography-mass spectrometry/mass spectrometry, and by PET imaging in the rat. In contrast, LSN2606428 was only evaluated in vitro; further evaluation was stopped due to its unfavorable pharmacological properties and binding affinity. 11 C-LY2428703 showed promising characteristics, including: (1) high potency for binding to human mGluR1 (IC 50 8.9 nM) with no significant affinity for other human mGlu receptors (mGluR2 through mGluR8); (2) binding to brain displaceable by administration of an mGluR1 antagonist; (3) only one major radiometabolite in both plasma and brain, with a negligible brain concentration (with 3.5 % of the total radioactivity in cerebellum) and no receptor affinity; (4) a large specific and displaceable signal in the mGluR1-rich cerebellum with no significant in vivo affinity for mGluR5, as shown by PET studies in rats; and (5) lack of substrate behavior for efflux transporters at the blood-brain barrier, as shown by PET studies conducted in wild-type and knockout mice. 11 C-LY2428703, a new PET radioligand for mGluR1 quantification, displayed promising characteristics both in vitro and in vivo in rodents. (orig.)

Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using (3)H-HOCPCA

DEFF Research Database (Denmark)

Klein, A B; Bay, T; Villumsen, I S

2016-01-01

analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version ((3)H-HOCPCA) to radioactively label the specific GHB high-affinity binding site and gain further insight into the density, distribution and developmental profile of this protein. We show that, in low nanomolar concentrations...... brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that (3)H-HOCPCA is a highly sensitive radioligand, offering advantages over...

A simplified radiometabolite analysis procedure for PET radioligands using a solid phase extraction with micellar medium

International Nuclear Information System (INIS)

Nakao, Ryuji; Halldin, Christer

2013-01-01

A solid phase extraction method has been developed for simple and high-speed direct determination of PET radioligands in plasma. Methods: This methodology makes use of a micellar medium and a solid-phase extraction cartridge for displacement of plasma protein bound radioligand and separation of PET radioligands from their radiometabolites without significant preparation. The plasma samples taken from monkey or human during PET measurements were mixed with a micellar eluent containing an anionic surfactant sodium dodecyl sulphate and loaded onto SPE cartridges. The amount of radioactivity corresponding to parent radioligand (retained on the cartridge) and its radioactive metabolites (eluted with micellar eluent) was measured. Results: Under the optimized conditions, excellent separation of target PET radioligands from their radiometabolites was achieved with a single elution and short run-time of 1 min. This method was successfully applied to study the metabolism for 11 C-labelled radioligands in human or monkey plasma. The amount of parent PET radioligands estimated by micellar solid phase extraction strongly corresponded with that determined by radio-LC. The improved throughput permitted the analysis of a large number of plasma samples (up to 13 samples per one PET study) for accurate estimation of metabolite-corrected input function during quantitative PET imaging studies. Conclusion: Solid phase extraction together with micellar medium is fast, sensitive and easy to use, and therefore it is an attractive alternative method to determine relative composition of PET radioligands in plasma

[111In-DOTA]LTT-SS28, a first pansomatostatin radioligand for in vivo targeting of somatostatin receptor-positive tumors.

Science.gov (United States)

Maina, Theodosia; Cescato, Renzo; Waser, Beatrice; Tatsi, Aikaterini; Kaloudi, Aikaterini; Krenning, Eric P; de Jong, Marion; Nock, Berthold A; Reubi, Jean Claude

2014-08-14

Radiolabeled pansomatostatin-like analogues are expected to enhance the diagnostic sensitivity and to expand the clinical indications of currently applied sst2-specific radioligands. In this study, we present the somatostatin mimic [DOTA]LTT-SS28 {[(DOTA)Ser1,Leu8,D-Trp22,Tyr25]SS28} and its 111In radioligand. [DOTA]LTT-SS28 exhibited a pansomatostatin-like profile binding with high affinity to all five hsst1-hsst5 subtypes (IC50 values in the lower nanomolar range). Furthermore, [DOTA]LTT-SS28 behaved as an agonist at hsst2, hsst3, and hsst5, efficiently stimulating internalization of the three receptor subtypes. Radioligand [111In-DOTA]LTT-SS28 showed good stability in the mouse bloodstream. It displayed strong and specific uptake in AR42J tumors 4 h postinjection (9.3±1.6% ID/g vs 0.3±0.0% ID/g during sst2 blockade) in mice. Significant and specific uptake was also observed in HEK293-hsst2-, HEK293-hsst3-, and HEK293-hsst5-expressing tumors (4.43±1.5, 4.88±1.1, and DOTA]LTT-SS28 specifically localizes in sst2-, sst3-, and sst5-expressing xenografts in mice showing promise for multi-sst1-sst5 targeted tumor imaging.

Characterisation of the human NMDA receptor subunit NR3A glycine binding site

DEFF Research Database (Denmark)

Nilsson, A; Duan, J; Mo-Boquist, L-L

2007-01-01

In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277...

Heterogeneous binding of sigma radioligands in the rat brain and liver

International Nuclear Information System (INIS)

Ross, S.B.

1991-01-01

The binding of four sigma receptor ligands, 3 H-(+)-N-allyl-N-normetazocine ( 3 H-(+)-SKF 10,047), 3 H-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ( 3 H-(+)-3-PPP), 3 H-haloperidol and 3 H-N,N'-di(o-totyl)guanidine ( 3 H-DTG), and the cytochrome P450IID6 ligand and dopamine uptake inhibitor 3 H-1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine ( 3 H-GBR 12935) to membranal preparations of rat liver or whole rat brain was examined regarding kinetical properties and inhibition by various compounds with affinity for sigma binding sites or cytochrome P-450. In rat brain the density of binding sites was increased in order (+)-SKF 10,047 3 H-(+)-SKF 10,047 there were quite marked differences between the ligands studied. Multiple binding sites were also indicated by the low Hill coefficients found for most of the compounds studied. It was found that the cytochrome P-450 inhibitor proadifen (SKF 525A), like haloperidol, was a potent inhibitor of the binding of 3 H-(+)-SKF 10,047, 3 H-(+)-3-PPP and 3 H-haloperidol to the liver and brain preparations, less active in inhibiting the binding of 3 H-DTG and least effective on the binding of 3 H-GBR 12935. Another cytochrome P-450 inhibitor, L-lobeline, was particularly potent in inhibiting the binding of 3 H-DTG but was also quite potent inhibitor of the binding of the other sigma ligands. It was less potent in inhibiting the binding of 3 H-GBR 12935. The binding of the latter ligand was potently inhibited by the analogous compound GBR 12909 but of the other compounds examined only L-lobeline, proadifen, haloperidol, DTG and (+)-3-PPP had IC50 values below 10 μM. The possibility that the sigma binding sites are identical with some subforms of cytochrome P-450 is discussed. (author)

Development of an in vitro binding assay for ecdysone receptor of mysid shrimp (Americamysis bahia)

Energy Technology Data Exchange (ETDEWEB)

Yokota, Hirofumi, E-mail: h-yokota@mail.kobe-c.ac.jp [Department of Biosphere Sciences, School of Human Sciences, Kobe College 4-1, Okadayama, Nishinomiya-shi, Hyogo 662-8505 (Japan); Eguchi, Sayaka [Department of Biosphere Sciences, School of Human Sciences, Kobe College 4-1, Okadayama, Nishinomiya-shi, Hyogo 662-8505 (Japan); Nakai, Makoto [Hita Laboratory, Chemicals Evaluation and Research Institute (CERI), 3-822, Ishii-machi, Hita-shi, Oita 877-0061 (Japan)

2011-10-15

Highlights: We successfully performed cDNA cloning of EcR and USP of mysid shrimp. We then expressed the ligand-binding domains of the corresponding receptor peptides. The translated peptides could bind to ecdysone agonists as heterodimers. These results indicate that they are functional hormone receptors of mysid shrimp. - Abstract: A global effort has been made to establish screening and testing methods that can identify the effects of endocrine-disrupting chemicals (EDCs) on invertebrates. The purpose of our study was to develop an in vitro receptor binding assay for ecdysone receptor (EcR) in mysid shrimp (Americamysis bahia). We cloned mysid shrimp EcR cDNA (2888 nucleotides) and ultraspiracle (USP) cDNA (2116 nucleotides), and determined that they encode predicted proteins of length 570 and 410 amino acids, respectively. The deduced amino acid sequences of these proteins shared 36-71% homology for EcR and 44-65% for USP with those of other arthropods. Phylogenetic analysis revealed that mysid shrimp EcR was classified into an independent cluster together with the EcRs of another mysid species, Neomysis integer and the cluster diverged early from those of the other taxonomic orders of crustaceans. We then expressed the ligand-binding domains (DEF regions) of mysid shrimp EcR (abEcRdef) and USP (abUSPdef) as glutathione S-transferase (GST)-fusion peptides in Escherichia coli. After purifying the fusion peptides by affinity chromatography and removing the GST labels, we subjected the peptides to a ligand-receptor binding assay. [{sup 3}H]-ponasterone A did not bind to abEcRdef or abUSPdef peptides alone but bound strongly to the abEcRdef/abUSPdef mixture with dissociation constant (K{sub d}) = 2.14 nM. Competitive binding assays showed that the IC{sub 50} values for ponasterone A, muristerone A, 20-hydroxyecdysone, and {alpha}-ecdysone were 1.2, 1.9, 35, and 1200 nM, respectively. In contrast, the IC{sub 50} values for two dibenzoylhydrazine ligands

Calibration and validation of the {sup 14}C-labelled polyethylene glycol-binding assay for tannins in tropical browse

Energy Technology Data Exchange (ETDEWEB)

Mlambo, V. [Animal Production Unit, FAO/IAEA Agriculture and Biotechnology Laboratory, Seibersdorf (Austria)]. E-mail: vmlambo@agric.uniswa.sz; Makkar, H.P.S. [Animal Production and Health Section, Joint FAO/IAEA Division of Nuclear Techniques in Agriculture and Food, International Atomic Energy Agency, Vienna (Austria)

2005-08-19

This study evaluates the radiolabelled polyethylene glycol (PEG)-binding procedure [Silanikove, N., Shinder, D., Gilboa, N., Eyal, M., Nitsan, Z., 1996. Polyethylene glycol-binding to plant samples as an assay for the biological effects of tannins: predicting the negative effects of tannins in Mediterranean browse on rumen degradation. J. Agric. Food Chem. 44, 3230-3234] for tannin analysis, using 27 tropical browse plants. In this method, the amount of PEG bound to a plant sample is assumed to be a reflection of its tannin content. The method was modified to exclude the use of non-tanniniferous substrate for estimating non-specific binding (NSB) in tannin-containing substrates. Non-specific binding values varied widely (0.4-2.8 mg PEG/100 mg DM tannin-free substrate) when the tannin-free substrate was changed from wheat straw to either rye grass or maize shoots. We therefore propose a modified radiolabelled PEG-binding method to estimate the level of PEG-binding (PEGb) to tannin-containing foliage without using tannin-free substrate to correct for non-specific binding. In this approach, incremental levels of each tanniniferous substrate were used to generate PEGb values. The resultant linear response was analysed and tannin activity was expressed as the slope of the response curve (PEGbSlope) observed for each substrate. The slope takes into account the non-specific binding in each substrate, thus PEGbSlope does not require correction for NSB using tannin-free samples. This approach improved the correlation between PEGb and the {sup 125}I-labelled bovine serum albumin precipitation assay. Relationships between the modified PEG-binding assay and radiolabelled bovine serum albumin assay, in vitro tannin bioassay and colorimetric assays are presented. (author)

The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays.

Science.gov (United States)

Hua, Boyang; Wang, Yanbo; Park, Seongjin; Han, Kyu Young; Singh, Digvijay; Kim, Jin H; Cheng, Wei; Ha, Taekjip

2018-03-13

Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9-RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.

Radiosynthesis and preliminary evaluation of 5-[{sup 123/125}I]iodo-3-(2(S)-azetidinylmethoxy)pyridine: a radioligand for nicotinic acetylcholine receptors

Energy Technology Data Exchange (ETDEWEB)

Horti, Andrew G.; Koren, Andrei O.; Lee, Kan Sam; Mukhin, Alexey G.; Vaupel, D. Bruce; Kimes, Alane S.; Stratton, Morgan; London, Edythe D. E-mail: elondon@intra.nida.nih.gov

1999-02-01

The radiochemical syntheses of 5-[{sup 125}I]iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-[{sup 125}I]-iodo-A-85380, [{sup 125}I]1) and 5-[{sup 123}I]-iodo-A-85380, [{sup 123}I]1, were accomplished by radioiodination of 5-trimethylstannyl-3-((1-tert-butoxycarbonyl-2(S) -azetidinyl)methoxy)pyridine, 2, followed by acidic deprotection. Average radiochemical yields of [{sup 125}I]1 and [{sup 123}I]1 were 40-55%; and the average specific radioactivities were 1,700 and 7,000 mCi/{mu}mol, respectively. Binding affinities of [{sup 125}I]1 and [{sup 123}I]1 in vitro (rat brain membranes) were each characterized by a K{sub d} value of 11 pM. Preliminary in vivo assay and ex vivo autoradiography of mouse brain indicated that [{sup 125}I]1 selectively labels nicotinic acetylcholine receptors (nAChRs) with very high affinity and specificity. These studies suggest that [{sup 123}I]1 may be useful as a radioligand for single photon emission computed tomography (SPECT) imaging of nAChRs.

Evaluation in vitro and in animals of a new {sup 11}C-labeled PET radioligand for metabotropic glutamate receptors 1 in brain

Energy Technology Data Exchange (ETDEWEB)

Zanotti-Fregonara, Paolo; Liow, Jeih-San; Zoghbi, Sami S.; Clark, David T.; Morse, Cheryl; Pike, Victor W. [National Institute of Mental Health, National Institutes of Health, Molecular Imaging Branch, Bethesda, MD (United States); Barth, Vanessa N.; Rhoads, Emily; Siuda, Edward; Heinz, Beverly A.; Nisenbaum, Eric; Dressman, Bruce; Joshi, Elizabeth; Luffer-Atlas, Debra; Fisher, Matthew J.; Masters, John J.; Goebl, Nancy; Kuklish, Steven L.; Tauscher, Johannes [Eli Lilly and Co., Indianapolis, IN (United States); Innis, Robert B. [National Institute of Mental Health, National Institutes of Health, Molecular Imaging Branch, Bethesda, MD (United States); National Institute of Mental Health, Molecular Imaging Branch, Bethesda, MD (United States)

2013-02-15

Two allosteric modulators of the group I metabotropic glutamate receptors (mGluR1 and mGluR5) were evaluated as positron emission tomography (PET) radioligands for mGluR1. LY2428703, a full mGluR1 antagonist (IC{sub 50} 8.9 nM) and partial mGluR5 antagonist (IC{sub 50} 118 nM), and LSN2606428, a full mGluR1 and mGluR5 antagonist (IC{sub 50} 35.3 nM and 10.2 nM, respectively) were successfully labeled with {sup 11}C and evaluated as radioligands for mGluR1. The pharmacology of LY2428703 was comprehensively assessed in vitro and in vivo, and its biodistribution was investigated by liquid chromatography-mass spectrometry/mass spectrometry, and by PET imaging in the rat. In contrast, LSN2606428 was only evaluated in vitro; further evaluation was stopped due to its unfavorable pharmacological properties and binding affinity. {sup 11}C-LY2428703 showed promising characteristics, including: (1) high potency for binding to human mGluR1 (IC{sub 50} 8.9 nM) with no significant affinity for other human mGlu receptors (mGluR2 through mGluR8); (2) binding to brain displaceable by administration of an mGluR1 antagonist; (3) only one major radiometabolite in both plasma and brain, with a negligible brain concentration (with 3.5 % of the total radioactivity in cerebellum) and no receptor affinity; (4) a large specific and displaceable signal in the mGluR1-rich cerebellum with no significant in vivo affinity for mGluR5, as shown by PET studies in rats; and (5) lack of substrate behavior for efflux transporters at the blood-brain barrier, as shown by PET studies conducted in wild-type and knockout mice. {sup 11}C-LY2428703, a new PET radioligand for mGluR1 quantification, displayed promising characteristics both in vitro and in vivo in rodents. (orig.)

Characterization of high specific activity [16 alpha-123I]Iodo-17 beta-estradiol as an estrogen receptor-specific radioligand capable of imaging estrogen receptor-positive tumors

International Nuclear Information System (INIS)

Pavlik, E.J.; Nelson, K.; Gallion, H.H.; van Nagell, J.R. Jr.; Donaldson, E.S.; Shih, W.J.; Spicer, J.A.; Preston, D.F.; Baranczuk, R.J.; Kenady, D.E.

1990-01-01

16 alpha-[123I]Iodo-17 beta-estradiol (16 alpha-[123I]E2) has been characterized for use as a selective radioligand for estrogen receptor (ERc) that is capable of generating in situ images of ERc-positive tumors. High specific activity 16 alpha-[123I]E2 (7,500-10,000 Ci/mmol) was used in all determinations. Radiochemical purity was determined by thin layer chromatography, and the selectivity of radioligand for ERc was evaluated using size exclusion high performance liquid chromatography on ERc prepared from rodent uteri. Efficiencies of radioidination approaching 100% were achieved, and excellent receptor selectivity was obtained even when the efficiency of radioiodination was as low as 10%. Low radiochemical purity was always associated with poor selectivity for ERc. No new radioligand species was generated during the course of radiodecay; however, reduced binding over time, even when increased activity was used to compensate for radiodecay, indicated that the formation of a radioinert competitor does occur. 16 alpha-[123I]E2 demonstrated stable, high affinity binding to ERc and was concentrated by ERc-positive tissues. After injecting 16 alpha-[123I]E2 in vivo, images of ERc-containing tissues were obtained, including rabbit reproductive tract and dimethylbenzanthracene-induced tumors. The demonstrations of ERc selectivity and image formation both indicate that 16 alpha-[123I]E2 should have promise as a useful new radiopharmaceutical for imaging ERc-positive cancers

Bindings of 3H-prazosin and 3H-yohimbine to alpha adrenoceptors in the guinea-pig stomach

International Nuclear Information System (INIS)

Taniguchi, T.; Nishikawa, H.

1988-01-01

Alpha adrenoceptor subtypes have been investigated by radioligand binding study in guinea-pig stomach using 3 H-prazosin and 3 H-yohimbine. The specific 3 H-prazosin binding to guinea-pig stomach was saturable and of high affinity with a Bmax of 33 fmol/mg protein. Specific 3 H-yohimbine binding to the tissue was also saturable and of high affinity with a Bmax of 150 fmol/mg protein. Adrenergic drugs competed for 3 H-prazosin binding in order of prazosin > phentolamine > methoxamine > norepinephrine > clonidine > epinephrine > yohimbine. These drugs competed for 3 H-yohimbine binding in order of yohimbine > phentolamine > clonidine > epinephrine > norepinephrine > prazosin > methoxamine. They also examined whether dopamine receptors exist in guinea-pig stomach, using radioligand binding study. Specific binding of 3 H-spiperone, 3 H-apomorphine, 3 H-dopamine and 3 H-domperidone was not detectable in the stomach. Dopaminergic drugs such as dopamine, haloperidol, domperidone and sulpiride competed for 3 H-prazosin binding in order of haloperidol > domperidone > dopamine > sulpiride. Metoclopramide, sulpiride and dopamine competed for 3 H-yohimbine binding in order of metoclopramide > sulpiride > dopamine

Preparation of a potential positron emission tomographic radioligand for the dopamine transporter

International Nuclear Information System (INIS)

Mueller, L.; Halldin, C.; Foged, C.; Karlsson, P.; Hall, H.; Swahn, C.G.; Suzdak, P.D.; Hohlweg, R.; Nielsen, E.B.; Frade, L.

1994-01-01

NNC 12-0722 (1-[2-(bis(4-fluorophenyl)-methoxy)ethyl]-4-methyl piperazine) is a new selective inhibitor of the dopamine transporter. [ 11 C]NNC 12-0722 was prepared by N-methylation of the desmethyl compound with [ 11 C]methyl iodide. The total radiochemical yield of [ 11 C]NNC 12-0722 was 40%-50% with an overall synthesis time of 30-35 min. The radiochemical purity was higher than 99% and the specific radioactivity about 1500 Ci/mmol (55 GBq/μmol). Autoradiographic examination of [ 11 C]NNC 12-0722 binding on whole hemisphere cryosections from human brain post mortem demonstrated specific binding in the caudate nucleus and putamen. In a positron emission tomographic examination of [ 11 C]NNC 12-0722 in a cynomolgus monkey there was a rapid uptake of radioactivity in the brain. In the striatum, a region with a high density of dopamine transporters, the radioactivity was two times higher than in the cerebellum. These results indicate that [ 11 C]NNC 12-0722 may be a useful radioligand for labelling of the dopamine transporter in man. (orig.)

Preclinical evaluation of an 18F-labelled beta1-adrenoceptor selective radioligand based on ICI 89,406.

Science.gov (United States)

Law, Marilyn P; Wagner, Stefan; Kopka, Klaus; Renner, Christiane; Pike, Victor W; Schober, Otmar; Schäfers, Michael

2010-05-01

Radioligand binding studies indicate a down-regulation of myocardial beta(1)-adrenoceptors (beta(1)-AR) in cardiac disease which may or may not be associated with a decrease in beta(2)-ARs. We have chosen ICI 89,406, a beta(1)-selective AR antagonist, as the lead structure to develop new beta(1)-AR radioligands for PET and have synthesised a fluoro-ethoxy derivative (F-ICI). (S)-N-[2-[3-(2-Cyano-phenoxy)-2-hydroxy-propylamino]-ethyl]-N'-[4-(2-[(18)F]fluoro-ethoxy)-phenyl]-urea ((S)-[(18)F]F-ICI) was synthesised. Myocardial uptake of radioactivity after intravenous injection of (S)-[(18)F]F-ICI into adult CD(1) mice or Wistar rats was assessed with positron emission tomography (PET) and postmortem dissection. Metabolism was assessed by high-performance liquid chromatography analysis of plasma and urine. The heart was visualised with PET after injection of (S)-[(18)F]F-ICI but neither unlabelled F-ICI nor propranolol (non-selective beta-AR antagonist) injected 15 min after (S)-[(18)F]F-ICI affected myocardial radioactivity. Ex vivo dissection demonstrated that predosing with propranolol or CGP 20712 (beta(1)-selective AR-antagonist) did not affect myocardial radioactivity. Radiometabolites rapidly appeared in plasma and both (S)-[(18)F]F-ICI and radiometabolites accumulated in urine. Myocardial uptake of (S)-[(18)F]F-ICI after intravenous injection was mainly at sites unrelated to beta(1)-ARs. (S)-[(18)F]F-ICI is not a suitable beta(1)-selective-AR radioligand for PET. (c) 2010 Elsevier Inc. All rights reserved.

Functional diagnostics for thyrotropin hormone receptor autoantibodies: bioassays prevail over binding assays.

Science.gov (United States)

Lytton, Simon David; Schluter, Anke; Banga, Paul J

2018-06-01

Autoantibodies to the thyrotropin hormone receptor (TSH-R) are directly responsible for the hyperthyroidism in Graves' disease and mediate orbital manifestations in Graves' orbitopathy (otherwise known as thyroid eye disease). These autoantibodies are heterogeneous in their function and collectively referred to as TRAbs. Measurement of TRAbs is clinically important for diagnosis of a variety of conditions and different commercial assays with high sensitivity and specificity are available for diagnostic purposes. This review provides overwhelming evidence that the TRAbs detected in binding assays by mainly the automated electrochemical luminescence immunoassays (ECLIA) do not distinguish TRAbs that stimulate the TSH-R (called TSIs or TSAbs) and TRAbs that just inhibit the binding of TSH without stimulating the TSH-R (called TBAbs). However, TSAbs and TBAbs have divergent pathogenic roles, and depending which fraction predominates cause different clinical symptoms and engender different therapeutic regimen. Therefore, diagnostic distinction of TSAbs and TBAbs is of paramount clinical importance. To date, only bioassays such as the Mc4 TSH-R bioassay (Thyretain TM , Quidel) and the Bridge assay (Immulite 2000, Siemens) can measure TSAbs, with only the former being able to distinguish between TSAbs and TBAbs. On this note, it is strongly recommended to only use the term TSI or TSAb when reporting the results of bioassays, whereas the results of automated TRAb binding assays should be reported as TRAbs (of undetermined functional significance). This review aims to present a technical and analytical account of leading commercial diagnostic methods of anti-TSH-R antibodies, a metaanalysis of their clinical performance and a perspective for the use of cell based TSH-R bioassays in the clinical diagnostics of Graves' disease.

Insulin-like growth factors: assay methods and their implications

International Nuclear Information System (INIS)

Guyda, H.J.; Posner, B.I.; Schiffrin, A.; Rappaport, R.; Postel-Vinay, M.C.; Corvol, M.T.

1981-01-01

The insulin-like growth factors (IGF's) are small molecular weight peptides (6-10 x 10 3 daltons) that circulate in blood plasma almost entirely bound to macromolecular carrier proteins. The growth-promoting and insulin-like activities of IGF's can be explained by the observed ability of these peptides to interact with the IGF receptor on the one hand and with the insulin receptor on the other. These observations have led to the establishment of radioreceptor assays (RRA's), competitive protein binding assays (CPBA's), and more recently radioimmunoassays (RIA's) for the IGF's that have different specificities. Because of their ease of performance and sensitivity, the radioligand assays have largely supplanted the biological assays originally utilized to identify and characterize these anabolic peptides. In this report the authors' studies are summarised which utilize a slightly acidic IGF which has been purified on the basis of its insulin-like activity in an insulin RRA and which was termed ILAs. They refer to purified insulin-like peptides that have the properties of a somatomedin by the generic term insulin-like growth factor (IGF). Somatomedin (SM) activity will be utilized to connote that activity in plasma or serum determined by bioassay. The competitive dose-response curves for IGF peptides in the insulin RRA as well as those in the ILAs RRA are presented. A combination of bioassays, RRA and RIA were employed to assess somatomedin activity and IGF peptide levels in a number of clinical circumstances. The correlations are discussed. (Auth.)

[11C]PE2I: a highly selective radioligand for PET examination of the dopamine transporter in monkey and human brain

International Nuclear Information System (INIS)

Halldin, Christer; Erixon-Lindroth, Nina; Pauli, Stefan; Chou, Yuan-Hwa; Okubo, Yoshiro; Karlsson, Per; Lundkvist, Camilla; Olsson, Hans; Farde, Lars; Guilloteau, Denis; Emond, Patrick

2003-01-01

The aim of this study was to explore the potential of a new selective dopamine transporter (DAT) compound as a radioligand for positron emission tomography (PET) examination of DAT in the human brain. The high affinity DAT compound N-(3-iodoprop-2E-enyl)-2β-carbomethoxy-3β-(4-methylphenyl)nortropane (PE2I) was radiolabelled by the O-methylation approach and the binding was characterised by PET in cynomolgus monkeys and a healthy man. Metabolite levels in plasma were measured by gradient high-performance liquid chromatography. O-methylation of the corresponding free acid precursor with [ 11 C]methyl triflate gave high radiochemical yield (80%) and specific radioactivity (55 GBq/μmol). [ 11 C]PE2I binding in cynomolgus monkeys was nine times higher in the striatum than in the cerebellum at peak equilibrium, which appeared 55-65 min after injection. Displacement and pretreatment measurements using unlabelled β-CIT, GBR 12909, cocaine, citalopram and maprotiline confirmed that [ 11 C]PE2I binds selectively to DAT. In a preliminary study in one human subject the radioactivity ratios of the striatum and substantia nigra to the cerebellum were 10 and 1.8, respectively, at peak equilibrium, which appeared at 40-50 min and 20 min, respectively, after injection. The fraction of the total radioactivity in monkey and human plasma representing unchanged [ 11 C]PE2I was 15-20% at 40 min after injection. The present characterisation of binding in monkey and man suggests that [ 11 C]PE2I is a suitable PET radioligand for quantitative regional examination of DAT in man. (orig.)

A radioreceptor assay of luteinizing hormone-releasing hormone receptor and characterization of LHRH binding to pituitary receptors in Shao duck

International Nuclear Information System (INIS)

Yang Peixin; Wu Meiwen; Chen Ziyuan

2000-01-01

The properties of Shao duck pituitary luteinizing hormone-releasing hormone (LHRH) receptors were analyzed in pituitary membrane preparation and isolated pituitary cells prepared by enzymatic dispersion with collagenase and trypsin, by using a super-agonist analog of (D-Lys 6 ) LHRH. High binding of 125 I-(D-Lys 6 ) LHRH to 10 6 cultured cells of Shao duck was observed after a 90 minute incubation at 4 degree C, while binding was significantly reduced after a 24h incubation. Binding of the radioligand was a function of tissue concentration of Shao duck pituitary membrane preparation, with a positive correlation over the range of 1-2 pituitary per-tube. Specific binding for 125 I-(D-Lys 6 ) LHRH increased with the increase in the amount of 125 I-(D-Lys 6 ) LHRH. The Scatchard analysis of data revealed a linear relationship between the amount of specific binding and the ratio of specific binding to free 1 '2 5 I(D-Lys 6 )LHRH, indicating a single class of high affinity sites. Equilibrium dissociation constant (Kd) was 0.34 nM in pituitary membrane preparation and 0.43 nM in isolated pituitary cells. Both Kd values were near and the maximum binding capacity (B max ) was great in isolated cells, suggesting no significant loss of the LHRH receptor population caused by the enzymatic procedure employed for cell dispersion in the present study. Addition of 9D-Lys 6 ) LHRH displaced bound 125 I-(D-Lys 6 ) LHRH. These results demonstrated the presence and provided characterization of LHRH receptors in Shao duck pituitary

An assay for the mannan-binding lectin pathway of complement activation

DEFF Research Database (Denmark)

Petersen, Steen Vang; Thiel, S; Jensen, L

2001-01-01

activation. Therefore, in a generally applicable complement activation assay specific for the MBL pathway, the activity of the classical pathway must be inhibited. This can be accomplished by exploiting the finding that high ionic strength buffers inhibit the binding of C1q to immune complexes and disrupt...

Dopaminergic modulation of mitral cell activity in the frog olfactory bulb: a combined radioligand binding-electrophysiological study

International Nuclear Information System (INIS)

Duchamp, A.; Moyse, E.; Delaleu, J.-C.; Coronas, V.; Duchamp-Viret, P.

1997-01-01

Dopamine content in the amphibian olfactory bulb is supplied by interneurons scattered among mitral cells in the external plexiform/mitral cell layer. In mammals, dopamine has been found to be involved in various aspects of bulbar information processing by influencing mitral cell odour responsiveness. Dopamine action in the bulb depends directly on the localization of its receptor targets, found to be mainly of the D 2 type in mammals. The present study assessed, in the frog, both the anatomical localization of D 2 -like, radioligand-labelled receptors of dopamine and the in vivo action of dopamine on unitary mitral cell activity in response to odours delivered over a wide range of concentrations. The [ 125 I]iodosulpride-labelled D 2 binding sites were visualized on frozen sagittal sections of frog brains by film radioautography. The sites were found to be restricted to the external plexiform/mitral cell layer; other layers of the olfactory bulb were devoid of specific labelling. Electrophysiological recordings of mitral unit activity revealed that dopamine or its agonist apomorphine induced a drastic reduction of spontaneous firing rate of mitral cells in most cases without altering odour intensity coding properties of these cells. Moreover, pre-treatment with the D 2 antagonist eticlopride blocked the dopamine-induced reduction of mitral cell spontaneous activity.In the frog olfactory bulb, both anatomical localization of D 2 -like receptors and functional data on dopamine involvement in information processing differ from those reported in mammals. This suggests a phylogenetic evolution of dopamine action in the olfactory bulb. In the frog, anatomical data perfectly corroborate electrophysiological results, together strongly suggesting a direct action of dopamine on mitral cells. In a physiologically operating system, such an action would result in a global improvement of signal-to-noise ratio. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights

[{sup 11}C]PE2I: a highly selective radioligand for PET examination of the dopamine transporter in monkey and human brain

Energy Technology Data Exchange (ETDEWEB)

Halldin, Christer; Erixon-Lindroth, Nina; Pauli, Stefan; Chou, Yuan-Hwa; Okubo, Yoshiro; Karlsson, Per; Lundkvist, Camilla; Olsson, Hans; Farde, Lars [Karolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, 17176, Stockholm (Sweden); Guilloteau, Denis; Emond, Patrick [INSERM U316 Universite Francois Rabelais, Tours (France)

2003-09-01

The aim of this study was to explore the potential of a new selective dopamine transporter (DAT) compound as a radioligand for positron emission tomography (PET) examination of DAT in the human brain. The high affinity DAT compound N-(3-iodoprop-2E-enyl)-2{beta}-carbomethoxy-3{beta}-(4-methylphenyl)nortropane (PE2I) was radiolabelled by the O-methylation approach and the binding was characterised by PET in cynomolgus monkeys and a healthy man. Metabolite levels in plasma were measured by gradient high-performance liquid chromatography. O-methylation of the corresponding free acid precursor with [{sup 11}C]methyl triflate gave high radiochemical yield (80%) and specific radioactivity (55 GBq/{mu}mol). [{sup 11}C]PE2I binding in cynomolgus monkeys was nine times higher in the striatum than in the cerebellum at peak equilibrium, which appeared 55-65 min after injection. Displacement and pretreatment measurements using unlabelled {beta}-CIT, GBR 12909, cocaine, citalopram and maprotiline confirmed that [{sup 11}C]PE2I binds selectively to DAT. In a preliminary study in one human subject the radioactivity ratios of the striatum and substantia nigra to the cerebellum were 10 and 1.8, respectively, at peak equilibrium, which appeared at 40-50 min and 20 min, respectively, after injection. The fraction of the total radioactivity in monkey and human plasma representing unchanged [{sup 11}C]PE2I was 15-20% at 40 min after injection. The present characterisation of binding in monkey and man suggests that [{sup 11}C]PE2I is a suitable PET radioligand for quantitative regional examination of DAT in man. (orig.)

Radio-ligand immunoassay for human hemoglobin variants

International Nuclear Information System (INIS)

Javid, J.; Pettis, P.K.; Miller, J.E.

1981-01-01

A quantitative method is described for the individual assay of human hemoglobin variants occurring singly or in mixture. The hemoglobin to be assayed is bound to specific antibody; the immune complex is attached to protein A-containing S. aureus and removed from the mixture. The hemoglobin thus isolated is quantified by its ability to bind radiolabeled haptoglobin. The technique is accurate and distinguishes among the 4 hemoglobins tested, namely Hb A, S, C and F. It has the advantage over conventional radioimmunoassay that a single probe, radiolabeled haptoglobin, is needed for the specific assay of any hemoglobin. (Auth.)

Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors.

Science.gov (United States)

Sakkal, Leon A; Rajkowski, Kyle Z; Armen, Roger S

2017-06-05

Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A 1 R, A 2A R, A 3 R) and muscarinic acetylcholine (M 1 R, M 5 R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M 1 R PAMs were predicted to bind in the analogous M 2 R PAM LY2119620 binding site. The M 5 R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Synthesis and binding affinity of an iodinated juvenile hormone

Energy Technology Data Exchange (ETDEWEB)

Prestwich, G.D.; Eng, W.S.; Robles, S.; Vogt, R.G.; Wisniewski, J.R.; Wawrzenczyk, C.

1988-01-25

The synthesis of the first iodinated juvenile hormone (JH) in enantiomerically enriched form is reported. This chiral compound, 12-iodo-JH I, has an iodine atom replacing a methyl group of the natural insect juvenile hormone, JH I, which is important in regulating morphogenesis and reproduction in the Lepidoptera. The unlabeled compound shows approximately 10% of the relative binding affinity for the larval hemolymph JH binding protein (JHBP) of Manduca sexta, which specifically binds natural /sup 3/H-10R,11S-JH I (labeled at 58 Ci/mmol) with a KD of 8 X 10(-8) M. It is also approximately one-tenth as biologically active as JH I in the black Manduca and epidermal commitment assays. The 12-hydroxy and 12-oxo compounds are poor competitors and are also biologically inactive. The radioiodinated (/sup 125/I)12-iodo-JH I can be prepared in low yield at greater than 2500 Ci/mmol by nucleophilic displacement using no-carrier-added /sup 125/I-labeled sodium iodide in acetone; however, synthesis using sodium iodide carrier to give the approximately 50 Ci/mmol radioiodinated ligand proceeds in higher radiochemical yield with fewer by-products and provides a radioligand which is more readily handled in binding assays. The KD of (/sup 125/I)12-iodo-JH I was determined for hemolymph JHBP of three insects: M. sexta, 795 nM; Galleria mellonella, 47 nM; Locusta migratoria, 77 nM. The selectivity of 12-iodo-JH I for the 32-kDa JHBP of M. sexta was demonstrated by direct autoradiography of a native polyacrylamide gel electrophoresis gel of larval hemolymph incubated with the radioiodinated ligand. Thus, the in vitro and in vivo activity of 12-iodo-JH I indicate that it can serve as an important new gamma-emitting probe in the search for JH receptor proteins in target tissues.

Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors.

Science.gov (United States)

Martínez-Pinilla, Eva; Varani, Katia; Reyes-Resina, Irene; Angelats, Edgar; Vincenzi, Fabrizio; Ferreiro-Vera, Carlos; Oyarzabal, Julen; Canela, Enric I; Lanciego, José L; Nadal, Xavier; Navarro, Gemma; Borea, Pier Andrea; Franco, Rafael

2017-01-01

The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB 2 receptors (CB 2 Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB 2 R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB 2 R. Using membrane preparations from CB 2 R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB 2 R where the synthetic cannabinoid, [ 3 H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB 2 R-selective compound, CM-157. The effect on binding to CB 2 R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the K D . CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB 2 R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors

Directory of Open Access Journals (Sweden)

Eva Martínez-Pinilla

2017-10-01

Full Text Available The mechanism of action of cannabidiol (CBD, the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB2 receptors (CB2Rs it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs; however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB2R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB2R. Using membrane preparations from CB2R-expressing HEK-293T (human embryonic kidney 293T cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB2R where the synthetic cannabinoid, [3H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB2R-selective compound, CM-157. The effect on binding to CB2R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the KD. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB2R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

Persistent Graves' hyperthyroidism despite rapid negative conversion of thyroid-stimulating hormone-binding inhibitory immunoglobulin assay results: a case report.

Science.gov (United States)

Ohara, Nobumasa; Kaneko, Masanori; Kitazawa, Masaru; Uemura, Yasuyuki; Minagawa, Shinichi; Miyakoshi, Masashi; Kaneko, Kenzo; Kamoi, Kyuzi

2017-02-06

Graves' disease is an autoimmune thyroid disorder characterized by hyperthyroidism, and patients exhibit thyroid-stimulating hormone receptor antibody. The major methods of measuring circulating thyroid-stimulating hormone receptor antibody include the thyroid-stimulating hormone-binding inhibitory immunoglobulin assays. Although the diagnostic accuracy of these assays has been improved, a minority of patients with Graves' disease test negative even on second-generation and third-generation thyroid-stimulating hormone-binding inhibitory immunoglobulins. We report a rare case of a thyroid-stimulating hormone-binding inhibitory immunoglobulin-positive patient with Graves' disease who showed rapid lowering of thyroid-stimulating hormone-binding inhibitory immunoglobulin levels following administration of the anti-thyroid drug thiamazole, but still experienced Graves' hyperthyroidism. A 45-year-old Japanese man presented with severe hyperthyroidism (serum free triiodothyronine >25.0 pg/mL; reference range 1.7 to 3.7 pg/mL) and tested weakly positive for thyroid-stimulating hormone-binding inhibitory immunoglobulins on second-generation tests (2.1 IU/L; reference range hyperthyroidism for more than 8 years, requiring 15 mg/day of thiamazole to correct. During that period, he tested negative on all first-generation, second-generation, and third-generation thyroid-stimulating hormone-binding inhibitory immunoglobulin assays, but thyroid scintigraphy revealed diffuse and increased uptake, and thyroid ultrasound and color flow Doppler imaging showed typical findings of Graves' hyperthyroidism. The possible explanations for serial changes in the thyroid-stimulating hormone-binding inhibitory immunoglobulin results in our patient include the presence of thyroid-stimulating hormone receptor antibody, which is bioactive but less reactive on thyroid-stimulating hormone-binding inhibitory immunoglobulin assays, or the effect of reduced levels of circulating thyroid

PET radioligands for imaging of Tau pathology: Current status

Energy Technology Data Exchange (ETDEWEB)

Choe, Yearn Seong [Dept. of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Lee, Kyung Han [Dept. of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul (Korea, Republic of)

2015-12-15

The incidence of Alzheimer’s disease (AD), a progressive neurodegenerative disorder, continues to soar with the rapid growth of the elderly population, thus creating an enormous social and economic burden. Although disease-modifying drugs to treat AD are not yet available, several candidate drugs are in clinical trials. Most of these drugs are expected to be effective at the early stages of the disease, and therefore the early and accurate diagnosis of AD will be a critical factor in efforts to improve the prognosis of patients with AD. This review focuses on lead radioligands developed to date and their preclinical data in order to facilitate the development of tau-specific positron emission tomography radioligands that are of great interest to the scientific community.

[3H]imidacloprid: synthesis of a candidate radioligand for the nicotinic acetylcholine receptor

International Nuclear Information System (INIS)

Latli, B.; Casida, J.E.

1992-01-01

Imidacloprid is an exceptionally potent insecticide known from physiological studies to act at the nicotinic acetylcholine receptor. To prepare [ 3 H]imidacloprid as a candidate radioligand, 6-chloronicotinoyl chloride was reduced with NaB 2 H 4 (in model studies) or NaB 3 H 4 in absolute ethanol to 2-chloro-5-pyridinylmethanol which was transformed to 2-chloro-5-chloromethylpyridine on refluxing with thionyl chloride. Coupling with 4,5-dihydro-N-nitro-1H-imidazol-2-amine then gave [ 2 H 2 ]imidacloprid incorporating about 95% of the deuterium or [ 3 H 2 ]imidacloprid (25 Ci/mmol) in 80% radiochemical yield. In studies not detailed here [ 3 H] imidacloprid was found to undergo high affinity, specific and saturable binding to a site in insect brain. (author)

FRET-based binding assay between a fluorescent cAMP analogue and a cyclic nucleotide-binding domain tagged with a CFP.

Science.gov (United States)

Romero, Francisco; Santana-Calvo, Carmen; Sánchez-Guevara, Yoloxochitl; Nishigaki, Takuya

2017-09-01

The cyclic nucleotide-binding domain (CNBD) functions as a regulatory domain of many proteins involved in cyclic nucleotide signalling. We developed a straightforward and reliable binding assay based on intermolecular fluorescence resonance energy transfer (FRET) between an adenosine-3', 5'-cyclic monophosphate analogue labelled with fluorescein and a recombinant CNBD of human EPAC1 tagged with a cyan fluorescence protein (CFP). The high FRET efficiency of this method (~ 80%) allowed us to perform several types of binding experiments with nanomolar range of sample using conventional equipment. In addition, the CFP tag on the CNBD enabled us to perform a specific binding experiment using an unpurified protein. Considering these advantages, this technique is useful to study poorly characterized CNBDs. © 2017 Federation of European Biochemical Societies.

Functional recombinant MHC class II molecules and high-throughput peptide-binding assays

DEFF Research Database (Denmark)

Justesen, Sune; Harndahl, Mikkel; Lamberth, Kasper

2009-01-01

BACKGROUND: Molecules of the class II major histocompability complex (MHC-II) specifically bind and present exogenously derived peptide epitopes to CD4+ T helper cells. The extreme polymorphism of the MHC-II hampers the complete analysis of peptide binding. It is also a significant hurdle......-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately....... CONCLUSION: We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools....

Synthesis and preliminary evaluation of [3H]PSB-0413, a selective antagonist radioligand for platelet P2Y12 receptors.

Science.gov (United States)

El-Tayeb, Ali; Griessmeier, Kerstin J; Müller, Christa E

2005-12-15

The selective antagonist radioligand [(3)H]2-propylthioadenosine-5'-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([(3)H]PSB-0413) was prepared by catalytic hydrogenation of its propargyl precursor with a high specific radioactivity of 74Ci/mmol. In preliminary saturation binding studies, [(3)H]PSB-0413 showed high affinity for platelet P2Y(12) receptors with a K(D) value of 4.57nM. Human platelets had a high density of P2Y(12) receptors exhibiting a B(max) value of 7.66pmol/mg of protein.

A radiometabolite study of the serotonin transporter PET radioligand [11C]MADAM

International Nuclear Information System (INIS)

Gourand, F.; Emond, P.; Bergström, J.P.; Takano, A.; Gulyás, B.; Guilloteau, D.; Barré, L.; Halldin, C.

2014-01-01

Introduction: 11 C]MADAM is a radioligand suitable for PET studies of the serotonin transporter (SERT). Metabolite analysis in human and non-human plasma samples using HPLC separation has shown that [ 11 C]MADAM was rapidly metabolized. A possible metabolic pathway is the S-oxidation which could lead to SOMADAM and SO 2 MADAM. In vitro evaluation of these two potential metabolites has shown that SOMADAM exhibited a good affinity for SERT and a good selectivity for SERT over NET and DAT. Methods: Comparative PET imaging studies in non-human primate brain with [ 11 C]MADAM and [ 11 C]SOMADAM were carried out, and plasma samples were analyzed using reverse phase HPLC. We have explored the metabolism of [ 11 C]MADAM in rat brain with a view to understand its possible interference for brain imaging with PET. Results: PET imaging studies in non-human primate brain using [ 11 C]SOMADAM indicated that this tracer does not bind with high amounts to brain regions known to be rich in SERT. The fraction of [ 11 C]SOMADAM in non-human primate plasma was approximately 5% at 4 min and 1% at 15 min after [ 11 C]MADAM injection. HPLC analysis of brain sample after [ 11 C]MADAM injection to rats demonstrated that [ 11 C]SOMADAM was not detected in the brain. Conclusions: 11 C]SOMADAM is not superior over [ 11 C]MADAM as a SERT PET radioligand. Nevertheless, [ 11 C]SOMADAM has been identified as a minor labeled metabolite of [ 11 C]MADAM measured in monkey plasma. [ 11 C]SOMADAM was not detected in rat brain

A radioisotope dilution assay for unlabelled vitamin B12-intrinsic factor complex employing the binding intrinsic factor antibody: probable evidence for two types of binding antibody

International Nuclear Information System (INIS)

Jacob, E.; O'Brien, H.A.W.; Mollin, D.L.

1977-01-01

A new radioisotope dilution assay for vitamin B 12 -intrinsic factor complex is described. The method is based on the use of the binding type intrinsic antibody (the binding reagent), which when combined with the intrinsic factor-vitamin B 12 complex (labelled ligand), is quantitatively adsorbed onto zirconium phosphate gel pH 6.25. The new assay has been shown to provide a measure of intrinsic factor comparable with other intrinsic factor assays, but it has the important advantage of being able to measure the unlabelled vitamin B 12 -intrinsic factor complex (unlabelled ligand), and will, therefore, be valuable in the study of physiological events in the gastrointestinal tract. During the study, it was found that there is some evidence for at least two types of binding intrinsic factor antibody: One which combines preferentially with the intrinsic factor-vitamin B 12 complex and one which combines equally well with this complex or with free intrinsic factor. (author)

A radioligand immunoassay for 1,25-dihydroxyvitamin D3 receptors using monoclonal antibody: detection of a phenotypic receptor variant in vitamin D-dependency rickets (type II) which does not bind hormone

International Nuclear Information System (INIS)

Pike, J.W.; Dokoh, Shigeharu; Liberman, U.A.; Eil, C.; Haussler, M.R.; Marx, S.J.

1984-01-01

Vitamin D-dependency rickets, type II (VDDRII), is a well recognized heritable disorder characterized by peripheral target organ resistance to 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), the hormonally active form of the vitamin. Recently, cultured skin fibroblasts obtained from a number of patients with VDDRII have been utilized to characterize the underlying molecular defects associated with this malady. Recently monoclonal antibodies to the vitamin D receptor have been generated, and a radioligand immunoassay (RLIA) for the detection of this molecule has been developed which is independent of its hormone-binding capacity. This report describes the application of the immunoassay in the detection of receptor-like molecules in fibroblasts derived from patients with VDDRII. The results indicate that the molecule is generally present in all patients, and provides a mechanism for individual responsiveness to pharmacologic treatment with vitamin D 3 metabolites. 8 refs.; 3 figs.; 1 table

Saturable binding of 35S-t-butylbicyclophosphorothionate to the sites linked to the GABA receptor and the interaction with gabaergic agents

International Nuclear Information System (INIS)

Wong, D.T.; Threlkeld, P.G.; Bymaster, F.P.; Squires, R.F.

1984-01-01

35 -S-t-Butylbicyclophosphorothionate ( 35 S-TBPS) binds in a concentration-saturable manner to specific sites on membranes from rat cerebral cortex. Using a filtration assay at 25 0 C, in 250 mM NaCl, specific binding of 35 S-TBPS constitutes about 84 to 94 percent of total binding, depending on radioligand concentrations. 35 S-TBPS binding is optimal in the presence of NaCl or NaBr and substantially less in the presence of NaI or NaF. It is sensitive to the treatment with 0.05 percent Triton X-100 but not to repeated freezing and thawing, procedures which increase 3 H-GABA binding. Pharmacological studies show that 35 S-TBPS binding is strongly inhibited by GABA-A receptor agonists (e.g., GABA and muscimol) and by the noncompetitive antagonist, picrotoxin, but not the competitive antagonist, bicuculline. Compounds which enhance binding of radioactive GABA and benzodiazepines, such as the pyrazolopyridines, cartazolate and trazolate, and a diaryl-triazine, LY81067, are also potent inhibitors of 35 S-TBPS binding, with LY81067 being the most effective. The effects of GABA, picrotoxin and LY81067 on the saturable binding of 35 S-TBPS in cortical membranes are compared. The present bindings are consistent with the interpretation that 35 S-TBPS binds, at or near the picrotoxin-sensitive anion recognition sites of the GABA/benzodiazepine/picrotoxin receptor complex

Aryl- and heteroaryl-substituted phenylalanines as AMPA receptor ligands

DEFF Research Database (Denmark)

Szymańska, Ewa; Chałupnik, Paulina; Johansen, Tommy Nørskov

2017-01-01

in radioligand binding assays at native rat ionotropic glutamate receptors. The most interesting compound in this series, (RS)-2-amino-3-(3'-hydroxy-5-(1H-pyrazol-4-yl)-[1,1'-biphenyl]-3-yl)propanoic acid 7e, showed the binding affinity of 4.6 µM for native AMPA receptors and almost 5-fold lower affinity...

Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture

International Nuclear Information System (INIS)

Dold, K.M.; Greenlee, W.F.

1990-01-01

A rapid and sensitive filtration assay for quantitating the specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to whole cells in culture is described. Cell monolayers are incubated with [3H]TCDD in the presence or absence of excess unlabeled ligand, detached from the culture dish with trypsin, filtered, and washed with cold (-78 degrees C) acetone to separate free and nonspecifically bound TCDD from specifically bound TCDD. TCDD receptor binding parameters were characterized in the murine hepatoma cell line Hepa1c1c7. The lower limit of detection of TCDD specific binding was in a sample equivalent to 10 micrograms of total cell protein. The equilibrium dissociation constant and stereospecificity for binding to the TCDD receptor were the same as those previously reported with other TCDD receptor assays on broken cell preparations. Analysis of binding in the murine hepatoma TCDD receptor variants TAO-c1BPrc1 and BPrc1 indicated that this assay will detect receptor number or affinity variants, but will not detect nuclear transfer deficient variants. The major advantage of the whole cell binding assay is that it provides the means to rapidly and reproducibly quantitate TCDD specific binding in small samples of whole cells in culture. In addition, this method eliminates loss or degradation of the receptor protein during the fractionation of cells required in previously reported methods. This method should prove useful in screening clonal cell populations for TCDD receptor number and affinity variants, and in screening for TCDD receptor binding activity in complementation studies of receptor deficient cells

Preparation of a homogeneous extract of human growth hormone, isohormone B, and its 125I-labelling for utilization in radioligand assays

International Nuclear Information System (INIS)

Santos, A.J. dos.

1985-01-01

Non-destructive polyacrylamide gel electrophoretic (PAGE) tecnique, with direct UV-densitometry, was set up to permit both qualitative and quantitative studies of human growth hormone (hGH) isohormone purification is presented. This tecnique was used on a preparative scale to obtain milligram amounts of the fundamental form of hGH, isohormone B(Ih-B). Reversed electrophoresis was employed to elute the protein band form the gel. Retention of bio-and immunoactivity was demonstrated via two separate experiments. An 'in vivo' bioassay, based on the weight increase of hypophysectomized rats with a 2 x 2 factorial assay design, was used to compare the true somatotrophic activity of an hGH preparation submitted to the purification process with that of a single control preparation. Retention of immunoactivity was confirmed by studying the antibody binding properties of purified and radioiodinated Ih-B and by determination of its absolute immunopotency against a calibrated secondary standard. Radioimmuno assay curves, determined using Ih-B, as a standard and labelled preparation, showed its applicability in setting up assays based on more homogeneous reagents. (Author) [pt

Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

Science.gov (United States)

Krainer, Georg; Keller, Sandro

2015-04-01

Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification of RNAIII-binding proteins in Staphylococcus aureus using tethered RNAs and streptavidin aptamers based pull-down assay.

Science.gov (United States)

Zhang, Xu; Zhu, Qing; Tian, Tian; Zhao, Changlong; Zang, Jianye; Xue, Ting; Sun, Baolin

2015-05-15

It has been widely recognized that small RNAs (sRNAs) play important roles in physiology and virulence control in bacteria. In Staphylococcus aureus, many sRNAs have been identified and some of them have been functionally studied. Since it is difficult to identify RNA-binding proteins (RBPs), very little has been known about the RBPs in S. aureus, especially those associated with sRNAs. Here we adopted a tRNA scaffold streptavidin aptamer based pull-down assay to identify RBPs in S. aureus. The tethered RNA was successfully captured by the streptavidin magnetic beads, and proteins binding to RNAIII were isolated and analyzed by mass spectrometry. We have identified 81 proteins, and expressed heterologously 9 of them in Escherichia coli. The binding ability of the recombinant proteins with RNAIII was further analyzed by electrophoresis mobility shift assay, and the result indicates that proteins CshA, RNase J2, Era, Hu, WalR, Pyk, and FtsZ can bind to RNAIII. This study suggests that some proteins can bind to RNA III in S. aureus, and may be involved in RNA III function. And tRSA based pull-down assay is an effective method to search for RBPs in bacteria, which should facilitate the identification and functional study of RBPs in diverse bacterial species.

Label-free detection of endocrine disrupting chemicals by integrating a competitive binding assay with a piezoelectric ceramic resonator.

Science.gov (United States)

Hu, Liang-sheng; Fong, Chi-Chun; Zou, Lan; Wong, Wing-Leung; Wong, Kwok-Yin; Wu, Rudolf S S; Yang, Mengsu

2014-03-15

A piezoelectric biosensor for detection of endocrine disrupting chemicals (EDCs) was developed by incorporating chemical/biochemical recognition elements on the ceramic resonator surface for competitive binding assays. A facile electrodeposition was employed to modify the sensor surface with Au nanoparticles, which increased the surface area and enhanced the binding capacity of the immobilized probes. Thiol-labeled long chain hydrocarbon with bisphenol A (BPA) as head group was synthesized and self-assembled on the Au nanoparticle surface as the sensing probes, which showed a linear response upon the binding of estrogen receptor (ER-α) ranging from 1 to 30 nM. Detection of estrone, 17β-estradiol and BPA was achieved by integrating a competitive binding assay with the piezoelectric sensor. In this detection scheme, different concentrations of EDCs were incubated with 30 nM of ER-α, and the un-bounded ER-α in the solution was captured by the probes immobilized on the ceramic resonator, which resulted in the frequency changes for different EDCs. The biosensor assay exhibited a linear response to EDCs with a low detection limit of 2.4-2.9 nM (S/N=3), and required only a small volume of sample (1.5 µl) with the assay time of 2h. The performance of the biosensor assay was also evaluated for rapid and facile determination of EDCs of environmental relevant concentrations in drinking water and seawater, and the recovery rate was in the range between 94.7% and 109.8%. © 2013 Elsevier B.V. All rights reserved.

"Assessment of human AT1 Binding Affinity of Some Novel 2-alkylthio-1-[4-(N-α-ethoxycarbonyl-nzylaminobenzyl-5-hydroxymethylimidazoles "

Directory of Open Access Journals (Sweden)

Setareh Badakhshannoory

2004-06-01

Full Text Available Antagonists of various components of the renin-angiotensin system have been the subject of many studies for the control of blood pressure. Compounds with a phenoxyphenylacetic acid moiety that mimic the structure of losartan which is a powerful competitive antagonist of angiotensin receptor, have shown to be effective. In this study, the affinity of some 2-alkylthio-1-[4-(N-α-ethoxycarbonylbenzylaminobenzyl]-5-hydroxymethyl imidazoles for the human AT1 receptor was assessed in a radioligand binding assay. It was found that an alkyl chain of appropriate length would be most suitable if situated on the imidazole ring. Furthermore, variations of the lower phenyl rings demonstrated that introduction of a methyl group in this position will account for the most desired effect.

( sup 3 H)(D-PEN sup 2 , D-PEN sup 5 ) enkephalin binding to delta opioid receptors on intact neuroblastoma-glioma (NG 108-15) hybrid cells

Energy Technology Data Exchange (ETDEWEB)

Knapp, R.J.; Yamamura, H.I. (Univ. of Arizona College of Medicine, Tucson (USA))

1990-01-01

({sup 3}H)(D-Pen{sup 2}, D-Pen{sup 5})enkephalin binding to intact NG 108-15 cells has been measured under physiological conditions of temperature and medium. The dissociation constant, receptor density, and Hill slope values measured under these conditions are consistent with values obtained by others using membranes prepared from these cells. Kinetic analysis of the radioligand binding to these cells show biphasic association and monophasic dissociation processes suggesting the presence of different receptor affinity states for the agonist. The data show that the binding affinity of ({sup 3}H)(D-Pen{sup 2}, D-Pen{sup 5})enkephalin under physiological conditions is not substantially different to that measured in 50 mM Tris buffer using cell membrane fractions. Unlike DPDPE, the {mu} opioid agonists morphine, normorphine, PL-17, and DAMGO, have much lower affinity for the {delta} receptor measured under these conditions than is observed by studies using 50 mM Tris buffer. The results described here suggest that this assay may serve as a useful model of {delta} opioid receptor binding in vivo.

Characterization of 12 GnRH peptide agonists - a kinetic perspective.

Science.gov (United States)

Nederpelt, Indira; Georgi, Victoria; Schiele, Felix; Nowak-Reppel, Katrin; Fernández-Montalván, Amaury E; IJzerman, Adriaan P; Heitman, Laura H

2016-01-01

Drug-target residence time is an important, yet often overlooked, parameter in drug discovery. Multiple studies have proposed an increased residence time to be beneficial for improved drug efficacy and/or longer duration of action. Currently, there are many drugs on the market targeting the gonadotropin-releasing hormone (GnRH) receptor for the treatment of hormone-dependent diseases. Surprisingly, the kinetic receptor-binding parameters of these analogues have not yet been reported. Therefore, this project focused on determining the receptor-binding kinetics of 12 GnRH peptide agonists, including many marketed drugs. A novel radioligand-binding competition association assay was developed and optimized for the human GnRH receptor with the use of a radiolabelled peptide agonist, [(125) I]-triptorelin. In addition to radioligand-binding studies, a homogeneous time-resolved FRET Tag-lite™ method was developed as an alternative assay for the same purpose. Two novel competition association assays were successfully developed and applied to determine the kinetic receptor-binding characteristics of 12 high-affinity GnRH peptide agonists. Results obtained from both methods were highly correlated. Interestingly, the binding kinetics of the peptide agonists were more divergent than their affinities with residence times ranging from 5.6 min (goserelin) to 125 min (deslorelin). Our research provides new insights by incorporating kinetic, next to equilibrium, binding parameters in current research and development that can potentially improve future drug discovery targeting the GnRH receptor. © 2015 The British Pharmacological Society.

Synthesis and Pharmacology of Halogenated δ-Opioid-Selective [D-Ala2]Deltorphin II Peptide Analogues

Science.gov (United States)

Pescatore, Robyn; Marrone, Gina F.; Sedberry, Seth; Vinton, Daniel; Finkelstein, Netanel; Katlowitz, Yitzchak E.; Pasternak, Gavril W.; Wilson, Krista R.; Majumdar, Susruta

2015-01-01

Deltorphins are naturally occurring peptides produced by the skin of the giant monkey frog (Phyllomedusa bicolor). They are δ-opioid receptor-selective agonists. Herein, we report the design and synthesis of a peptide, Tyr-D-Ala-(pI)Phe-Glu-Ile-Ile-Gly-NH2 3 (GATE3-8), based on the [D-Ala2]deltorphin II template, which is δ-selective in in vitro radioligand binding assays over the μ- and κ-opioid receptors. It is a full agonist in [35S]GTPγS functional assays and analgesic when administered supraspinally to mice. Analgesia of 3 (GATE3-8) is blocked by the selective δ receptor antagonist naltrindole, indicating that the analgesic action of 3 is mediated by the δ-opioid receptor. We have established a radioligand in which 125I isincorporated into 3 (GATE3-8). The radioligand has a KD of 0.1 nM in Chinese hamster ovary (CHO) cells expressing the δ receptor. Additionally, a series of peptides based on 3 (GATE3-8) was synthesized by incorporating various halogens in the para position on the aromatic ring of Phe3. The peptides were characterized for binding affinity at the μ-, δ-, and κ-opioid receptors, which showed a linear correlation between binding affinity and the size of the halogen substituent. These peptides may be interesting tools for probing δ-opioid receptor pharmacology. PMID:25844930

Synthesis and pharmacology of halogenated δ-opioid-selective [d-Ala(2)]deltorphin II peptide analogues.

Science.gov (United States)

Pescatore, Robyn; Marrone, Gina F; Sedberry, Seth; Vinton, Daniel; Finkelstein, Netanel; Katlowitz, Yitzchak E; Pasternak, Gavril W; Wilson, Krista R; Majumdar, Susruta

2015-06-17

Deltorphins are naturally occurring peptides produced by the skin of the giant monkey frog (Phyllomedusa bicolor). They are δ-opioid receptor-selective agonists. Herein, we report the design and synthesis of a peptide, Tyr-d-Ala-(pI)Phe-Glu-Ile-Ile-Gly-NH2 3 (GATE3-8), based on the [d-Ala(2)]deltorphin II template, which is δ-selective in in vitro radioligand binding assays over the μ- and κ-opioid receptors. It is a full agonist in [(35)S]GTPγS functional assays and analgesic when administered supraspinally to mice. Analgesia of 3 (GATE3-8) is blocked by the selective δ receptor antagonist naltrindole, indicating that the analgesic action of 3 is mediated by the δ-opioid receptor. We have established a radioligand in which (125)I is incorporated into 3 (GATE3-8). The radioligand has a KD of 0.1 nM in Chinese hamster ovary (CHO) cells expressing the δ receptor. Additionally, a series of peptides based on 3 (GATE3-8) was synthesized by incorporating various halogens in the para position on the aromatic ring of Phe(3). The peptides were characterized for binding affinity at the μ-, δ-, and κ-opioid receptors, which showed a linear correlation between binding affinity and the size of the halogen substituent. These peptides may be interesting tools for probing δ-opioid receptor pharmacology.

PET imaging of lung inflammation with [18F]FEDAC, a radioligand for translocator protein (18 kDa.

Directory of Open Access Journals (Sweden)

Akiko Hatori

Full Text Available PURPOSE: The translocator protein (18 kDa (TSPO is highly expressed on the bronchial and bronchiole epithelium, submucosal glands in intrapulmonary bronchi, pneumocytes and alveolar macrophages in human lung. This study aimed to perform positron emission tomography (PET imaging of lung inflammation with [(18F]FEDAC, a specific TSPO radioligand, and to determine cellular sources enriching TSPO expression in the lung. METHODS: An acute lung injury model was prepared by intratracheal administration of lipopolysaccharide (LPS to rat. Uptake of radioactivity in the rat lungs was measured with small-animal PET after injection of [(18F]FEDAC. Presence of TSPO was examined in the lung tissue using Western blot and immunohistochemical assays. RESULTS: The uptake of [(18F]FEDAC increased in the lung with the progress of inflammation by treatment with LPS. Pretreatment with a TSPO-selective ligand PK11195 showed a significant decrease in the lung uptake of [(18F]FEDAC due to competitive binding to TSPO. TSPO expression was elevated in the inflamed lung section and its level responded to the [(18F]FEDAC uptake and severity of inflammation. Increase of TSPO expression was mainly found in the neutrophils and macrophages of inflamed lungs. CONCLUSION: From this study we conclude that PET with [(18F]FEDAC may be a useful tool for imaging TSPO expression and evaluating progress of lung inflammation. Study on human lung using [(18F]FEDAC-PET is promising.

Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

International Nuclear Information System (INIS)

Sørensen, Thomas Just; Thyrhaug, Erling; Szabelski, Mariusz; Gryczynski, Ignacy; Gryczynski, Zygmunt; Luchowski, Rafal; Laursen, Bo W

2013-01-01

Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20–200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes. (paper)

Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

Science.gov (United States)

Just Sørensen, Thomas; Thyrhaug, Erling; Szabelski, Mariusz; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Laursen, Bo W.

2013-06-01

Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.

Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-alpha binding assay: protocol optimization and intralaboratory assay performance as initial steps towards validation.

Science.gov (United States)

Freyberger, Alexius; Wilson, Vickie; Weimer, Marc; Tan, Shirlee; Tran, Hoai-Son; Ahr, Hans-Jürgen

2010-08-01

Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-alpha (ERalpha) binding assay is available, although hr-ERalpha is available from several sources. In a joint effort, US EPA and Bayer Schering Pharma with funding from the EU-sponsored 6th framework project, ReProTect, developed a model protocol for such a binding assay. Important features of this assay are the use of a full length hr-ERalpha and performance in a 96-well plate format. A full length hr-ERalpha was chosen, as it was considered to provide the most accurate and human-relevant results, whereas truncated receptors could perform differently. Besides three reference compounds [17beta-estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERalpha [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o,p'-DDT, nonylphenol, n-butylparaben, and corticosterone] were used to explore the performance of the assay. Three independent experiments per compound were performed on different days, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and receptor preparation were freshly prepared for each experiment. The ERalpha binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERalpha ligand binding domain. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the assay was robust and reliably ranked compounds with strong, weak, and no affinity for the ERalpha with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as

Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization.

Science.gov (United States)

Machida, Kazuya; Liu, Bernard

2017-01-01

Recognition of phosphotyrosine-containing sequences by SH2 domains confers specificity in tyrosine kinase pathways. By assessing interactions between isolated SH2 domains and their binding proteins, it is possible to gain insight into otherwise inaccessible complex cellular systems. Far-Western, pull-down, and fluorescence polarization (FP) have been frequently used for characterization of phosphotyrosine signaling. Here, we outline standard protocols for these established assays using recombinant SH2 domain, emphasizing the importance of appropriate sample preparation and assay controls.

Choice of procedure conditions for radioimmunoassay of aflatoxin B1 using 125I as a marker and dextran-coated charcoal as a separation matrix

International Nuclear Information System (INIS)

Fukal, L.; Sova, Z.; Rauch, P.; Kas, J.

1987-01-01

Assay conditions for the radioimmunoassay for aflatoxin B 1 using 125 I-radiolabel and dextran-coated charcoal for the separation of free and bound radioligand were optimized. Casein was chosen as the best protecting protein. The most suitable incubation conditions are at 4 deg C for 18 h in darkness, radioligand sorption on the dextran-coated charcoal takes 30 min at 4 deg C and the antiserum is diluted in order to reach zero specific binding in the range between 35 and 50%. (author)

Development of two fluorine-18 labeled PET radioligands targeting PDE10A and in vivo PET evaluation in nonhuman primates.

Science.gov (United States)

Stepanov, Vladimir; Takano, Akihiro; Nakao, Ryuji; Amini, Nahid; Miura, Shotaro; Hasui, Tomoaki; Kimura, Haruhide; Taniguchi, Takahiko; Halldin, Christer

2018-02-01

Phosphodiesterase 10A (PDE10A) is a member of the PDE enzyme family that degrades cyclic adenosine and guanosine monophosphates (cAMP and cGMP). Based on the successful development of [ 11 C]T-773 as PDE10A positron emission tomography (PET) radioligand, in this study our aim was to develop and evaluate fluorine-18 analogs of [ 11 C]T-773. [ 18 F]FM-T-773-d 2 and [ 18 F]FE-T-773-d 4 were synthesized from the same precursor used for 11 C-labeling of T-773 in a two-step approach via 18 F-fluoromethylation and 18 F-fluoroethylation, respectively, using corresponding deuterated synthons. A total of 12 PET measurements were performed in seven non-human primates. First, baseline PET measurements were performed using High Resolution Research Tomograph system with both [ 18 F]FM-T-773-d 2 and [ 18 F]FE-T-773-d 4 ; the uptake in whole brain and separate brain regions, as well as the specific binding and tissue ratio between putamen and cerebellum, was examined. Second, baseline and pretreatment PET measurements using MP-10 as the blocker were performed for [ 18 F]FM-T-773-d 2 including arterial blood sampling with radiometabolite analysis in four NHPs. Both [ 18 F]FM-T-773-d 2 and [ 18 F]FE-T-773-d 4 were successfully radiolabeled with an average molar activity of 293 ± 114 GBq/μmol (n=8) for [ 18 F]FM-T-773-d 2 and 209 ± 26 GBq/μmol (n=4) for [ 18 F]FE-T-773-d 4 , and a radiochemical yield of 10% (EOB, n=12, range 3%-16%). Both radioligands displayed high brain uptake (~5.5% of injected radioactivity for [ 18 F]FM-T-773-d 2 and ~3.5% for [ 18 F]FE-T-773-d 4 at the peak) and a fast washout. Specific binding reached maximum within 30 min for [ 18 F]FM-T-773-d 2 and after approximately 45 min for [ 18 F]FE-T-773-d 4 . [ 18 F]FM-T-773-d 2 data fitted well with kinetic compartment models. BP ND values obtained indirectly through compartment models were correlated well with those obtained by SRTM. BP ND calculated with SRTM was 1.0-1.7 in the putamen. The occupancy with 1

[123I]epidepride binding to cerebellar dopamine D2/D3 receptors is displaceable: implications for the use of cerebellum as a reference region

DEFF Research Database (Denmark)

Pinborg, Lars H; Videbaek, Charlotte; Ziebell, Morten

2007-01-01

The low density of cerebellar dopamine D(2)/D(3) receptors provides the basis for using the cerebellum as a representation of free- and non-specifically bound radioligand in positron emission tomography (PET) and single photon emission computed tomography (SPECT) studies. With the development...... of ultra high-affinity dopamine D(2)/D(3) ligands like [(123)I]epidepride, [(18)F]fallypride, and [(11)C]FLB-457, quantification of extrastriatal low density receptor populations including the cerebellum is possible with important implications for calculation of binding parameters. [(123)I...... [(123)I]epidepride binding to dopamine D(2)/D(3) receptors in the cerebellum. Using the cerebellum as a representation of free and non-specifically bound radioligand and neglecting the specifically bound component may lead to results that erroneously imply that antipsychotic drugs bind to extrastriatal...

Acute S-ketamine application does not alter cerebral [18F]altanserin binding: a pilot PET study in humans

International Nuclear Information System (INIS)

Matusch, A.; Rota Kops, E.; Winz, O.H.; Elmenhorst, D.; Herzog, H.; Hurlemann, R.; Zilles, K.; Bauer, A.

2007-01-01

Modeling short-term psychotic states with subanaesthetic doses of ketamine provides substantial experimental evidence in support of the glutamate hypothesis of schizophrenia. Ketamine exerts its pharmacological effects both directly via interactions with glutamate receptors and indirectly by stimulating presynaptic release of endogenous serotonin (5-HT). The aim of this feasibility study was to examine whether acute ketamine-induced 5-HT release interferes with the binding of the 5-HT 2A receptor (5-HT 2A R) radioligand [ 18 F]altanserin and positron emission tomography (PET). Two subjects treated with ketamine and one subject treated with placebo underwent [ 18 F]altanserin PET at distribution equilibrium conditions. Robust physiological, psychopathological and cognitive effects were present at ketamine plasma concentrations exceeding 100 μg/l during >70 min. Notwithstanding, we observed stable radioligand binding (changes ±95 % CI of -1.0 ± 1.6 % and +4.1 ± 1.8 % versus -1.2 ± 2.6 %) in large cortical regions presenting high basal uptake of both, [ 18 F]altanserin and ketamine. Marginal decreases of 4 % of radioligand binding were observed in the frontal lobe, and 8 % in a posteriorly specified frontomesial subregion. This finding is not compatible with a specific radioligand displacement from 5-HT2 AR which should occur proportionally throughout the whole brain. Instead, the spatial pattern of these minor reductions was congruent with ketamine-induced increases in cerebral blood flow observed in a previous study using [ 15 O]butanol PET. This may caused by accelerated clearance of unspecifically bound [ 18 F]altanserin from cerebral tissue with increased perfusion. In conclusion, this study suggests that [ 18 F]altanserin PET is not sensitive to acute neurotransmitter fluctuations under ketamine. Advantageously, the stability of [ 18 F]altanserin PET towards acute influences is a prerequisite for its future use to detect sub-acute and chronic effects of

Synthesis, radiolabelling, and evaluation of [11C]PB212 as a radioligand for imaging sigma-1 receptors using PET.

Science.gov (United States)

Spinelli, Francesco; Haider, Ahmed; Toscano, Annamaria; Pati, Maria Laura; Keller, Claudia; Berardi, Francesco; Colabufo, Nicola Antonio; Abate, Carmen; Ametamey, Simon M

2018-01-01

The Sigma-1 receptor (Sig-1R) has been described as a pluripotent modulator of distinct physiological functions and its involvement in various central and peripheral pathological disorders has been demonstrated. However, further investigations are required to understand the complex role of the Sig-1R as a molecular chaperon. A specific PET radioligand would provide a powerful tool in Sig-1R related studies. As part of our efforts to develop a Sig-1R PET radioligand that shows antagonistic properties, we investigated the suitability of 1-(4-(6-methoxynaphthalen-1-yl)butyl)-4-methylpiperidine (designated PB212) for imaging Sig-1R. PB212 is a Sig-1R antagonist and exhibits subnanomolar affinity ( K i = 0.030 nM) towards Sig-1R as well as good to excellent selectivity over Sig-2R. The radiolabelling of [ 11 C]PB212 was accomplished by O-methylation of the phenolic precursor using [ 11 C]MeI. In vitro autoradiography with [ 11 C]PB212 on WT and Sig-1R KO mouse brain tissues revealed high non-specific binding, however using rat spleen tissues from CD1 mice and Wistar rats, high specific binding was observed. The spleen is known to have a high expression of Sig-1R. In vivo PET experiments in Wistar rats also showed high accumulation of [ 11 C]PB212 in the spleen. Injection of Sig-1R binding compounds, haloperidol (1 mg/kg) or fluspidine (1 mg/kg) shortly before [ 11 C]PB212 administration induced a drastic reduction of radiotracer accumulation, confirming the specificity of [ 11 C]PB212 towards Sig-1R in the spleen. The results obtained herein indicate that although [ 11 C]PB212 is not suitable for imaging Sig-1R in the brain, it is a promising candidate for the detection and quantification of Sig-1Rs in the periphery.

Synthesis and biochemical evaluation of tritium-labeled 1-methyl-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-1H-indazole-3-carboxa mide, a useful radioligand for 5HT3 receptors

International Nuclear Information System (INIS)

Robertson, D.W.; Bloomquist, W.; Cohen, M.L.; Reid, L.R.; Schenck, K.; Wong, D.T.

1990-01-01

The advent of potent, highly selective 5HT3 receptor antagonists has stimulated considerable interest in 5HT3 receptor mediated physiology and pharmacology. To permit detailed biochemical studies regarding interaction of the indazole class of serotonin (5HT) antagonists with 5HT3 receptors in multiple tissues, we synthesized 1-methyl-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-1H-indazole- 3-carboxamide (LY278584, compound 9) in high specific activity, tritium-labeled form. This radioligand was selected as a synthetic target because of its potency as a 5HT3-receptor antagonist, its selectivity for this receptor viz a viz other 5HT-receptor subtypes, and the ability to readily incorporate three tritia via the indazole N-CH3 substituent. Alkylation of N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-1H-indazole-3-carboxamide (8) with sodium hydride and tritium-labeled iodomethane, followed by HPLC purification, resulted in [3H]-9 with a radiochemical purity of 99% and a specific activity of 80.5 Ci/mmol. This radioligand bound with high affinity to a single class of saturable recognition sites in membranes isolated from cerebral cortex of rat brain. The Kd was 0.69 nM and the Bmax was 16.9 fmol/mg of protein. The specific binding was excellent, and accounted for 83-93% of total binding at concentrations of 2 nM or less. The potencies of known 5HT3-receptor antagonists as inhibitors of [3H]-9 binding correlated well with their pharmacological receptor affinities as antagonists of 5HT-induced decreases in heart rate and contraction of guinea pig ileum, suggesting the central recognition site for this radioligand may be extremely similar to or identical with peripheral 5HT3 receptors

Tritium labelling and characterization of the potent imidazoline I1 receptor antagonist [5,7-{sup 3}H] ({+-})-efaroxan at high specific activity

Energy Technology Data Exchange (ETDEWEB)

Egan, J.A.; Filer, C.N. E-mail: crist.filer@perkinelmer.com

2003-06-01

({+-})-Efaroxan 1 is a selective antagonist at the imidazoline I1 receptor. [{sup 3}H] ({+-})-Efaroxan was required to explore its mechanism of action via receptor binding assay, and the radioligand was prepared by means of catalytic dehalogenation of a dibrominated precursor with tritium.

Insulin-like growth factor I and II in 14 animal species and man as determined by three radioligand assays and two bioassays

Energy Technology Data Exchange (ETDEWEB)

Zangger, I.; Zapf, J.; Froesch, E.R.

1987-01-01

Insulin-like growth factor I and II (IGF I and II) were determined by five different assays in human serum, in the sera of ten mammalian species and in chicken, turtle, and frog serum. Sera of all tested mammals contain two different IGFs corresponding to human immunoreactive IGF I and receptor reactive IGF II. Receptor reactive IGF II of most animal species does not show significant cross-reactivity in the RIA for human IGF II. IGF activity was also detected in sera of non-mammals, such as chicken and turtles, but not in frog serum. The IGF values obtained with the different assay system corresponded rather well: there is a good correlation between the values obtained in the protein binding and the fat cell assay, and between the results of the latter assays and the sum of immunoreactive IGF I and receptor reative IGF II. The results suggest that those regions in the IGF I and II molecules which are responsible for reactivity with the type I IGF and the insulin receptor have not essentially changed during evolution. Similarly, the C-region, which mainly determines the immunological properties of IGFs, appears to have remained relatively constant in the IGF I, but not in the IGF II molecule.

Evaluation of the superselective radioligand [123I]PE2I for imaging of the dopamine transporter in SPECT

DEFF Research Database (Denmark)

Ziebell, Morten

a B/I ratio of [123I]PE2I. This B/I ratio (2.7h) gave rise to steady state conditions and excellent reproducibility. Further, manual delineation of ROI directly on SPECT images performed equally well to a MRI-defined probability map based ROI delineation in terms of intrasubject variability of binding......Imaging of the dopamine transporter (DAT) with Single Photon Emission Computer Tomography (SPECT) has increasingly been used as a biomarker for the integrity of presynaptic dopaminergic nerve cells in patients with movement disorders. 123-I-labelled N-(3-iodoprop-2E-enyl)-2-β-carbomethoxy-3β-(4...... potential of DAT. Finally the in vivo SERT binding in DAT images obtained with [123I]FP-CIT was significant as compared to the [123I]PE2I image. [123I]PE2I is a super selective SPECT DAT radioligand with optimal kinetic properties for accurate quantification of the DAT availability in striatum. Apart from...

Evaluation of the superselective radioligand [123I]PE2I for imaging of the dopamine transporter in SPECT

DEFF Research Database (Denmark)

Ziebell, Morten

2011-01-01

a B/I ratio of [123I]PE2I. This B/I ratio (2.7h) gave rise to steady state conditions and excellent reproducibility. Further, manual delineation of ROI directly on SPECT images performed equally well to a MRI-defined probability map based ROI delineation in terms of intrasubject variability of binding......Imaging of the dopamine transporter (DAT) with Single Photon Emission Computer Tomography (SPECT) has increasingly been used as a biomarker for the integrity of presynaptic dopaminergic nerve cells in patients with movement disorders. 123-I-labelled N-(3-iodoprop-2E-enyl)-2-ß-carbomethoxy-3ß-(4...... potential of DAT. Finally the in vivo SERT binding in DAT images obtained with [123I]FP-CIT was significant as compared to the [123I]PE2I image. [123I]PE2I is a super selective SPECT DAT radioligand with optimal kinetic properties for accurate quantification of the DAT availability in striatum. Apart from...

Affinities and densities of high-affinity [3H]muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

International Nuclear Information System (INIS)

Butterworth, R.F.; Lavoie, J.; Giguere, J.F.; Pomier-Layrargues, G.

1988-01-01

The integrity of GABA-A receptors and of central benzodiazepine receptors was evaluated in membrane preparations from prefrontal cortex and caudate nuclei obtained at autopsy from nine cirrhotic patients who died in hepatic coma and an equal number of age-matched control subjects. Histopathological studies revealed Alzheimer Type II astrocytosis in all cases in the cirrhotic group; controls were free from neurological, psychiatric or hepatic diseases. Binding to GABA-A receptors was studied using [ 3 H]muscimol as radioligand. The integrity of central benzodiazepine receptors was evaluated using [ 3 H]flunitrazepam and [ 3 H]Ro15-1788. Data from saturation binding assays was analyzed by Scatchard plot. No modifications of either affinities (Kd) or densities (Bmax) of [ 3 H]muscimol of central benzodiazepine binding sites were observed. These findings do not support recent suggestions that alterations of either high-affinity GABA or benzodiazepine receptors play a significant role in the pathogenesis of hepatic encephalopathy

Zinc blotting assay for detection of zinc binding prolamin in barley (Hordeum vulgare) grain

DEFF Research Database (Denmark)

Uddin, Mohammad Nasir; Nielsen, Ane Langkilde-Lauesen; Vincze, Eva

2014-01-01

In plants, zinc is commonly found bound to proteins. In barley (Hordeum vulgare), major storage proteins are alcohol-soluble prolamins known as hordeins, and some of them have the potential to bind or store zinc. 65Zn overlay and blotting techniques have been widely used for detecting zinc......-binding protein. However, to our knowledge so far this zinc blotting assay has never been applied to detect a prolamin fraction in barley grains. A radioactive zinc (65ZnCl2) blotting technique was optimized to detect zinc-binding prolamins, followed by development of an easy-to-follow nonradioactive colorimetric...... zinc blotting method with a zinc-sensing dye, dithizone. Hordeins were extracted from mature barley grain, separated by SDS-PAGE, blotted on a membrane, renatured, overlaid, and probed with zinc; subsequently, zinc-binding specificity of certain proteins was detected either by autoradiography or color...

GMP-140 binds to a glycoprotein receptor on human neutrophils: Evidence for a lectin-like interaction

International Nuclear Information System (INIS)

Moore, K.L.; Varki, A.; McEver, R.P.

1991-01-01

GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism

Radiopeptide internalisation and externalisation assays: Cell viability and radioligand integrity

International Nuclear Information System (INIS)

Raza Naqvi, Syed Ali; Sosabowski, Jane K.; Ahamad Nagra, Saeed; Ishfaq, Malik M.; Mather, Stephen J.; Matzow, Torkjel

2011-01-01

Various aspects of radiopeptide receptor-mediated cell internalisation and externalisation assays were assessed, including the integrity of externalised peptides and the effect of varying the pH and incubation time of the acid wash step (to remove surface receptor-bound ligand) on efficacy and cell viability. The observed intact proportion of externalised peptide was 5-10%, and acid wash buffers with pH 2.8 or below were found to be detrimental to cell viability and integrity, particularly following prolonged incubation times.

Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

International Nuclear Information System (INIS)

Gil, D.W.; Wolfe, B.B.

1986-01-01

Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands [ 3 H]quinuclidinyl benzilate or [ 3 H]PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of [ 3 H]quinuclidinyl benzilate in a biphasic manner

A new graphic plot analysis for determination of neuroreceptor binding in positron emission tomography studies.

Science.gov (United States)

Ito, Hiroshi; Yokoi, Takashi; Ikoma, Yoko; Shidahara, Miho; Seki, Chie; Naganawa, Mika; Takahashi, Hidehiko; Takano, Harumasa; Kimura, Yuichi; Ichise, Masanori; Suhara, Tetsuya

2010-01-01

In positron emission tomography (PET) studies with radioligands for neuroreceptors, tracer kinetics have been described by the standard two-tissue compartment model that includes the compartments of nondisplaceable binding and specific binding to receptors. In the present study, we have developed a new graphic plot analysis to determine the total distribution volume (V(T)) and nondisplaceable distribution volume (V(ND)) independently, and therefore the binding potential (BP(ND)). In this plot, Y(t) is the ratio of brain tissue activity to time-integrated arterial input function, and X(t) is the ratio of time-integrated brain tissue activity to time-integrated arterial input function. The x-intercept of linear regression of the plots for early phase represents V(ND), and the x-intercept of linear regression of the plots for delayed phase after the equilibrium time represents V(T). BP(ND) can be calculated by BP(ND)=V(T)/V(ND)-1. Dynamic PET scanning with measurement of arterial input function was performed on six healthy men after intravenous rapid bolus injection of [(11)C]FLB457. The plot yielded a curve in regions with specific binding while it yielded a straight line through all plot data in regions with no specific binding. V(ND), V(T), and BP(ND) values calculated by the present method were in good agreement with those by conventional non-linear least-squares fitting procedure. This method can be used to distinguish graphically whether the radioligand binding includes specific binding or not.

Bacteroides gingivalis-Actinomyces viscosus cohesive interactions as measured by a quantitative binding assay

International Nuclear Information System (INIS)

Schwarz, S.; Ellen, R.P.; Grove, D.A.

1987-01-01

There is limited evidence, mostly indirect, to suggest that the adherence of Bacteroides gingivalis to teeth may be enhanced by the presence of gram-positive dental plaque bacteria like Actinomyces viscosus. The purpose of this study was to carry out direct quantitative assessments of the cohesion of B gingivalis and A. viscosus by using an in vitro assay modeled on the natural sequence in which these two species colonize the teeth. The assay allowed comparisons to be made of the adherence of 3 H-labeled B. gingivalis 2561 and 381 to saliva-coated hydroxyapatite beads (S-HA) and A. viscosus WVU627- or T14V-coated S-HA (actinobeads) in equilibrium and kinetics binding studies. A series of preliminary binding studies with 3H-labeled A. viscosus and parallel studies by scanning electron microscopy with unlabeled A. viscosus were conducted to establish a protocol by which actinobeads suitable for subsequent Bacteroides adherence experiments could be prepared. By scanning electron microscopy, the actinobeads had only small gaps of exposed S-HA between essentially irreversibly bound A. viscosus cells. Furthermore, B. gingivalis cells appeared to bind preferentially to the Actinomyces cells instead of the exposed S-HA. B. gingivalis binding to both S-HA and actinobeads was saturable with at least 2 X 10(9) to 3 X 10(9) cells per ml, and equilibrium with saturating concentrations was reached within 10 to 20 min. B. gingivalis always bound in greater numbers to the actinobeads than to S-HA. These findings provide direct measurements supporting the concept that cohesion with dental plaque bacteria like A. viscosus may foster the establishment of B. gingivalis on teeth by enhancing its adherence

(D-Pen2,4 prime -125I-Phe4,D-Pen5)enkephalin: A selective high affinity radioligand for delta opioid receptors with exceptional specific activity

Energy Technology Data Exchange (ETDEWEB)

Knapp, R.J.; Sharma, S.D.; Toth, G.; Duong, M.T.; Fang, L.; Bogert, C.L.; Weber, S.J.; Hunt, M.; Davis, T.P.; Wamsley, J.K. (Department of Pharmacology, University of Arizona, College of Medicine, Tucson (United States))

1991-09-01

(D-Pen2,4{prime}-125I-Phe4,D-Pen5)enkephalin ((125I)DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. (125I)DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 {plus minus} 67 pM and a receptor density (Bmax) value of 36.4 {plus minus} 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM (125I) DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 {plus minus} 0.88 {times} 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM (3H) DPDPE and 0.8 nM (3H) (D-Pen2,4{prime}-Cl-Phe4, D-Pen5)enkephalin, respectively. The dissociation rate of (125I)DPDPE (0.917 {plus minus} 0.117 {times} 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of (125I)DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes (125I)DPDPE a valuable new radioligand for studies of delta opioid receptors.

Iodine-123 labelled nor-β-CIT binds to the serotonin transporter in vivo as assessed by biodistribution studies in rats

International Nuclear Information System (INIS)

Booij, J.; Knol, R.J.J.; Reneman, L.; De Bruin, K.; Van Royen, E.A.; Janssen, A.G.M.

1998-01-01

Iodine-123 labelled 2β-carbomethoxy-3β-(4-iodophenyl)nortropane (nor-β-CIT), a radioiodinated cocaine analogue, was evaluated as an agent for the in vivo labelling of serotonin transporters by biodistribution studies in rats. Intravenous injection of [ 123 I]nor-β-CIT resulted in high accumulation of radioactivity in brain areas with high densities of serotonin (hypothalamus) and dopamine transporters (striatum), although the binding was less pronounced in the hypothalamus. While binding of [ 123 I]nor-β-CIT in the hypothalamus was blocked significantly by fluvoxamine (a selective serotonin transporter blocker) but not by GBR12,909 (a selective dopamine transporter blocker), the opposite was observed in the striatum. The results of this study indicate that [ 123 I]nor-β-CIT, although not being a selective radioligand, binds specifically to serotonin transporters in the hypothalamus in vivo and thus suggest that [ 123 I]nor-β-CIT promises to be a suitable radioligand for single-photon emission tomography imaging of serotonin transporters in humans. (orig.)

Radiopeptide internalisation and externalization assays: cell viability and radioligand integrity.

Science.gov (United States)

Naqvi, Syed Ali Raza; Sosabowski, Jane K; Nagra, Saeed Ahamad; Ishfaq, Malik M; Mather, Stephen J; Matzow, Torkjel

2011-01-01

Various aspects of radiopeptide receptor-mediated cell internalisation and externalization assays were assessed, including the integrity of externalized peptides and the effect of varying the pH and incubation time of the acid wash step (to remove surface receptor-bound ligand) on efficacy and cell viability. The observed intact proportion of externalized peptide was 5-10%, and acid wash buffers with pH 2.8 or below were found to be detrimental to cell viability and integrity, particularly following prolonged incubation times. Copyright © 2010 Elsevier Ltd. All rights reserved.

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

Energy Technology Data Exchange (ETDEWEB)

Costantini, Lindsey M. [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Irvin, Susan C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Kennedy, Steven C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Guo, Feng [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Goldstein, Harris; Herold, Betsy C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Snapp, Erik L., E-mail: erik-lee.snapp@einstein.yu.edu [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States)

2015-02-15

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

International Nuclear Information System (INIS)

Costantini, Lindsey M.; Irvin, Susan C.; Kennedy, Steven C.; Guo, Feng; Goldstein, Harris; Herold, Betsy C.; Snapp, Erik L.

2015-01-01

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells

Specific binding component of the 'inactive' stereoisomer (S,S)-[125I]IQNB to rat brain muscarinic receptors in vivo

International Nuclear Information System (INIS)

Boulay, Sheila F.; McRee, R. Carter; Cohen, Victor I.; Sood, Virendar K.; Zeeberg, Barry R.; Reba, Richard C.

1996-01-01

In vivo nonspecific binding can be estimated using the inactive stereoisomer of a receptor radioligand. However, the binding of the inactive stereoisomer may be partially specific. Specific binding of the inactive (S,S)-[ 125 I]IQNB was estimated from the inhibition induced by a competing nonradioactive ligand. This technique differed from the usual approach, since it was used to study the inactive rather than the active stereoisomer. The results indicate that there is substantial specific binding for (S,S)-[ 125 I]IQNB

[{sup 11}C]NNC 22-0215, a metabolically stable dopamine D{sub 1} radioligand for PET

Energy Technology Data Exchange (ETDEWEB)

Foged, Christian; Halldin, Christer; Swahn, Carl-Gunnar; Ginovart, Nathalie; Karlsson, Per; Lundkvist, Camilla; Farde, Lars

1998-07-01

NNC 22-0215 has been found to be a metabolically stable dopamine D{sub 1} antagonist with high affinity and selectivity for D{sub 1} receptors in vitro. We prepared [{sup 11}C]NNC 22-0215 with a specific radioactivity of about 50 GBq/{mu}mol at time of administration. In PET experiments with [{sup 11}C]NNC 22-0215 there was a rapid uptake of radioactivity in the cynomolgus monkey brain (1.8% of total radioactivity injected). Radioactivity accumulated most markedly in the striatum and the neocortex. The striatum to cerebellum ratio was about 4, with specific binding that remained at a plateau level from 50 min to 100 min after injection. Binding in the striatum and neocortex was markedly displaced by SCH 23390, whereas binding in the cerebellum was not reduced. Metabolite studies showed that about 80% of the radioactivity in the monkey plasma represented unchanged radioligand 30 min after injection. The rate of metabolism in monkey plasma in vivo was also determined for a series of structurally related {sup 11}C-labelled benzazepines, previously used as dopamine D{sub 1} receptor ligands for PET. Results indicate a significantly slower rate of metabolism for [{sup 11}C]NNC 22-0215 than for any of the previously labelled benzazepines. Thus [{sup 11}C]NNC 22-0215 has potential for imaging of selective binding to the dopamine D{sub 1} receptors in the human brain with high count rates at time of equilibrium.

Tritium labelling and characterization of the cognition enhancing drug tacrine using several precursors

Energy Technology Data Exchange (ETDEWEB)

Egan, Judith A.; Nugent, Richard P.; Filer, Crist N. E-mail: crist.filer@perkinelmer.com

2002-12-01

Tacrine and its analogues have shown promise as cognition enhancers and potential chemotherapeutic agents for Alzheimer's disease. [{sup 3}H]tacrine was required to explore its mechanism of action via receptor binding assay and the radioligand was prepared by means of catalytic dehalogenation of several brominated precursors.

Two novel assays for the detection of haemin-binding properties of antimalarials evaluated with compounds isolated from medicinal plants.

Science.gov (United States)

Steele, J C P; Phelps, R J; Simmonds, M S J; Warhurst, D C; Meyer, D J

2002-07-01

Forty-two compounds isolated from nine plants used within South America for the treatment of malaria were tested for haemin binding using two novel, rapid screening methods. The data obtained were analysed with respect to IC(50) values for in vitro toxicity to Plasmodium falciparum trophozoites. One method, a multiwell assay based on the inhibition of the interaction of haemin with glutathione (GSH), is sensitive in the 10 microM range, takes c. 1 h and is suitable for either a high throughput screen or rapid assay during natural product isolation. Of 19 compounds showing antiplasmodial activity (IC(50) 40% inhibition of GSH-haemin reaction. The sensitivity and specificity of the assay were 0.85 and 0.82, respectively. The positive predictive value was 0.81 and the negative predictive value 0.86. A more sensitive assay (0.1 microM range) is based on the reversal by haemin-binding compounds of the haemin inhibition of the L-dopachrome-methyl ester tautomerase activity of human macrophage migration inhibitory factor. This assay gives a better idea of the affinity of interaction and uses very small amounts of test compound. The log[RI(50)] of eight of the compounds that tested positive in the above assays together with those of quinine and chloroquine showed a positive correlation with log[antiplasmodial IC(50)] for strain T9-96 (r = 0.824) and strain K1 (r = 0.904). Several of the antimalarial compounds that bind haemin are isoquinolines, a class not shown previously to interact with haemin.

A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases.

Science.gov (United States)

Qi, Jun; Kizjakina, Karina; Robinson, Reeder; Tolani, Karishma; Sobrado, Pablo

2012-06-01

N-Hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and bacteria. NMOs catalyze the hydroxylation of sine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington's and Alzheimer's diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin-dependent monooxygenases. Fluorescently labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a K(d) value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K(d) values of 2.1 ± 0.2 and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we show that this assay can be used to identify inhibitors of NMOs. A Z' factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration. Copyright © 2012 Elsevier Inc. All rights reserved.

Radioiododestannylation. Convenient synthesis of a stable penicillin derivative for rapid penicillin binding protein (PBP) assay

International Nuclear Information System (INIS)

Blaszczak, L.C.; Halligan, N.G.; Seitz, D.E.

1989-01-01

Radioiodination of p-(trimethylstannyl)penicillin V with [ 125 I]Na using a modification of the chloramine-T method is simple, high yielding, and site-specific. The structure and penicillin binding protein (PBP) affinity of p-[ 125 I]-penicillin V (IPV) are similar to penicillin G and the product can be used directly without purification in the PBP assay. Because of the high degree of stability toward autoradiolysis and equivalent PBP binding affinity, IPV can be used in place of [ 3 H]-penicillin G or [ 14 C]-penicillin G for these experiments. (author)

Specific binding of atrial natriuretic factor in brain microvessels

International Nuclear Information System (INIS)

Chabrier, P.E.; Roubert, P.; Braquet, P.

1987-01-01

Cerebral capillaries constitute the blood-brain barrier. Studies of specific receptors (neurotransmitters or hormones) located on this structure can be performed by means of radioligand-binding techniques on isolated brain microvessels. The authors examined on pure bovine cerebral microvessel preparations the binding of atrial natriuretic factor (ANF), using 125 I-labeled ANF. Saturation and competition experiments demonstrated the presence of a single class of ANF-binding sites with high affinity and with a binding capacity of 58 fmol/mg of protein. The binding of 125 I-labeled ANF to brain microvessels is specific, reversible, and time dependent, as is shown by association-dissociation experiments. The demonstration of specific ANF-binding sites on brain microvessels supposes a physiological role of ANF on brain microvasculature. The coexistence of ANF and angiotensin II receptors on this cerebrovascular tissue suggests that the two circulating peptides may act as mutual antagonists in the regulation of brain microcirculation and/or blood-brain barrier function

Azadioxatriangulenium (ADOTA+): A long fluorescence lifetime fluorophore for large biomolecule binding assay

Science.gov (United States)

Sørensen, Thomas Just; Thyrhaug, Erling; Szabelski, Mariusz; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Laursen, Bo W.

2013-01-01

Of the many optical bioassays available, sensing by fluorescence anisotropy have great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is in the order of 20–200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatics dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecules assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immuniglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time by more than 75 %, and a change in the steady-state anisotropy increase of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay for detecting binding events involving biomolecules of far larger size than what is possible with the other red emitting organic dyes. PMID:24058730

Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay

DEFF Research Database (Denmark)

Nyholm, H C; Nielsen, Anette Lynge; Lyndrup, J

1995-01-01

We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA......, and cytosolic progesterone receptors (PR) measured by LBA were also studied. While ERc concentrations determined by LBA and EIA were highly correlated (r: 0.94), ERc values detected by LBA were approximately twice those found by EIA (median values of ERc: 155 vs. 64 fmol/mg cytosol protein, DCC vs. EIA......). The percentages of ERc positive tumors were 89% by LBA and 77% by EIA. The median fraction of total ER present as ERn was 63%. PR levels correlated positively with ERn concentrations (r: 0.73). We explore possible reasons why greater concentrations of ERc are determined by estradiol binding than by the ER-EIA kit...

Value of 111In-DOTA-lanreotide and 111In-DOTA-DPhe1-Tyr3-octreotide in differentiated thyroid cancer: results of in vitro binding studies and in vivo comparison with 18F-FDG PET

International Nuclear Information System (INIS)

Rodrigues, Margarida; Virgolini, Irene; Traub-Weidinger, Tatjana; Leimer, Maria; Li, Shuren; Dudczak, Robert; Andreae, Fritz; Angelberger, Peter

2005-01-01

Radioiodine-negative thyroid cancer presents diagnostic and therapeutic difficulties, warranting the implementation of new imaging and treatment strategies. The purpose of this study was twofold. First, we investigated in vitro the binding characteristics of 111 In-DOTA-lanreotide ( 111 In-DOTA-LAN) and 111 In-DOTA-DPhe 1 -Tyr 3 -octreotide ( 111 In-DOTA-TOC) to cells derived from differentiated thyroid cancer (DTC). Second, we evaluated the value of somatostatin receptor (SSTR) scintigraphy with these radioligands, as compared with 18 F-fluorodeoxyglucose (FDG) positron emission tomography (PET), for the detection of tumour lesions in DTC patients. Binding of 111 In-DOTA-LAN and 111 In-DOTA-TOC to cells isolated from surgically removed thyroid tissue was evaluated in vitro by performing saturation and displacement studies. Eighteen DTC patients with elevated thyroglobulin (12 radioiodine-negative, six radioiodine-positive) were investigated with 111 In-DOTA-LAN, 111 In-DOTA-TOC and 18 F-FDG PET scans. Large numbers of SSTR binding sites for 111 In-DOTA-LAN and 111 In-DOTA-TOC were found on the cells investigated. Both SSTR radioligands exhibited a high binding affinity for these SSTR binding sites. 111 In-DOTA-LAN and 111 In-DOTA-TOC scintigraphy detected 37 and 33 lesions, respectively, in 17 (94%) patients each, whereas 18 F-FDG PET revealed 30 lesions in 15 (83%) patients. Uptake of both SSTR radioligands was found in several radioiodine-negative sites. No striking differences in lesion imaging by 111 In-DOTA-LAN and 111 In-DOTA-TOC were found. In both radioiodine-negative and radioiodine-positive patients, more lesions were SSTR-positive/ 18 F-FDG-negative than were 18 F-FDG-positive/SSTR-negative. Adding a SSTR scan with these radioligands to the diagnostic work-up increases the diagnostic capacity in DTC, and should be considered particularly in radioiodine-negative patients with elevated thyroglobulin levels. (orig.)

Use of PET and the radioligand [carbonyl-11C]WAY-100635 in psychotropic drug development

International Nuclear Information System (INIS)

Andree, Bengt; Halldin, Christer; Thorberg, Seth-Olov; Sandell, Johan; Farde, Lars

2000-01-01

Positron-emission tomography (PET) provides potential in neuropsychiatric drug development by expanding knowledge of drug action in the living human brain and reducing time consumption and costs. The 5-hydroxytryptamine 1A (5-HT 1A ) receptor is of central interest as a target for the treatment of anxiety, depression, and schizophrenia. Research on the clinical significance of the 5-HT 1A receptor now benefits from the highly selective radioligand [carbonyl- 11 C]WAY-100635 (WAY) for quantitative determination of 5-HT 1A receptors in the primate and human brain in vivo using PET. In this paper, three studies are reviewed to demonstrate the suitability of WAY as radioligand for quantification of central 5-HT 1A receptors in brain and as an applicable tool for drug development. In the first study a monkey model was used to characterize WAY binding. It was confirmed that the reference ligand 8-OH-DPAT and psychoactive drugs such as buspirone and pindolol occupies 5-HT 1A receptors in the primate brain. Pindolol is an β-adrenoreceptor antagonist with a high affinity to 5-HT 1A receptors. This drug has been suggested in combination with selective serotonin reuptake inhibitors for the treatment of depression and was given to healthy males in the second study. Pindolol induced a marked inhibition of central 5-HT 1A receptors as calculated by the ratio-analysis method and simplified reference tissue model, 2 h after administration of 10 mg as a single oral dose. This observation suggests that pindolol may have a role for the suggested potentiation of selective serotonin reuptake inhibitor treatment of depression. The third study was on robalzotan (NAD-299), a recently developed 5-HT 1A receptor antagonist and putative drug with implications for the treatment of depression. In the cynomolgus monkey brain, robalzotan in the dose range 2-100 μg/kg IV occupied 5-HT 1A receptors in a dose-dependent and saturable manner with a maximal calculated occupancy of 70-80%. The

[11C]-MeJDTic: a novel radioligand for κ-opioid receptor positron emission tomography imaging

International Nuclear Information System (INIS)

Poisnel, Geraldine; Oueslati, Farhana; Dhilly, Martine; Delamare, Jerome; Perrio, Cecile; Debruyne, Daniele; Barre, Louisa

2008-01-01

Introduction: Radiopharmaceuticals that can bind selectively the κ-opioid receptor may present opportunities for staging clinical brain disorders and evaluating the efficiency of new therapies related to stroke, neurodegenerative diseases or opiate addiction. The N-methylated derivative of JDTic (named MeJDTic), which has been recently described as a potent and selective antagonist of κ-opioid receptor in vitro, was labeled with carbon-11 and evaluated for in vivo imaging the κ-opioid receptor in mice. Methods: [ 11 C]-MeJDTic was prepared by methylation of JDTic with [ 11 C]-methyl triflate. The binding of [ 11 C]-MeJDTic to κ-opioid receptor was investigated ex vivo by biodistribution and competition studies using nonfasted male CD1 mice. Results: [ 11 C]-MeJDTic exhibited a high and rapid distribution in peripheral organs. The uptake was maximal in lung where the κ receptor is largely expressed. [ 11 C]-MeJDTic rapidly crossed the blood-brain barrier and accumulated in the brain regions of interest (hypothalamus). The parent ligand remained the major radioactive compound in brain during the experiment. Chase studies with U50,488 (a κ referring agonist), morphine (a μ agonist) and naltrindole (a δ antagonist) demonstrated that this uptake was the result of specific binding to the κ-opioid receptor. Conclusion: These findings suggested that [ 11 C]-MeJDTic appeared to be a promising selective 'lead' radioligand for κ-opioid receptor PET imaging

Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

International Nuclear Information System (INIS)

Waser, Beatrice; Reubi, Jean Claude

2014-01-01

Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the 125 iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer 125 I-GLP-1(7-36)amide. Receptor autoradiography studies with 125 I-GLP-1(7-36)amide agonist or 125 I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. The antagonist 125 I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer 125 I-GLP-1(7-36)amide. For comparison, 125 I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. The GLP-1 receptor antagonist exendin(9-39) labelled with 125 I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. (orig.)

Stage-specific adhesion of Leishmania promastigotes to sand fly midguts assessed using an improved comparative binding assay.

Directory of Open Access Journals (Sweden)

Raymond Wilson

2010-09-01

Full Text Available The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s that mediate binding is not fully understood.To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti. The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed.The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms, but is absent in the early blood meal and final stages (procyclic

Evaluation of σ-1 receptor radioligand 18F-FTC-146 in rats and squirrel monkeys using PET

DEFF Research Database (Denmark)

James, Michelle L; Shen, Bin; Nielsen, Carsten Haagen

2014-01-01

. Pretreatment with known S1R compounds, haloperidol, or BD1047, before radioligand administration, significantly attenuated (18)F-FTC-146 accumulation in all rat brain regions by approximately 85% (P ...-FTC-146 was observed in rat plasma. Preliminary monkey PET/MRI studies demonstrated specific accumulation of (18)F-FTC-146 in the brain (mainly in cortical structures, cerebellum, and vermis) that could be attenuated by pretreatment with haloperidol. HPLC of monkey plasma suggested radioligand metabolism...

Iodine-123 labelled nor-{beta}-CIT binds to the serotonin transporter in vivo as assessed by biodistribution studies in rats

Energy Technology Data Exchange (ETDEWEB)

Booij, J.; Knol, R.J.J.; Reneman, L.; De Bruin, K.; Van Royen, E.A. [Dept. of Nuclear Medicine, Univ. of Amsterdam (Netherlands); Janssen, A.G.M. [Amersham Cygne and Eindhoven University of Technology (Netherlands)

1998-12-01

Iodine-123 labelled 2{beta}-carbomethoxy-3{beta}-(4-iodophenyl)nortropane (nor-{beta}-CIT), a radioiodinated cocaine analogue, was evaluated as an agent for the in vivo labelling of serotonin transporters by biodistribution studies in rats. Intravenous injection of [{sup 123}I]nor-{beta}-CIT resulted in high accumulation of radioactivity in brain areas with high densities of serotonin (hypothalamus) and dopamine transporters (striatum), although the binding was less pronounced in the hypothalamus. While binding of [{sup 123}I]nor-{beta}-CIT in the hypothalamus was blocked significantly by fluvoxamine (a selective serotonin transporter blocker) but not by GBR12,909 (a selective dopamine transporter blocker), the opposite was observed in the striatum. The results of this study indicate that [{sup 123}I]nor-{beta}-CIT, although not being a selective radioligand, binds specifically to serotonin transporters in the hypothalamus in vivo and thus suggest that [{sup 123}I]nor-{beta}-CIT promises to be a suitable radioligand for single-photon emission tomography imaging of serotonin transporters in humans. (orig.) With 1 fig., 2 tabs., 15 refs.

[3H]PN200-110 and [3H]ryanodine binding and reconstitution of ion channel activity with skeletal muscle membranes

International Nuclear Information System (INIS)

Hamilton, S.L.; Alvarez, R.M.; Fill, M.; Hawkes, M.J.; Brush, K.L.; Schilling, W.P.; Stefani, E.

1989-01-01

Skeletal muscle membranes derived either from the tubular (T) network or from the sarcoplasmic reticulum (SR) were characterized with respect to the binding of the dihydropyridine, [ 3 H]PN200-110, and the alkaloid, [ 3 H]ryanodine; polypeptide composition; and ion channel activity. Conditions for optimizing the binding of these radioligands are discussed. A bilayer pulsing technique is described and is used to examine the channels present in these membranes. Fusion of T-tubule membranes into bilayers revealed the presence of chloride channels and dihydropyridine-sensitive calcium channels with three distinct conductances. The dihydropyridine-sensitive channels were further characterized with respect to their voltage dependence. Pulsing experiments indicated that two different populations of dihydropyridine-sensitive channels existed. Fusion of heavy SR vesicles revealed three different ion channels; the putative calcium release channel, a potassium channel, and a chloride channel. Thus, this fractionation procedure provides T-tubules and SR membranes which, with radioligand binding and single channel recording techniques, provide a useful tool to study the characteristics of skeletal muscle ion channels and their possible role in excitation-contraction coupling

Cocaine- and amphetamine-regulated transcript (CART) peptide specific binding in pheochromocytoma cells PC12

Czech Academy of Sciences Publication Activity Database

Maletínská, Lenka; Maixnerová, Jana; Matyšková, Resha; Haugvicová, Renata; Šloncová, Eva; Elbert, Tomáš; Slaninová, Jiřina; Železná, Blanka

2007-01-01

Roč. 559, 2/3 (2007), s. 109-114 ISSN 0014-2999 R&D Projects: GA ČR GA303/05/0614 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514; CEZ:AV0Z50200510 Keywords : radioligand binding * CART * PC12 cells * food intake Subject RIV: CE - Biochemistry Impact factor: 2.376, year: 2007

The assay of estrogen receptors in three components of human breast cancer tissue

International Nuclear Information System (INIS)

Lu Hanping; Gui Zhining

1992-01-01

The binding capacities of estrogen receptors in nuclear matrix, nuclei and cytosol of human breast cancer tissue (EmR, EnR, EcR) were estimated with radioligand binding assay of receptors. The average B max values of these components in 21 breast cancer specimens are 417.54 ± 170.95, 147.75 ± 98.32, 7.34 ± 5.33 fmol/mg protein, and those in 10 normal breast tissue specimens are 42.33 ± 8.49, 25.05 ± 7.81, 5.91 ± 2.28 fmol/mg protein. Comparing the cancer and normal breast tissues, there is significant difference in B max values of EmR and EnR (P max values of EcR (P > 0.10). The EmR/EnR value of 21 breast cancer tissue is 0.65 ± 0.10, and that of 10 normal breast tissue is 0.42 ± 0.04. There is statistical difference between the cancer and normal. 10 of 13 (77%) patients, who are EcR-positive, have higher EmR/EnR values (≥0.50). The results suggest that estrogen receptors are mainly located at the nuclear matrix, ER levels in nucleus, especially in nuclear matrix of breast cancer tissue are valuable parameters and may be useful for predicting whether the patient will be responsible to endocrine therapy

Interaction of xenobiotics with estrogen receptors α and β and a putative plasma sex hormone-binding globulin from channel catfish (Ictalurus punctatus)

Science.gov (United States)

Gale, William L.; Patino, Reynaldo; Maule, Alec G.

2004-01-01

Estrogens are important regulators of physiological functions. Although environmental contaminants (xenoestrogens) which interfere with estrogen signaling are of increasing concern, there is only limited information about their ability to interact with estrogen-binding proteins (SHBG) or receptors (ER). Recombinant ER?? and ?? were obtained after transient transfection of COS-7 cells with channel catfish ER cDNA. Plasma from adult female channel catfish was the source of SHBG. Tritiated estradiol ( 3H-E2) was used in standard radioligand-binding assays to characterize the binding properties of channel catfish SHBG (ccfSHBG) and to estimate the inhibition constants for various estrogenic compounds. Binding of 3H-E2 to ccfSHBG was saturable and of high affinity with a Kd (??SE) of 1.9??0.14nM and a Bmax of 14.3??2.4pmol/mg protein (n=3 assays). Additionally, ccfSHBG displayed binding specificity for androgens and estrogens. Endosulfan, 4-nonylphenol, and 4-octylphenol displaced 3H-E2 binding to ccfSHBG albeit only at very high concentrations, whereas dieldrin and atrazine showed little displacement activity even at the highest concentrations used. The synthetic estrogen ethynylestradiol had higher affinity than E2 for ccfSHBG. This finding differs from results with human and rainbow trout SHBG. The alkylphenolic compounds (4-octylphenol and 4-nonylphenol) displayed some ability to displace 3H-E2 binding from ER?? and ?? at high concentrations, but dieldrin and atrazine had little binding activity for both ER subtypes and endosulfan for ER??. The xenobiotics tested generally showed equivalent or greater affinity for ER?? than ER??, whereas natural estrogens had much greater affinity for ER?? than ER??. These observations suggest that results of studies using fish tissue ER extracts must be interpreted with caution, since both ER subtypes may be present, and that the binding of xenoestrogens to SHBG must be taken into account for proper assessment of endocrine

5-HT radioligands for human brain imaging with PET and SPECT

DEFF Research Database (Denmark)

Paterson, Louise M; Kornum, Birgitte R; Nutt, David J

2013-01-01

The serotonergic system plays a key modulatory role in the brain and is the target for many drug treatments for brain disorders either through reuptake blockade or via interactions at the 14 subtypes of 5-HT receptors. This review provides the history and current status of radioligands used...

The serotonin transporter in rhesus monkey brain: comparison of DASB and citalopram binding sites

International Nuclear Information System (INIS)

Zeng Zhizhen; Chen, T.-B.; Miller, Patricia J.; Dean, Dennis; Tang, Y.S.; Sur, Cyrille; Williams, David L.

2006-01-01

We have characterized the interaction of the serotonin transporter ligand [ 3 H]-N,N-dimethyl-2-(2-amino-4-cyanophenylthio)-benzylamine (DASB) with rhesus monkey brain in vitro using tissue homogenate binding and autoradiographic mapping. [ 3 H]-DASB, a tritiated version of the widely used [ 11 C] positron emission tomography tracer, was found to selectively bind to a single population of sites with high affinity (K d =0.20±0.04 nM). The serotonin transporter density (B max ) obtained for rhesus frontal cortex was found to be 66±8 fmol/mg protein using [ 3 H]-DASB, similar to the B max value obtained using the reference radioligand [ 3 H]-citalopram, a well-characterized and highly selective serotonin reuptake inhibitor (83±22 fmol/mg protein). Specific binding sites of both [ 3 H]-DASB and [ 3 H]-citalopram were similarly and nonuniformly distributed throughout the rhesus central nervous system, in a pattern consistent with serotonin transporter localization reported for human brain. Regional serotonin transporter densities, estimated from optical densities of the autoradiographic images, were well correlated between the two radioligands. Finally, DASB and fluoxetine showed dose-dependent full inhibition of [ 3 H]-citalopram binding in a competition autoradiographic study, with K i values in close agreement with those obtained from rhesus brain homogenates. This side-by-side comparison of [ 3 H]-DASB and [ 3 H]-citalopram binding sites in rhesus tissue homogenates and in adjacent rhesus brain slices provides additional support for the use of [ 11 C]-DASB to assess the availability and distribution of serotonin transporters in nonhuman primates

Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

Science.gov (United States)

Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

2017-06-20

Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

Hormone assay

International Nuclear Information System (INIS)

Eisentraut, A.M.

1977-01-01

An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

Synthesis and in vitro evaluation of no-carrier-added 2-(3-(4-(4-[{sup 18}F]fluorobenzyl)piperazin-1-yl)propyl)benzo[d]thiazole, a potential dopamine D{sub 4} receptor radioligand

Energy Technology Data Exchange (ETDEWEB)

Li, Gu-Cai; Zhang, Ru [Hunan Institute of Engineering, Hunan Xiangtan (China). College of Chemistry and Chemical Engineering; Xia, Jiao-yun [Changsha Univ. of Science and Technology (China). School of Chemistry and Biology Engineering

2016-07-01

The dopamine D{sub 4} receptor has been shown to play important roles in some central nervous system pathologies. Specific radioligands for the D{sub 4} receptor may be useful to understand the function of the D{sub 4} receptor and its correlations with various disorders. 2-(3-(4-(4-[{sup 18}F]Fluorobenzyl)piperazin-1-yl)propyl)benzo[d]thiazole ([{sup 18}F]4) was synthesized through a one-pot two-step procedure with total yield 18.6% (decay corrected). The specific activity of the radioligand was 112 GBq/μmol and its radiochemical purity was >95.0%. Its affinity and selectivity for dopamine D{sub 2}-like receptors were measured through in vitro receptor binding evaluation and the K{sub i} value for the D{sub 4} receptor was determined to be 2.9±0.2 nM, and its selectivity for the dopamine D{sub 4} receptor is 709-fold versus D{sub 2long} receptor, 823-fold versus D{sub 3} receptor. The partition coefficient (Log D) of it was determined to be 2.6±0.1 through octanol-water partition experiment. The ligand presents desirable combination of lipophilicity, affinity and selectivity for the dopamine D{sub 4} receptor. The results suggested that the radioligand shows promises for the in vivo study of the dopamine D{sub 4} receptor.

Use of PET and the radioligand [carbonyl-{sup 11}C]WAY-100635 in psychotropic drug development

Energy Technology Data Exchange (ETDEWEB)

Andree, Bengt; Halldin, Christer; Thorberg, Seth-Olov; Sandell, Johan; Farde, Lars

2000-07-01

Positron-emission tomography (PET) provides potential in neuropsychiatric drug development by expanding knowledge of drug action in the living human brain and reducing time consumption and costs. The 5-hydroxytryptamine{sub 1A} (5-HT{sub 1A}) receptor is of central interest as a target for the treatment of anxiety, depression, and schizophrenia. Research on the clinical significance of the 5-HT{sub 1A} receptor now benefits from the highly selective radioligand [carbonyl-{sup 11}C]WAY-100635 (WAY) for quantitative determination of 5-HT{sub 1A} receptors in the primate and human brain in vivo using PET. In this paper, three studies are reviewed to demonstrate the suitability of WAY as radioligand for quantification of central 5-HT{sub 1A} receptors in brain and as an applicable tool for drug development. In the first study a monkey model was used to characterize WAY binding. It was confirmed that the reference ligand 8-OH-DPAT and psychoactive drugs such as buspirone and pindolol occupies 5-HT{sub 1A} receptors in the primate brain. Pindolol is an {beta}-adrenoreceptor antagonist with a high affinity to 5-HT{sub 1A} receptors. This drug has been suggested in combination with selective serotonin reuptake inhibitors for the treatment of depression and was given to healthy males in the second study. Pindolol induced a marked inhibition of central 5-HT{sub 1A} receptors as calculated by the ratio-analysis method and simplified reference tissue model, 2 h after administration of 10 mg as a single oral dose. This observation suggests that pindolol may have a role for the suggested potentiation of selective serotonin reuptake inhibitor treatment of depression. The third study was on robalzotan (NAD-299), a recently developed 5-HT{sub 1A} receptor antagonist and putative drug with implications for the treatment of depression. In the cynomolgus monkey brain, robalzotan in the dose range 2-100 {mu}g/kg IV occupied 5-HT{sub 1A} receptors in a dose-dependent and saturable

[{sup 11}C]-MeJDTic: a novel radioligand for {kappa}-opioid receptor positron emission tomography imaging

Energy Technology Data Exchange (ETDEWEB)

Poisnel, Geraldine; Oueslati, Farhana; Dhilly, Martine; Delamare, Jerome [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France); Perrio, Cecile [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France)], E-mail: perrio@cyceron.fr; Debruyne, Daniele [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France)], E-mail: debruyne@cyceron.fr; Barre, Louisa [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France)

2008-07-15

Introduction: Radiopharmaceuticals that can bind selectively the {kappa}-opioid receptor may present opportunities for staging clinical brain disorders and evaluating the efficiency of new therapies related to stroke, neurodegenerative diseases or opiate addiction. The N-methylated derivative of JDTic (named MeJDTic), which has been recently described as a potent and selective antagonist of {kappa}-opioid receptor in vitro, was labeled with carbon-11 and evaluated for in vivo imaging the {kappa}-opioid receptor in mice. Methods: [{sup 11}C]-MeJDTic was prepared by methylation of JDTic with [{sup 11}C]-methyl triflate. The binding of [{sup 11}C]-MeJDTic to {kappa}-opioid receptor was investigated ex vivo by biodistribution and competition studies using nonfasted male CD1 mice. Results: [{sup 11}C]-MeJDTic exhibited a high and rapid distribution in peripheral organs. The uptake was maximal in lung where the {kappa} receptor is largely expressed. [{sup 11}C]-MeJDTic rapidly crossed the blood-brain barrier and accumulated in the brain regions of interest (hypothalamus). The parent ligand remained the major radioactive compound in brain during the experiment. Chase studies with U50,488 (a {kappa} referring agonist), morphine (a {mu} agonist) and naltrindole (a {delta} antagonist) demonstrated that this uptake was the result of specific binding to the {kappa}-opioid receptor. Conclusion: These findings suggested that [{sup 11}C]-MeJDTic appeared to be a promising selective 'lead' radioligand for {kappa}-opioid receptor PET imaging.

Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor.

Science.gov (United States)

Thorarensen, Atli; Sarver, Ronald W; Tian, Fang; Ho, Andrea; Romero, Donna L; Marotti, Keith R

2007-08-15

In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA. Displacement of dansylsarcosine from HSA by 6 indicated it competed with compounds that bound at site II (ibuprofen binding site) on HSA. Analog 6 also shifted the NMR peaks of an HSA bound oleic acid molecule that itself was affected by compounds that bound at site II. In addition to binding at site II, 6 interacted at site I (warfarin binding site) as indicated by displacement of dansylamide and the shifting of NMR peaks of an HSA bound oleic acid molecule affected by warfarin site binding. Additional evidence for multiple site interaction was discovered when a percentage of 6 could be displaced by either ibuprofen or phenylbutazone. A competition assay was established using 6 to determine relative affinities of other antibacterial inhibitors for HSA.

One-step preparation of [2,3-{sup 3}H]1-aminocyclo-propanecarboxylic acid: a useful ligand for strychnine-insensitive glycine receptors

Energy Technology Data Exchange (ETDEWEB)

Lewin, A.H.; Lamb, P.B. [Research Triangle Inst., Research Triangle Park, NC (United States); Popik, P.; Skolnick, P. [National Insts. of Health, Bethesda, MD (United States)

1994-11-01

Catalytic hydrogenation of 1-aminocyclopropenecarboxylic acid under tritium gas afforded [2,3-{sup 3}H]1-aminocyclopropanecarboxylic acid with specific activity 26 Ci/mmol, determined by a combination of {sup 1}H and {sup 3}H NMR. Pilot radioligand binding assays indicate this compound will be a useful prove for the NMDA receptor-associated strychnine-insensitive glycine receptor. (author).

High-affinity cannabinoid binding site in brain: A possible marijuana receptor

International Nuclear Information System (INIS)

Nye, J.S.

1988-01-01

The mechanism by which delta 9 tetrahydrocannabinol (delta 9 THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5'-Trimethylammonium-delta 8 THC (TMA) is a positively charged analog of delta- 8 THC modified on the 5' carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of [ 3 H]-5'-trimethylammonium-delta- 8 THC ([ 3 H]TMA) to rat neuronal membranes. [ 3 H]TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of [ 3 H]TMA binding activity of approximately 60,000 daltons apparent molecular weight

Parallel force assay for protein-protein interactions.

Science.gov (United States)

Aschenbrenner, Daniela; Pippig, Diana A; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E

2014-01-01

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

Energy Technology Data Exchange (ETDEWEB)

Waser, Beatrice; Reubi, Jean Claude [University of Berne, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, PO Box 62, Berne (Switzerland)

2014-06-15

Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the {sup 125}iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer {sup 125}I-GLP-1(7-36)amide. Receptor autoradiography studies with {sup 125}I-GLP-1(7-36)amide agonist or {sup 125}I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. The antagonist {sup 125}I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer {sup 125}I-GLP-1(7-36)amide. For comparison, {sup 125}I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. The GLP-1 receptor antagonist exendin(9-39) labelled with {sup 125}I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. (orig.)

The typing of Staphylococcus epidermidis by a lectin-binding assay

DEFF Research Database (Denmark)

Jarløv, J O; Hansen, J E; Rosdahl, V T

1992-01-01

A new typing method for Staphylococcus epidermidis was developed. Four biotinylated lectins--wheat germ agglutinin (WGA), soy bean agglutinin (SBA), lentil agglutinin (LCA) and Concanavalin A (ConA)--were added to immobilised whole cells of coagulase-negative staphylococci (CNS) in microtitration...... plates. The amount of bound lectin was measured by peroxidase-conjugated avidin followed by a peroxidase reaction. The method was compared to antibiotic-resistance analysis, phage typing, plasmid DNA profiles and slime production. A total of 113 isolates of CNS from 21 patients was investigated and 71...... strains of CNS, including 64 strains of S. epidermidis, were detected if all typing methods were taken into consideration. If only one typing method was used the highest discriminatory power among the S. epidermidis isolates was obtained with the lectin-binding assay which allowed 49 different strains...

Establishment and characterization of a new and stable collagen-binding assay for the assessment of von Willebrand factor activity

Science.gov (United States)

Ni, Y; Nesrallah, J; Agnew, M; Geske, F J; Favaloro, E J

2013-01-01

Introduction Laboratory diagnosis of von Willebrand disease (VWD) requires determination of both von Willebrand factor (VWF) protein levels and activity. Current VWF activity tests include the ristocetin cofactor assay and the collagen-binding assay (VWF:CB). The goal of this investigation is to characterize a new collagen-binding assay and to determine its effectiveness in identifying VWD. Methods Analytical studies were carried out to characterize the performance of a new VWF:CB ELISA. Additionally, samples from a normal population were tested as were well-characterized type 1 and type 2 VWD samples. Results Repeatability and within-laboratory precision studies resulted in coefficients of variation (CVs) of ≤11%. A linear range of 1–354% (0.01–3.54 IU/mL) was determined, along with a limit of detection and a lower limit of quantitation of 1.6% and 4.0% (0.016 and 0.04 IU/mL), respectively. Samples tested from apparently healthy individuals resulted in a normal range of 54–217% (0.54–2.17 IU/mL). Known VWD type 1 and type 2 samples were also analyzed by the ELISA, with 99% of samples having VWF:CB below the normal reference range and an estimated 96% sensitivity and 87% specificity using a VWF collagen-binding/antigen cutoff ratio of 0.50. Conclusion This new VWF:CB ELISA provides an accurate measure of collagen-binding activity that aids in the diagnosis and differentiation of type 1 from type 2 VWD. PMID:23107512

Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

Science.gov (United States)

Brand, Luise H.; Fischer, Nina M.; Harter, Klaus; Kohlbacher, Oliver; Wanke, Dierk

2013-01-01

WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function. PMID:23975197

PK of immunoconjugate anticancer agent CMD-193 in rats: ligand-binding assay approach to determine in vivo immunoconjugate stability.

Science.gov (United States)

Hussain, Azher; Gorovits, Boris; Leal, Mauricio; Fluhler, Eric

2014-01-01

Antibody-drug conjugates (ADCs) are a new generation of anticancer therapeutics. The objective of this manuscript is to propose a methodology that can be used to assess the stability of the ADCs by using the PK data obtained by ligand-binding assays that measure various components of ADCs. The ligand-binding assays format of different components of ADCs provided unique valuable PK information. The mathematical manipulation of the bioanalytical data provided an insight into the in vivo integrity, indicating that the loading of the calicheamicin on the G193 antibody declines in an apparent slow first-order process. This report demonstrates the value of analyzing various components of the ADC and their PK profiles to better understand the disposition and in vivo stability of ADCs.

Potential of [11C]DASB for measuring endogenous serotonin with PET: binding studies

International Nuclear Information System (INIS)

Lundquist, Pinelopi; Wilking, Helena; Hoeglund, A. Urban; Sandell, Johan; Bergstroem, Mats; Hartvig, Per; Langstroem, Bengt

2005-01-01

The serotonin transporter radioligand [ 11 C]-3-amino-4-(2-dimethylaminomethylphenylsulfanyl)-benzonitrile, or [ 11 C]DASB, was examined in order to assess its potential for measuring fluctuations in endogenous serotonin concentrations with positron emission tomography. Binding characteristics of [ 11 C]DASB and the propensity for serotonin to displace the tracer were explored in rat brain homogenates. Experiments showed that serotonin displaced [ 11 C]DASB in vitro. Ex vivo experiments performed after tranylcypromine injection (3 or 15 mg/kg) showed a dose-dependent trend in radioactivity uptake and suggested that serotonin may compete with [ 11 C]DASB for transporter binding

[123I]Iodobenzamide binding to the rat dopamine D2 receptor in competition with haloperidol and endogenous dopamine - an in vivo imaging study with a dedicated small animal SPECT

International Nuclear Information System (INIS)

Nikolaus, Susanne; Larisch, Rolf; Wirrwar, Andreas; Jamdjeu-Noune, Marlyse; Antke, Christina; Beu, Markus; Mueller, Hans-Wilhelm; Schramm, Nils

2005-01-01

This study assessed [ 123 I]iodobenzamide binding to the rat dopamine D 2 receptor in competition with haloperidol and endogenous dopamine using a high-resolution small animal SPECT. Subsequent to baseline quantifications of D 2 receptor binding, imaging studies were performed on the same animals after pre-treatment with haloperidol and methylphenidate, which block D 2 receptors and dopamine transporters, respectively. Striatal baseline equilibrium ratios (V 3 '' ) of [ 123 I]iodobenzamide binding were 1.42±0.31 (mean±SD). After pre-treatment with haloperidol and methylphenidate, V 3 '' values decreased to 0.54±0.46 (p 123 I]iodobenzamide binding induced by pre-treatment with haloperidol reflects D 2 receptor blockade, whereas the decrease in receptor binding induced by pre-treatment with methylphenidate can be interpreted in terms of competition between [ 123 I]IBZM and endogenous dopamine. Findings show that multiple in vivo measurements of [ 123 I]iodobenzamide binding to D 2 receptors in competition with exogenous and endogenous ligands are feasible in the same animal. This may be of future relevance for the in vivo evaluation of novel radioligands as well as for studying the interrelations between pre- and/or postsynaptic radioligand binding and different levels of endogenous dopamine. (orig.)

Pharmacology of morphine and morphine-3-glucuronide at opioid, excitatory amino acid, GABA and glycine binding sites

Energy Technology Data Exchange (ETDEWEB)

Bartlett, S.E.; Smith, M.T. (Department of Pharmacy, The University of Queensland (Australia)); Dood, P.R. (Clinical Research Centre, Royal Brisbane Hospital Foundation, Brisbane (Australia))

1994-07-01

Morphine in high doses and its major metabolite, morphine-3-glucuronide, cause CNS excitation following intrathecal and intracerebroventricular administration by an unknown mechanism. This study investigated whether morphine and morphine-3-glucuronide interact at major excitatory (glutamate), major inhibitory (GABA or glycine), or opioid binding sites. Homogenate binding assays were performed using specific radioligands. At opioid receptors, morphine-3-glucuronide and morphine caused an equipotent sodium shift, consistent with morphine-3-glucuronide behaving as an agonist. This suggests that morphine-3-glucuronide-mediated excitation is not caused by an interaction at opioid receptors. Morphine-3-glucuronide and morphine caused a weak inhibition of the binding of [sup 3]H-MK801 (non-competitive antagonist) and [sup 125]I-ifenprodil (polyamine site antagonist), but at unphysiologically high concentrations. This suggests that CNS excitation would not result from an interaction of morphine-3-glucuronide and high-dose morphine with these sites on the NMDA receptor. Morphine-3-glucuronide and morphine inhibited the binding of [sup 3]H-muscimol (GABA receptor agonist), [sup 3]H-diazepam and [sup 3]H-flunitraxepam (benzodiazepine agonists) binding very weakly, suggesting the excitatory effects of morphine-3-glucuronide and high-dose morphine are not elicited through GABA[sub A] receptors. Morphine-3-glucuronide and high-dose morphine did not prevent re-uptake of glutamate into presynaptic nerve terminals. In addition, morphine-3-glucuronide and morphine did not inhibit the binding of [sup 3]H-strychnine (glycine receptor antagonist) to synaptic membranes prepared from bovine spinal cord. It is concluded that excitation caused by high-dose morphine and morphine-3-glucuronide is not mediated by an interaction with postsynaptic amino acid receptors. (au) (30 refs.).

The serotonin transporter in rhesus monkey brain: comparison of DASB and citalopram binding sites

Energy Technology Data Exchange (ETDEWEB)

Zeng Zhizhen [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States)]. E-mail: zhizhen_zeng@merck.com; Chen, T.-B. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Miller, Patricia J. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Dean, Dennis [Labeled Compound Synthesis Group, Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900 (United States); Tang, Y.S. [Labeled Compound Synthesis Group, Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900 (United States); Sur, Cyrille [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Williams, David L. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States)

2006-05-15

We have characterized the interaction of the serotonin transporter ligand [{sup 3}H]-N,N-dimethyl-2-(2-amino-4-cyanophenylthio)-benzylamine (DASB) with rhesus monkey brain in vitro using tissue homogenate binding and autoradiographic mapping. [{sup 3}H]-DASB, a tritiated version of the widely used [{sup 11}C] positron emission tomography tracer, was found to selectively bind to a single population of sites with high affinity (K {sub d}=0.20{+-}0.04 nM). The serotonin transporter density (B {sub max}) obtained for rhesus frontal cortex was found to be 66{+-}8 fmol/mg protein using [{sup 3}H]-DASB, similar to the B {sub max} value obtained using the reference radioligand [{sup 3}H]-citalopram, a well-characterized and highly selective serotonin reuptake inhibitor (83{+-}22 fmol/mg protein). Specific binding sites of both [{sup 3}H]-DASB and [{sup 3}H]-citalopram were similarly and nonuniformly distributed throughout the rhesus central nervous system, in a pattern consistent with serotonin transporter localization reported for human brain. Regional serotonin transporter densities, estimated from optical densities of the autoradiographic images, were well correlated between the two radioligands. Finally, DASB and fluoxetine showed dose-dependent full inhibition of [{sup 3}H]-citalopram binding in a competition autoradiographic study, with K {sub i} values in close agreement with those obtained from rhesus brain homogenates. This side-by-side comparison of [{sup 3}H]-DASB and [{sup 3}H]-citalopram binding sites in rhesus tissue homogenates and in adjacent rhesus brain slices provides additional support for the use of [{sup 11}C]-DASB to assess the availability and distribution of serotonin transporters in nonhuman primates.

Biochemical studies of mouse brain tubulin: colchicine binding (DEAE-cellulose filter) assay and subunits (α and β) biosynthesis and degradation (in newborn brain)

International Nuclear Information System (INIS)

Tse, C.F.

1978-01-01

A DEAE-cellulose filter assay, measuring [ 3 H]colchicine bound to colchicine binding protein (CBP) absorbed on filter discs, has been modified to include lM sucrose in the incubation medium for complexing colchicine to CBP in samples before applying the samples to filter discs (single point assay). Due to the much greater stability of colchicine binding capacity in the presence of lM sucrose, multiple time-point assays and least squares linear regression analysis were not necessary for accurate determination of CBP in hybrid mouse brain at different stages of development. The highest concentrations of CBP were observed in the 160,000g supernatant and pellet of newborn brain homogenate. Further studies of the modified filter assay documented that the assay has an overall counting efficiency of 27.3%, that DEAE-cellulose filters bind and retain all tubulin in the assay samples, and that one molecule of colchicine binds approximately one molecule of tubulin dimer. Therefore, millimoles of colchicine bound per milligram total protein can be used to calculate tubulin content. With this technique tubulin content of brain supernatant was found to be 11.9% for newborn, and 7.15% for 11 month old mice. Quantitative densitometry was also used to measure mouse brain supernatant actin content for these two stages. In vivo synthesis and degradation rates of tubulin α and β subunits of two day mouse brain 100,000g supernatant were studied after intracerebral injection of [ 3 H]leucine. Quantitative changes of the ratio of tritium specific activities of tubulin α and β subunits with time were determined. The pattern of change was biphasic. During the first phase the ratio decreased; during the second phase the ratio increased continuously. An interpretation consistent with all the data in this study is that the α subunit is synthesized at a more rapid rate than the β subunit

Parallel force assay for protein-protein interactions.

Directory of Open Access Journals (Sweden)

Daniela Aschenbrenner

Full Text Available Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

Allosteric regulation by oleamide of the binding properties of 5-hydroxytryptamine7 receptors.

Science.gov (United States)

Hedlund, P B; Carson, M J; Sutcliffe, J G; Thomas, E A

1999-12-01

Oleamide belongs to a family of amidated lipids with diverse biological activities, including sleep induction and signaling modulation of several 5-hydroxytryptamine (5-HT) receptor subtypes, including 5-HT1A, 5-HT2A/2C, and 5-HT7. The 5-HT7 receptor, predominantly localized in the hypothalamus, hippocampus, and frontal cortex, stimulates cyclic AMP formation and is thought to be involved in the regulation of sleep-wake cycles. Recently, it was proposed that oleamide acts at an allosteric site on the 5-HT7 receptor to regulate cyclic AMP formation. We have further investigated the interaction between oleamide and 5-HT7 receptors by performing radioligand binding assays with HeLa cells transfected with the 5-HT7 receptor. Methiothepin, clozapine, and 5-HT all displaced specific [3H]5-HT (100 nM) binding, with pK(D) values of 7.55, 7.85, and 8.39, respectively. Oleamide also displaced [3H]5-HT binding, but the maximum inhibition was only 40% of the binding. Taking allosteric (see below) cooperativity into account, a K(D) of 2.69 nM was calculated for oleamide. In saturation binding experiments, oleamide caused a 3-fold decrease in the affinity of [3H]5-HT for the 5-HT7 receptor, without affecting the number of binding sites. A Schild analysis showed that the induced shift in affinity of [3H]5-HT reached a plateau, unlike that of a competitive inhibitor, illustrating the allosteric nature of the interaction between oleamide and the 5-HT7 receptor. Oleic acid, the product of oleamide hydrolysis, had a similar effect on [3H]5-HT binding, whereas structural analogs of oleamide, trans-9,10-octadecenamide, cis-8,9-octadecenamide, and erucamide, did not alter [3H]5-HT binding significantly. The findings support the hypothesis that oleamide acts via an allosteric site on the 5-HT7 receptor regulating receptor affinity.

Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

Directory of Open Access Journals (Sweden)

Kathleen Ingenhoven

2017-07-01

Full Text Available ObjectiveTo develop and validate a method for the detection of binding anti-drug antibodies (ADAs against interferon beta (IFN-β in human serum as part of a European initiative (ABIRISK aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minimize the risk.MethodA two-tiered bridging enzyme-linked immunosorbent assay (ELISA format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled Streptavidin and TMB substrate. Confirmation assay: Screen “putative positive” samples are tested in the presence of excess drug (preincubation of sera with 0.3 µg/mL of soluble IFN-β and percentage of inhibition is calculated.ResultsThe assay is precise, and the sensitivity of the assay was confirmed to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control.ConclusionAn ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its clinical relevance and may allow for the development of predictive tools, key aims within the ABIRISK consortium.

Laboratory automation of high-quality and efficient ligand-binding assays for biotherapeutic drug development.

Science.gov (United States)

Wang, Jin; Patel, Vimal; Burns, Daniel; Laycock, John; Pandya, Kinnari; Tsoi, Jennifer; DeSilva, Binodh; Ma, Mark; Lee, Jean

2013-07-01

Regulated bioanalytical laboratories that run ligand-binding assays in support of biotherapeutics development face ever-increasing demand to support more projects with increased efficiency. Laboratory automation is a tool that has the potential to improve both quality and efficiency in a bioanalytical laboratory. The success of laboratory automation requires thoughtful evaluation of program needs and fit-for-purpose strategies, followed by pragmatic implementation plans and continuous user support. In this article, we present the development of fit-for-purpose automation of total walk-away and flexible modular modes. We shared the sustaining experience of vendor collaboration and team work to educate, promote and track the use of automation. The implementation of laboratory automation improves assay performance, data quality, process efficiency and method transfer to CRO in a regulated bioanalytical laboratory environment.

Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

Science.gov (United States)

Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram

2002-02-01

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

Insect Ryanodine Receptor: Distinct But Coupled Insecticide Binding Sites for [N-C3H3]Chlorantraniliprole, Flubendiamide, and [3H]Ryanodine

OpenAIRE

Isaacs, André K.; Qi, Suzhen; Sarpong, Richmond; Casida, John E.

2012-01-01

Radiolabeled anthranilic diamide insecticide [N-C3H3]chlorantraniliprole was synthesized at high specific activity and compared with phthalic diamide insecticide flubendiamide and [3H]ryanodine in radioligand binding studies with house fly muscle membranes to provide the first direct evidence with a native insect ryanodine receptor that the major anthranilic and phthalic diamide insecticides bind at different allosterically coupled sites, i.e. there are three distinct Ca2+-release channel tar...

In vivo and in vitro binding assay of 153Sm-EDTMP

International Nuclear Information System (INIS)

Chen Daming; Wang Yuqing; Jin Xiaohai; Fan Hongqiang; Bai Hongsheng; Jia Bin; Zhang Jingming

1999-01-01

With the waters ultra hydrogel TM 120 μm hplc column (7.7 mm x 300 mm), several experiments have been finished, including the in vitro binding assay of 153 Sm-EDTMP, 153 SmCl 3 with the Cys, BSA, mouse plasma; HPLC analysis of the urine and the extracting solution of liver homogenate after having injected the 153 Sm-EDTMP and 153 SmCl 3 2h; HPLC analysis of the production ( 153 Sm-EDTMP) radiation self-decomposition with large dose. For the HPLC analysis, the condition is the mobile phase of 0.85 mol/mL PBS (pH7.5), flow rate of 0.5 mL/min, sampling of 15 μL. The results are following: (1) The 153 SmCl 3 not only is able to bind with the mouse plasma in vitro, but also is able to be absorbed by liver in vivo; (2) 153 Sm-EDTMP is not bind with the mouse plasma, the Cys and BSA in vitro and vivo; 153 Sm-EDTMP is not found in the extracted solution of liver homogenate at n(EDTMP): n(Sm) ≥ 5:1; 153 Sm-EDTMP is not decomposed in the urine, 1 53 Sm-EDTMP is stable in vivo; (3) 153 Sm-EDTMP radiation self-decomposition is not detected with large dose in the term of validity (6 d), but two small degradation peaks have been found in the production solution after 60 d, the radiochemistry purity of production is always great than 98% during the period

Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

Science.gov (United States)

Ketelslegers, J M; Catt, K J

1978-07-03

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.

99Tcm labelling and in vitro binding of dextran-somatostatin conjugate

International Nuclear Information System (INIS)

Cui Haiping; China Inst. of Atomic Energy, Beijing; Zhai Shizhen; Du Jin; Beijing Univ., Beijing

2006-01-01

Natural somatostatin and dextran-20 are used to synthesis somatostatin-dextran (SMS-Dx 20 ). The in vitro somatostatin receptor competition binding study of somatostatin-dextran is carried out by using rate brain cortex membranes (express somatostatin receptor type 2) and 125 I-Tyr 3 -Octreotide as a radioligand. The somatostatin-dextran conjugate is then labelled with 99 Tc m using SnCl 2 as reduce agent and tested for its in vitro binding properties. The somatostatin-dextran conjugate shows high somatostatin receptor binding affinity, i.e. in the same IC 50 value as the reference ligand Octreotide (IC 50 ∼5.95 nmol/L). The labelling efficiency is more than 85%. The specific binding of 99 Tc m labeled somatostatin-dextran conjugate is 25%-40%. The somatostatin-dextran conjugate is worthy of further investigation for 99 Tc m radiolabeling with diagnostic possibilities for somatostatin receptor positive tumors. (authors)

Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

International Nuclear Information System (INIS)

James, I.F.; Goldstein, A.

1984-01-01

A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, [ 3 H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [ 3 H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites

Effects of saw palmetto extract on micturition reflex of rats and its autonomic receptor binding activity.

Science.gov (United States)

Oki, Tomomi; Suzuki, Mayumi; Nishioka, Yasuhiko; Yasuda, Akio; Umegaki, Keizo; Yamada, Shizuo

2005-04-01

We examined the effects of saw palmetto extract (SPE) on the rat micturition reflex and on autonomic receptors in the lower urinary tract. The effect of SPE was examined on cystometrograms of anesthetized rats induced by intravesical infusion of saline or 0.1% acetic acid. SHR/NDmc-cp (cp/cp) rats received repeat oral administration of SPE and nighttime urodynamic function was determined. The autonomic receptor binding activity of SPE in the rat bladder and prostate was examined by radioligand binding assay. Intraduodenal administration of SPE (60 mg/kg) in anesthetized rat cystometry caused a significant increase in the micturition interval, micturition volume and bladder capacity during intravesical saline infusion. Also, similar administration of SPE at doses of 12 and 20 mg/kg significantly reversed the shortened micturition interval as well as the decreased micturition volume and bladder capacity due to 0.1% acetic acid infusion in a dose dependent manner. In conscious SHR/NDmc-cp (cp/cp) rats repeat oral administration of SPE (6 mg/kg daily) constantly increased the micturition interval and concomitantly decreased voiding frequency. SPE inhibited specific binding of [H]NMS ([N-methyl-H]scopolamine methyl chloride) (bladder) and [H]prazosin (prostate) with IC50 values of 46.1 and 183 microg/ml, respectively. SPE significantly alleviates urodynamic symptoms in hyperactive rat bladders by increasing bladder capacity and subsequently prolonging the micturition interval. Our data may support the clinical efficacy of SPE for the treatment of lower urinary tract symptoms.

Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC.

Science.gov (United States)

Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Gapsys, Vytautas; Ucurum, Zöhre; de Groot, Bert L; Fotiadis, Dimitrios

2016-09-13

Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.

Dopamine receptors in the guinea-pig heart. A binding study

International Nuclear Information System (INIS)

Sandrini, M.; Benelli, A.; Baraldi, M.

1984-01-01

The binding of dopaminergic agonists and antagonists to guinea-pig myocardial membrane preparations was studied using 3 H-dopamine and 3 H-spiperone as radioligand. 3 H-Dopamine bound specifically to heart membranes while 3 H-spiperone did not. A Scatchard analysis of 3 H-dopamine binding showed a curvilinear plot indicating the presence of two dopamine receptor populations that we have termed high- (K/sub d/ = 1.2 nM, B/sub mx/ = 52.9 fmol/mg prot.) and low- (K/sub d/ = 11.8 nM, B/sub mx/ = 267.3 fmol/gm prot.) affinity binding sites, respectively. The charactization of the high-affinity component of 3 H-dopamine binding indicated that the binding is rapid, saturable, stereospecific, pH- and temperature-dependent, and displaced by dopaminergic agonists and antagonists known to act similarly in vivo. The finding that pretreatment with dibenamine (which has been described as an α-adrenoceptor irreversible blocker) did not affect the binding of dopamine to cardiac membrane preparations suggests that α-adrenoceptors and dopamine receptors have separate recognition sites in the heart. It is concluded that 3 H-dopamine binds to specific dopamine receptors in the heart of guinea-pigs

Derisking the Cu-Mediated 18F-Fluorination of Heterocyclic Positron Emission Tomography Radioligands.

Science.gov (United States)

Taylor, Nicholas J; Emer, Enrico; Preshlock, Sean; Schedler, Michael; Tredwell, Matthew; Verhoog, Stefan; Mercier, Joel; Genicot, Christophe; Gouverneur, Véronique

2017-06-21

Molecules labeled with fluorine-18 ( 18 F) are used in positron emission tomography to visualize, characterize and measure biological processes in the body. Despite recent advances in the incorporation of 18 F onto arenes, the development of general and efficient approaches to label radioligands necessary for drug discovery programs remains a significant task. This full account describes a derisking approach toward the radiosynthesis of heterocyclic positron emission tomography (PET) radioligands using the copper-mediated 18 F-fluorination of aryl boron reagents with 18 F-fluoride as a model reaction. This approach is based on a study examining how the presence of heterocycles commonly used in drug development affects the efficiency of 18 F-fluorination for a representative aryl boron reagent, and on the labeling of more than 50 (hetero)aryl boronic esters. This set of data allows for the application of this derisking strategy to the successful radiosynthesis of seven structurally complex pharmaceutically relevant heterocycle-containing molecules.

Value of {sup 111}In-DOTA-lanreotide and {sup 111}In-DOTA-DPhe{sup 1}-Tyr{sup 3}-octreotide in differentiated thyroid cancer: results of in vitro binding studies and in vivo comparison with {sup 18}F-FDG PET

Energy Technology Data Exchange (ETDEWEB)

Rodrigues, Margarida; Virgolini, Irene [Lainz Hospital, Institute of Nuclear Medicine, Vienna (Austria); University Clinic for Nuclear Medicine, Innsbruck (Austria); Traub-Weidinger, Tatjana [University Clinic for Nuclear Medicine, Innsbruck (Austria); University Hospital, Department of Nuclear Medicine, Vienna (Austria); Leimer, Maria; Li, Shuren; Dudczak, Robert [University Hospital, Department of Nuclear Medicine, Vienna (Austria); Andreae, Fritz [University Clinic for Nuclear Medicine, Innsbruck (Austria); Angelberger, Peter [Austrian Research Center, Department of Radiochemistry, Seibersdorf (Austria)

2005-10-01

Radioiodine-negative thyroid cancer presents diagnostic and therapeutic difficulties, warranting the implementation of new imaging and treatment strategies. The purpose of this study was twofold. First, we investigated in vitro the binding characteristics of {sup 111}In-DOTA-lanreotide ({sup 111}In-DOTA-LAN) and {sup 111}In-DOTA-DPhe{sup 1}-Tyr{sup 3}-octreotide ({sup 111}In-DOTA-TOC) to cells derived from differentiated thyroid cancer (DTC). Second, we evaluated the value of somatostatin receptor (SSTR) scintigraphy with these radioligands, as compared with {sup 18}F-fluorodeoxyglucose (FDG) positron emission tomography (PET), for the detection of tumour lesions in DTC patients. Binding of {sup 111}In-DOTA-LAN and {sup 111}In-DOTA-TOC to cells isolated from surgically removed thyroid tissue was evaluated in vitro by performing saturation and displacement studies. Eighteen DTC patients with elevated thyroglobulin (12 radioiodine-negative, six radioiodine-positive) were investigated with {sup 111}In-DOTA-LAN, {sup 111}In-DOTA-TOC and {sup 18}F-FDG PET scans. Large numbers of SSTR binding sites for {sup 111}In-DOTA-LAN and {sup 111}In-DOTA-TOC were found on the cells investigated. Both SSTR radioligands exhibited a high binding affinity for these SSTR binding sites. {sup 111}In-DOTA-LAN and {sup 111}In-DOTA-TOC scintigraphy detected 37 and 33 lesions, respectively, in 17 (94%) patients each, whereas {sup 18}F-FDG PET revealed 30 lesions in 15 (83%) patients. Uptake of both SSTR radioligands was found in several radioiodine-negative sites. No striking differences in lesion imaging by {sup 111}In-DOTA-LAN and {sup 111}In-DOTA-TOC were found. In both radioiodine-negative and radioiodine-positive patients, more lesions were SSTR-positive/{sup 18}F-FDG-negative than were {sup 18}F-FDG-positive/SSTR-negative. Adding a SSTR scan with these radioligands to the diagnostic work-up increases the diagnostic capacity in DTC, and should be considered particularly in radioiodine

Iodine-123 N-methyl-4-iododexetimide: a new radioligand for single-photon emission tomographic imaging of myocardial muscarinic receptors

International Nuclear Information System (INIS)

Hicks, R.J.; Kassiou, M.; Eu, P.; Katsifis, A.G.; Garra, M.; Power, J.; Najdovski, L.; Lambrecht, R.M.

1995-01-01

Cardiac muscarinic receptor ligands suitable for positron emission tomography have previously been characterised. Attempts to develop radioligands of these receptors suitable for single-photon emission tomographic (SPET) imaging have not been successful due to high lung retention and high non-specific binding of previously investigated potential tracers. The purpose of this study was to evaluate the biodistribution and in vivo imaging characteristics of a new radiopharmaceutical, [ 123 I]N-methyl-4-iododexetimide. Biodistribution studies performed in rats showed high cardiac uptake (2.4% ID/g) 10 min after injection with a heart to lung activity ratio of 5:1. Specificity and stereoselectivity of cardiac binding were demonstrated using blocking experiments in rats. Dynamic imaging studies in anaesthetised greyhounds demonstrated rapid and high myocardial uptake and low lung binding with stable heart to lung activity ratios of >2.5:1 between 10 and 30 min, making SPET imaging feasible. Administration of an excess of an unlabelled muscarinic antagonist, methyl-quinuclidinyl benzylate rapidly displaced myocardial activity to background levels and the pharmacologically inactive enantiomer, [ 123 I]N-methyl-4-iodolevetimide, had no detectable cardiac uptake, indicating specific and stereoselective muscarinic receptor binding. SPET revealed higher activity in the inferior than in the anterior wall, this being consistent with previously described regional variation of cardiac parasympathetic innervation. [ 123 I]N-methyl-4-iododexetimide shows promise as an imaging agent for muscarinic receptor distribution in the heart and may be helpful in evaluating diverse cardiac diseases associated with altered muscarinic receptor function, including heart failure and diabetic heart disease. (orig.)

Drug binding affinities and potencies are best described by a log-normal distribution and use of geometric means

International Nuclear Information System (INIS)

Stanisic, D.; Hancock, A.A.; Kyncl, J.J.; Lin, C.T.; Bush, E.N.

1986-01-01

(-)-Norepinephrine (NE) is used as an internal standard in their in vitro adrenergic assays, and the concentration of NE which produces a half-maximal inhibition of specific radioligand binding (affinity; K/sub I/), or half-maximal contractile response (potency; ED 50 ) has been measured numerous times. The goodness-of-fit test for normality was performed on both normal (Gaussian) or log 10 -normal frequency histograms of these data using the SAS Univariate procedure. Specific binding of 3 H-prazosin to rat liver (α 1 -), 3 H rauwolscine to rat cortex (α 2 -) and 3 H-dihydroalprenolol to rat ventricle (β 1 -) or rat lung (β 2 -receptors) was inhibited by NE; the distributions of NE K/sub I/'s at all these sites were skewed to the right, with highly significant (p 50 's of NE in isolated rabbit aorta (α 1 ), phenoxybenzamine-treated dog saphenous vein (α 2 ) and guinea pig atrium (β 1 ). The vasorelaxant potency of atrial natriuretic hormone in histamine-contracted rabbit aorta also was better described by a log-normal distribution, indicating that log-normalcy is probably a general phenomenon of drug-receptor interactions. Because data of this type appear to be log-normally distributed, geometric means should be used in parametric statistical analyses

Synthesis and evaluation of iodine-123 labelled tricyclic tropanes as radioligands for the serotonin transporter

International Nuclear Information System (INIS)

Quinlivan, Mitchell; Mattner, Filomena; Papazian, Vahan; Zhou, Jia; Katsifis, Andrew; Emond, Patrick; Chalon, Sylvie; Kozikowski, Alan; Guilloteau, Denis; Kassiou, Michael

2003-01-01

The tricyclic tropane analogues (1S,3S,6R,10S)-(Z)-10-(benzoyloxymethyl)-9-(3-chloro-4-iodobenzylidene)-7 -azatricyclo[4.3.1.0 3,7 ]decane, 1, and (1S,3S,6R,10S)-(Z)-9-(3-chloro-4-iodobenzylidene)-7-azatricyclo[4.3.1.0 3,7 ] = decane-10-carboxylic acid methyl ester, 2, have been shown to be potent and selective serotonin transporter (SERT) ligands. They possess nanomolar affinity for the SERT (Ki = 0.06 nM and 1.8 nM respectively) and are suitable for radiolabelling using iodine-123. In the present study we prepared [ 123 I]1 and [ 123 I]2 from the appropriate tributylstannane precursors using acidic media with chloramine-T as the oxidising agent. The radiochemical yield obtained for [ 123 I]1 varied between 50-60% while for [ 123 I]2 the range was 65-80%. Both radioligands were obtained with radiochemical purity > 97% and specific activity estimated to be > 185 GBq/μmol. The biodistribution of [ 123 I]1 demonstrated low degree of brain penetration at 5 min (0.14%ID/g) with a homogenous distribution. The radioactivity cleared quickly from all brain regions with no preferential localization. In comparison, [ 123 I]2 demonstrated on average a higher brain uptake at 5 min (0.5%ID/g). However the distribution of radioactivity was homogenous and cleared to levels similar to [ 123 I]1 at 1 hr post-injection. Pre-administration of citalopram failed to show any significant inhibition of [ 123 I]2 uptake in the rat brain. The high lipophilicity of 1 and 2 (HPLC-derived log P 7.4 values of 6.41 and 4.25 respectively) and in vivo metabolism, seen by high thyroid uptake would explain the absence of any specific binding observed in the rat brain. In view of these results [ 123 I]1 and [ 123 I]2 do not appear to be suitable radioligands for in vivo studies of the SERT

Synthesis and evaluation of iodine-123 labelled tricyclic tropanes as radioligands for the serotonin transporter

Energy Technology Data Exchange (ETDEWEB)

Quinlivan, Mitchell; Mattner, Filomena; Papazian, Vahan; Zhou, Jia; Katsifis, Andrew; Emond, Patrick; Chalon, Sylvie; Kozikowski, Alan; Guilloteau, Denis; Kassiou, Michael E-mail: mkassiou@med.usyd.edu.au

2003-10-01

The tricyclic tropane analogues (1S,3S,6R,10S)-(Z)-10-(benzoyloxymethyl)-9-(3-chloro-4-iodobenzylidene)-7 -azatricyclo[4.3.1.0{sup 3,7}]decane, 1, and (1S,3S,6R,10S)-(Z)-9-(3-chloro-4-iodobenzylidene)-7-azatricyclo[4.3.1.0{sup 3,7}] = decane-10-carboxylic acid methyl ester, 2, have been shown to be potent and selective serotonin transporter (SERT) ligands. They possess nanomolar affinity for the SERT (Ki = 0.06 nM and 1.8 nM respectively) and are suitable for radiolabelling using iodine-123. In the present study we prepared [{sup 123}I]1 and [{sup 123}I]2 from the appropriate tributylstannane precursors using acidic media with chloramine-T as the oxidising agent. The radiochemical yield obtained for [{sup 123}I]1 varied between 50-60% while for [{sup 123}I]2 the range was 65-80%. Both radioligands were obtained with radiochemical purity > 97% and specific activity estimated to be > 185 GBq/{mu}mol. The biodistribution of [{sup 123}I]1 demonstrated low degree of brain penetration at 5 min (0.14%ID/g) with a homogenous distribution. The radioactivity cleared quickly from all brain regions with no preferential localization. In comparison, [{sup 123}I]2 demonstrated on average a higher brain uptake at 5 min (0.5%ID/g). However the distribution of radioactivity was homogenous and cleared to levels similar to [{sup 123}I]1 at 1 hr post-injection. Pre-administration of citalopram failed to show any significant inhibition of [{sup 123}I]2 uptake in the rat brain. The high lipophilicity of 1 and 2 (HPLC-derived log P{sub 7.4} values of 6.41 and 4.25 respectively) and in vivo metabolism, seen by high thyroid uptake would explain the absence of any specific binding observed in the rat brain. In view of these results [{sup 123}I]1 and [{sup 123}I]2 do not appear to be suitable radioligands for in vivo studies of the SERT.

Binding assays with streptavidin-functionalized superparamagnetic nanoparticles and biotinylated analytes using fluxgate magnetorelaxometry

International Nuclear Information System (INIS)

Heim, Erik; Ludwig, Frank; Schilling, Meinhard

2009-01-01

Binding assays based on the magnetorelaxation of superparamagnetic nanoparticles as markers are presented utilizing a differential fluxgate system. As ligand and receptor, streptavidin and biotin, respectively, are used. Superparamagnetic nanoparticles are functionalized with streptavidin and bound to two types of biotinylated analytes: agarose beads and bovine serum (BSA) proteins. The size difference of the two analytes causes a different progress of the reaction. As a consequence, the analysis of the relaxation signal is carried out dissimilarly for the two analytes. In addition, we studied the reaction kinetics of the two kinds of analytes with the fluxgate system.

Identification and characterization of alpha 1 adrenergic receptors in the canine prostate using [125I]-Heat

International Nuclear Information System (INIS)

Lepor, H.; Baumann, M.; Shapiro, E.

1987-01-01

We have recently utilized radioligand receptor binding methods to characterize muscarinic cholinergic and alpha adrenergic receptors in human prostate adenomas. The primary advantages of radioligand receptor binding methods are that neurotransmitter receptor density is quantitated, the affinity of unlabelled drugs for receptor sites is determined, and receptors can be localized using autoradiography on slide-mounted tissue sections. Recently, [ 125 I]-Heat, a selective and high affinity ligand with high specific activity (2200 Ci/mmole) has been used to characterize alpha 1 adrenergic receptors in the brain. In this study alpha 1 adrenergic receptors in the dog prostate were characterized using [ 125 I]-Heat. The Scatchard plots were linear indicating homogeneity of [ 125 I]-Heat binding sites. The mean alpha 1 adrenergic receptor density determined from these Scatchard plots was 0.61 +/- 0.07 fmol/mg. wet wt. +/- S.E.M. The binding of [ 125 I]-Heat to canine prostate alpha 1 adrenergic binding sites was of high affinity (Kd = 86 +/- 19 pM). Steady state conditions were reached following an incubation interval of 30 minutes and specific binding and tissue concentration were linear within the range of tissue concentrations assayed. The specificity of [ 125 I]-Heat for alpha 1 adrenergic binding sites was confirmed by competitive displacement assays using unlabelled clonidine and prazosin. Retrospective analysis of the saturation experiments demonstrated that Bmax can be accurately calculated by determining specific [ 125 I]-Heat binding at a single ligand concentration. [ 125 I]-Heat is an ideal ligand for studying alpha 1 adrenergic receptors in the prostate and its favorable properties should facilitate the autoradiographic localization of alpha 1 adrenergic receptors in the prostate

Biochemical studies of mouse brain tubulin: colchicine binding (DEAE-cellulose filter) assay and subunits ( α and β) biosynthesis and degradation (in newborn brain)

Energy Technology Data Exchange (ETDEWEB)

Tse, Cek-Fyne [Univ. of Rochester, NY (United States)

1978-01-01

A DEAE-cellulose filter assay, measuring (³H)colchicine bound to colchicine binding protein (CBP) absorbed on filter discs, has been modified to include lM sucrose in the incubation medium for complexing colchicine to CBP in samples before applying the samples to filter discs (single point assay). Due to the much greater stability of colchicine binding capacity in the presence of lM sucrose, multiple time-point assays and least squares linear regression analysis were not necessary for accurate determination of CBP in hybrid mouse brain at different stages of development. The highest concentrations of CBP were observed in the 160,000g supernatant and pellet of newborn brain homogenate. Further studies of the modified filter assay documented that the assay has an overall counting efficiency of 27.3%, that DEAE-cellulose filters bind and retain all tubulin in the assay samples, and that one molecule of colchicine binds approximately one molecule of tubulin dimer. Therefore, millimoles of colchicine bound per milligram total protein can be used to calculate tubulin content. With this technique tubulin content of brain supernatant was found to be 11.9% for newborn, and 7.15% for 11 month old mice. Quantitative densitometry was also used to measure mouse brain supernatant actin content for these two stages. In vivo synthesis and degradation rates of tubulin ..cap alpha.. and ..beta.. subunits of two day mouse brain 100,000g supernatant were studied after intracerebral injection of (³H)leucine. Quantitative changes of the ratio of tritium specific activities of tubulin ..cap alpha.. and ..beta.. subunits with time were determined. The pattern of change was biphasic. During the first phase the ratio decreased; during the second phase the ratio increased continuously. An interpretation consistent with all the data in this study is that the ..cap alpha.. subunit is synthesized at a more rapid rate than the ..beta.. subunit. (ERB)

Comparative study on collagen-binding enzyme-linked immunosorbent assay and ristocetin cofactor activity assays for detection of functional activity of von Willebrand factor.

Science.gov (United States)

Turecek, Peter L; Siekmann, Jürgen; Schwarz, Hans Peter

2002-04-01

For more than two decades, the ristocetin cofactor (RCo) assay, which measures the von Willebrand factor (vWF)-mediated agglutination of platelets in the presence of the antibiotic ristocetin, has been the most common method for measuring the functional activity of vWF. There is, however, general agreement among clinical analysts that this method has major practical disadvantages in performance and reproducibility. Today, collagen-binding assays (CBA) based on the enzyme-linked immunosorbent assay (ELISA) technique that measure the interaction of vWF and collagen are an alternative analytic procedure based on a more physiological function than that of the RCo procedure. We used both assay systems in a comparative study to assess the functional activity of vWF in plasma as well as in therapeutic preparations. We measured RCo activities of plasma from healthy donors and patients with different types of von Willebrand disease (vWD) and of vWF as a drug substance in factor (F) VIII/vWF concentrates using both the aggregometric and the macroscopic methods. In addition, we measured collagen-binding activity (vWF:CB) using a recently developed commercially available CBA system. To investigate the relation between the structure and the functional activity of vWF, we isolated vWF species with different numbers of multimers from FVIII/vWF concentrates by affinity chromatography on immobilized heparin. The vWF:RCo and vWF:CB of the different fractions were measured, and the multimeric structure of vWF was analyzed by sodium dodecyl sulfate (SDS) agarose gel electrophoresis. (vWF:CB and vWF:RCo are part of the nomenclature proposed by the International Society on Thrombosis and Hemostasis Scientific and Standardization Committee [ISTH SSC] subcommittee on von Willebrand factor, in Maastricht, Germany, June 16, 2000.) Measurement of functional vWF activity by CBA can be carried out with substantially higher interassay reproducibility than can measurement of RCo. Both assay

Development of [3H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([3H]PSB-12150): A Useful Tool for Studying GPR17

Science.gov (United States)

2014-01-01

The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [3H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities. PMID:24900835

Development of [(3)H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([(3)H]PSB-12150): A Useful Tool for Studying GPR17.

Science.gov (United States)

Köse, Meryem; Ritter, Kirsten; Thiemke, Katharina; Gillard, Michel; Kostenis, Evi; Müller, Christa E

2014-04-10

The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [(3)H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities.

Clinical experience with a radioreceptor assay for TSH-binding inhibiting immunoglobulins (TBII)

International Nuclear Information System (INIS)

Heberling, H.J.; Bierwolf, B.; Lohmann, D.

1988-01-01

The aim was evaluate the clinical value of a commercial kit for determination of TSH-binding inhibiting immunoglobulin (TBII). 47 of 50 patients with untreated hyperthyroid Graves' disease were TBII positive (sensitivity 94%). TBII was in the normal range in all normal volunteers and in patients with simple goiter, thyroid cancer and in most cases of nonimmunogenic hyperthyreoidism (19 of 22). After 12 months antithyroid drug therapy with methimazole of 21 patients the prevalence of positive TBII findings was 28%. In contrast to this, 50 percent of the patients had increased microsomal antibodies at the end of therapy. The determination of TBII by TRAK assay proved to be a sensitive, specific and practical method. The assay can be used to differentiate between hyperthyreoidism of autoimmune or nonimmunogenic origin. Even so this method seems to be helpful for the follow-up during medical treatment of patients with Graves' disease. The results indicate that persistence of increased TBII levels are markers of active Graves' disease and suggest that in this situation ablative measures should be performed. Normalization of TBII on the end of a longstanding antithyroid therapy does not exclude the possibility of relapse in the further course. (author)

Neurofunctional imaging of the pancreas utilizing the cholinergic PET radioligand [18F]4-fluorobenzyltrozamicol

International Nuclear Information System (INIS)

Clark, P.B.; Gage, H.D.; Brown-Proctor, C.; Buchheimer, N.; Morton, K.A.; Calles-Escandon, J.; Mach, R.H.

2004-01-01

The pancreas is one of the most heavily innervated peripheral organs in the body. Parasympathetic and sympathetic neurons terminate in the pancreas and provide tight control of endocrine and exocrine functions. The aim of this study was to determine whether the pancreas can be imaged with a radioligand that binds to specific neuroreceptors. Using fluorine-18 4-fluorobenzyltrozamicol (FBT), which binds to the presynaptic vesicular acetylcholine transporter, positron emission tomography scans were performed in four adult mice, two adult rhesus monkeys, and one adult human. In these mammals, the pancreas is intensely FBT avid, with uptake greater than in any other organ at 30, 60, and 90 min. The maximum standardized uptake value (SUV) ratios of pancreas to liver, for example, ranged from 1.4 to 1.7 in rhesus monkeys (mean 1.6; median 1.7) and from 1.9 to 4.7 (mean 3.24; median 3.02) in mice. The maximum SUV ratio of pancreas to liver in the human was 1.8. These data suggest that neuroreceptor imaging of the pancreas in vivo is feasible in animal models and humans. This imaging could allow researchers to interrogate functions under control of the autonomic nervous system in the pancreas, with applications possible in transplanted and native pancreata. Also, as beta cell function is intimately related to parasympathetic cholinergic input, FBT activity in the pancreas may correlate with insulin-producing beta cell mass. This could ultimately provide a method of in vivo imaging in animal models and humans for diabetes research. (orig.)

QSAR of estrogen receptor modulators: exploring selectivity requirements for ER(alpha) versus ER(beta) binding of tetrahydroisoquinoline derivatives using E-state and physicochemical parameters.

Science.gov (United States)

Mukherjee, Subhendu; Saha, Achintya; Roy, Kunal

2005-02-15

Considering importance of developing selective estrogen receptor modulators (SERMs), the present paper explores selectivity requirements of tetrahydroisoquinoline derivatives for binding with ER(alpha) versus ER(beta) receptors using E-state index and physicochemical parameters. The best model [n=21, Q(2)=0.512, R(a)(2)=0.613, R=0.819, F=11.6 (df 3,17)] for ER(alpha) binding data obtained from radioligand binding assay showed importance of C(1), C(15) and lipophilicity (logP) while the best model [n=21, Q(2)=0.768, R(a)(2)=0.796, R=0.904, F=40.1 (df 2,18)] for ER(beta) binding data showed importance of C(1) and molar refractivity (MR). While modeling ER(alpha)/ER(beta) selectivity [n=21, Q(2)=0.695, R(a)(2)=0.739, R=0.882, F=19.8 (df 3,17)], C(1), C(15) and molar refractivity were found to be significant contributors. The data obtained from cellular transcription assay were also modeled. In case of ER(alpha), the best equation involving E-state values of C(1) and C(14) and logP explained 62.1% of the variance while the best equation for ER(beta) involving E-state values of C(1) and C(15) and MR explained 64.6% of the variance of the response variable. In case of ER(alpha)/ER(beta) selectivity, the best equation involving E-state values of O(8), C(14) and N(27) showed 48.3% explained variance, which increased to 63.5% on deletion of single outlier. From the analysis it appears that the nitrogen atom of the aminoethoxyphenyl substituent and 6-hydroxy substituent of the tetrahydroisoquinoline nucleus play important roles for ER(alpha)/ER(beta) selectivity in addition to R(1) and R(2) substituents.

A FRET-based high throughput screening assay to identify inhibitors of anthrax protective antigen binding to capillary morphogenesis gene 2 protein.

Directory of Open Access Journals (Sweden)

Michael S Rogers

Full Text Available Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA, a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2 protein and tumor endothelial marker 8 (TEM8. Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein-protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.

The 5-HT1A Receptor PET Radioligand 11C-CUMI-101 Has Significant Binding to α1-Adrenoceptors in Human Cerebellum, Limiting Its Use as a Reference Region.

Science.gov (United States)

Shrestha, Stal S; Liow, Jeih-San; Jenko, Kimberly; Ikawa, Masamichi; Zoghbi, Sami S; Innis, Robert B

2016-12-01

Prazosin, a potent and selective α 1 -adrenoceptor antagonist, displaces 25% of 11 C-CUMI-101 ([O-methyl- 11 C]2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)dione) binding in monkey cerebellum. We sought to estimate the percentage contamination of 11 C-CUMI-101 binding to α 1 -adrenoceptors in human cerebellum under in vivo conditions. In vitro receptor-binding techniques were used to measure α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors in human, monkey, and rat cerebellum. Binding potential (maximum number of binding sites × affinity [(1/dissociation constant]) was determined using in vitro homogenate binding assays in human, monkey, and rat cerebellum. 3 H-prazosin was used to determine the maximum number of binding sites, as well as the dissociation constant of 3 H-prazosin and the inhibition constant of CUMI-101. α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors were similar across species. Cerebellar binding potentials were 3.7 for humans, 2.3 for monkeys, and 3.4 for rats. Reasoning by analogy, 25% of 11 C-CUMI-101 uptake in human cerebellum reflects binding to α 1 -adrenoceptors, suggesting that the cerebellum is of limited usefulness as a reference tissue for quantification in human studies. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Autoantibodies Enhance Agonist Action and Binding to Cardiac Muscarinic Receptors in Chronic Chagas’ Disease

Science.gov (United States)

Hernández, Ciria C.; Nascimento, José H.; Chaves, Elen A.; Costa, Patrícia C.; Masuda, Masako O.; Kurtenbach, Eleonora; Campos de Carvalho, Antônio C.; Giménez, Luis E.

2009-01-01

Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M2-muscarinic acetylcholine receptors (M2AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M2AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M2AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [3H]-N-methyl scopolamine ([3H]-NMS) in allosterism binding assays. A peptide corresponding to the M2AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [3H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [3H]-NMS dissociation right shifted from an IC50 of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 × 10−8, 1.33 × 10−7, and 2.0 × 10−7 mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M2AChRs as a positive cooperativity effect on acetylcholine action. PMID:18702010

Platelet binding and biodistribution of [99mTc]rBitistatin in animal species and humans

International Nuclear Information System (INIS)

Knight, Linda C.; Romano, Jan E.; Bright, Lewis T.; Agelan, Alexis; Kantor, Steven; Maurer, Alan H.

2007-01-01

Introduction: 99m Tc recombinant bitistatin (rBitistatin) is a radioligand for α IIb β 3 (glycoproteins IIb/IIIa) receptor on platelets and is being developed as a diagnostic radiopharmaceutical for in vivo imaging of acute thrombi and emboli. Prior to the first administration of [ 99m Tc]rBitistatin to human subjects, its biodistribution and effects on platelets were evaluated in animals. This paper reports findings in animal studies in comparison with initial findings in normal human subjects. Methods: [ 99m Tc]rBitistatin was administered to mice, guinea pigs and dogs to assess time-dependent organ distribution, urinary excretion and blood disappearance rates. Blood samples were analyzed to determine radioligand binding to circulating platelets and the extent of plasma protein binding. The effect of [ 99m Tc]rBitistatin on circulating platelet count was determined. These factors were also determined in normal human subjects who received [ 99m Tc]rBitistatin as part of a Phase I clinical trial. Results: The main organs that accumulated [ 99m Tc]rBitistatin were kidneys, liver and spleen in all animal species and humans. The main organs seen on human images were the kidneys and spleen. Liver uptake was fainter, and soft-tissue background was low. [ 99m Tc]rBitistatin bound to circulating platelets in blood, with a higher percentage of binding to platelets in guinea pigs and dogs compared to that in humans. Plasma protein binding was low and of little consequence in view of platelet binding. The main route of excretion was through the urine. [ 99m Tc]rBitistatin did not affect platelet counts in humans or dogs. Conclusions: [ 99m Tc]rBitistatin, when administered at low doses for imaging, has no adverse effects on platelets and has the qualitative biodistribution predicted by animal studies. [ 99m Tc]rBitistatin was found to bind to circulating platelets in humans, suggesting that it will be able to bind to activated platelets in vivo in patients with acute

Quantitative analyses of regional [{sup 11}C]PE2I binding to the dopamine transporter in the human brain: a PET study

Energy Technology Data Exchange (ETDEWEB)

Jucaite, Aurelija [Karolinska Institutet, Department of Woman and Child Health, Stockholm (Sweden); Odano, Ikuo [Niigata University, Department of Sensory and Integrative Medicine, Asahimachi-dori Niigata (Japan); Olsson, Hans; Pauli, Stefan; Halldin, Christer; Farde, Lars [Karolinska Institutet, Psychiatry Section, Department of Clinical Neuroscience, Stockholm (Sweden)

2006-06-15

The dopamine transporter (DAT) is a plasma membrane protein of central interest in the pathophysiology of neuropsychiatric disorders and is known to be a target for psychostimulant drugs. [{sup 11}C]PE2I is a new radioligand which binds selectively and with moderate affinity to central DAT, as has been demonstrated in vitro by autoradiography and in vivo by positron emission tomography (PET). The aims of the present PET study were to quantify regional [{sup 11}C]PE2I binding to DAT in the human brain and to compare quantitative methods with regard to suitability for applied clinical studies. One PET measurement was performed in each of eight healthy male subjects. The binding potential (BP) values were obtained by applying kinetic compartment analysis, which uses the metabolite-corrected arterial plasma curve as an input function. They were compared with the BP values quantified by two reference tissue approaches, using cerebellum as a reference region representing free and non-specific radioligand binding. The radioactivity concentration was highest in the striatum, lower in the midbrain and very low in the cerebellum. The regional [{sup 11}C]PE2I binding could be interpreted by kinetic compartment models. However, the BP values in the striatum obtained by the compartment analyses were about 30% higher than the BP values obtained using reference tissue methods. We suggest that the difference may be explained by the inaccurate metabolite correction, small amounts of radioactive metabolites that could account for the presence of non-specific binding in the cerebellum and insufficient data acquisition time. (orig.)

Brain serotonin 4 receptor binding is inversely associated with verbal memory recall

DEFF Research Database (Denmark)

Stenbæk, Dea S; Fisher, Patrick M; Ozenne, Brice

2017-01-01

the association between cerebral 5-HT 4R binding and affective verbal memory recall. METHODS: Twenty-four healthy volunteers were scanned with the 5-HT 4R radioligand [11C]SB207145 and positron emission tomography, and were tested with the Verbal Affective Memory Test-24. The association between 5-HT 4R binding...... and affective verbal memory was evaluated using a linear latent variable structural equation model. RESULTS: We observed a significant inverse association across all regions between 5-HT 4R binding and affective verbal memory performances for positive (p = 5.5 × 10-4) and neutral (p = .004) word recall......BACKGROUND: We have previously identified an inverse relationship between cerebral serotonin 4 receptor (5-HT 4R) binding and nonaffective episodic memory in healthy individuals. Here, we investigate in a novel sample if the association is related to affective components of memory, by examining...

Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

DEFF Research Database (Denmark)

Ingenhoven, Kathleen; Kramer, Daniel; Jensen, Poul Erik Hyldgaard

2017-01-01

to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control. CONCLUSION: An ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its......OBJECTIVE: To develop and validate a method for the detection of binding anti-drug antibodies (ADAs) against interferon beta (IFN-β) in human serum as part of a European initiative (ABIRISK) aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization...... to minimize the risk. METHOD: A two-tiered bridging enzyme-linked immunosorbent assay (ELISA) format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled...

Evaluation of in vivo selective binding of [11C]doxepin to histamine H1 receptors in five animal species

International Nuclear Information System (INIS)

Ishiwata, Kiichi; Kawamura, Kazunori; Wang Weifang; Tsukada, Hideo; Harada, Norihiro; Mochizuki, Hideki; Kimura, Yuichi; Ishii, Kenji; Iwata, Ren; Yanai, Kazuhiko

2004-01-01

The specific binding of [ 11 C]doxepin, which has been used as a radioligand for mapping histamine H 1 receptors in human brain by positron emission tomography, was evaluated in five animal species. In mice the [ 11 C]doxepin uptake was reduced by treatment with cold doxepin and two H 1 receptor antagonists, but not with H 2 /H 3 antagonists. The specific binding evaluated with treatment with (+)-chlorpheniramine (H 1 antagonist) was in the range of 10-30% in mouse, rat, rabbit, and monkey, but was not detected in guinea pig

Radioiodinated SB 207710 as a radioligand in vivo: imaging of brain 5-HT{sub 4} receptors with SPET

Energy Technology Data Exchange (ETDEWEB)

Pike, Victor W. [MRC Cyclotron Unit, Imperial College School of Medicine, Hammersmith Hospital, Ducane Road, W12 0NN, London (United Kingdom); PET Radiopharmaceutical Sciences Section, Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Building 10, Room B3 C346A, 10 Center Drive, MD 20892-1003, Bethesda (United States); Halldin, Christer; Nobuhara, Kenji; Swahn, Carl-Gunnar; Karlsson, Per; Olsson, Hans; Larsson, Stig; Schnell, Per-Olof; Farde, Lars [Department of Clinical Neuroscience, Psychiatry Section, Karolinska Institutet, Karolinska Hospital, 17176, Stockholm (Sweden); Hiltunen, Julka [MAP Medical Technologies, Oy, Tikkakoski (Finland); Mulligan, Rachel S. [MRC Cyclotron Unit, Imperial College School of Medicine, Hammersmith Hospital, Ducane Road, W12 0NN, London (United Kingdom); Institute of Psychiatry, SE 8AF, De Crespigny Park, Denmark Hill, London (United Kingdom); Centre for PET, Austin and Repatriation Medical Centre, Studley Road, Melbourne VIC 3084 (Australia); Hume, Susan P.; Hirani, Ella [MRC Cyclotron Unit, Imperial College School of Medicine, Hammersmith Hospital, Ducane Road, W12 0NN, London (United Kingdom); Imaging Research Solutions Ltd., Cyclotron Building, Hammersmith Hospital, Ducane Road, W12 0NN, London (United Kingdom); Whalley, Jaqueline [MRC Cyclotron Unit, Imperial College School of Medicine, Hammersmith Hospital, Ducane Road, W12 0NN, London (United Kingdom); Pilowsky, Lyn S. [Institute of Psychiatry, SE 8AF, De Crespigny Park, Denmark Hill, London (United Kingdom); Institute of Nuclear Medicine, Royal Free and University College, Medical School, Mortimer Street, W1N 8AA, London (United Kingdom); Ell, Peter J. [Institute of Nuclear Medicine, Royal Free and University College, Medical School, Mortimer Street, W1N 8AA, London (United Kingdom)

2003-11-01

Single-photon emission tomography (SPET) and positron emission tomography (PET), when coupled to suitable radioligands, are uniquely powerful for investigating the status of neurotransmitter receptors in vivo. The serotonin subtype-4 (5-HT{sub 4}) receptor has discrete and very similar distributions in rodent and primate brain. This receptor population may play a role in normal cognition and memory and is perhaps perturbed in some neuropsychiatric disorders. SB 207710 [(1-butyl-4-piperidinylmethyl)-8-amino-7-iodo-1,4-benzodioxan-5-carboxylate] is a selective high-affinity antagonist at 5-HT{sub 4} receptors. We explored radioiodinated SB 207710 as a possible radioligand for imaging 5-HT{sub 4} receptors in vivo. Rats were injected intravenously with iodine-125 labelled SB 207710, euthanised at known times and dissected to establish radioactivity content in brain tissues. Radioactivity entered brain but cleared rapidly and to a high extent from blood and plasma. Between 45 and 75 min after injection, the ratios of radioactivity concentration in each of 12 selected brain tissues to that in receptor-poor cerebellum correlated with previous measures of 5-HT{sub 4} receptor density distribution in vitro. The highest ratio was about 3.4 in striatum. SB 207710 was labelled with iodine-123 by an iododestannylation procedure. A cynomolgus monkey was injected intravenously with [{sup 123}I]SB 207710 and examined by SPET. Maximal whole brain uptake of radioactivity was 2.3% of the injected dose at 18 min after radioligand injection. Brain images acquired between 9 and 90 min showed high radioactivity uptake in 5-HT{sub 4} receptor-rich regions, such as striatum, and low uptake in receptor-poor cerebellum. At 169 min the ratio of radioactivity concentration in striatum to that in cerebellum was 4.0. In a second SPET experiment, the cynomolgus monkey was pretreated with a selective 5-HT{sub 4} receptor antagonist, SB 204070, at 20 min before [{sup 123}I]SB 207710 injection

[3H]QNB binding and contraction of rabbit colonic smooth muscle cells

International Nuclear Information System (INIS)

Ringer, M.J.; Hyman, P.E.; Kao, H.W.; Hsu, C.T.; Tomomasa, T.; Snape, W.J. Jr.

1987-01-01

The authors used radioligand binding and studies of cell contraction to characterize muscarinic receptors on dispersed smooth muscle cells from rabbit proximal and distal colon. Cells obtained after serial incubations in collagenase were used to measure binding of tritiated quinuclidinyl benzilate ([ 3 H]QNB). At 37 degree C, specific [ 3 H]QNB binding was saturable and linearly related to cell number. Nonlinear regression analysis was used to determine the affinity of [ 3 H]QNB for its receptor. The IC 50 for the muscarinic agonists bethanechol and oxotremorine were 80 and 0.57 μM, respectively. Hill coefficients were 0.67 for both, suggesting more complex interaction involving receptors of different affinities. In studies of cell contraction, bethanechol stimulated a dose-dependent decrease in cell length with half the maximal contraction occurring at 100 pM. These results suggest that (1) contraction is mediated by binding of bethanechol to M 2 -muscarinic receptors and that (2) there are a large number of spare receptors in colonic smooth muscle

Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

Science.gov (United States)

Murase, Hirotaka; Noguchi, Tomoharu; Sasaki, Shigeki

2018-06-01

Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg 2+ , which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat. Copyright © 2018 Elsevier Ltd. All rights reserved.

Photolabeling of brain membrane proteins by lysergic acid diethylamide

International Nuclear Information System (INIS)

Mahon, A.C.; Hartig, P.R.

1982-01-01

3 H-Lysergic acid diethylamide ( 3 H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a sub-population of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. 3 H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays

Preclinical evaluation of [{sup 18}F]2FNQ1P as the first fluorinated serotonin 5-HT{sub 6} radioligand for PET imaging

Energy Technology Data Exchange (ETDEWEB)

Becker, Guillaume [Universite Claude Bernard Lyon 1, CNRS INSERM, Lyon Neuroscience Research Center, Lyon (France); Hospices Civils de Lyon, Lyon (France); Colomb, Julie [Universite Claude Bernard Lyon 1, CNRS, Institute of Chemistry and Biochemistry, Villeurbanne (France); Sgambato-Faure, Veronique; Tremblay, Leon [Universite Claude Bernard Lyon 1, CNRS, Cognitive Neuroscience Center, Bron (France); Billard, Thierry [Universite Claude Bernard Lyon 1, CNRS, Institute of Chemistry and Biochemistry, Villeurbanne (France); CERMEP-Imaging Platform, Groupement Hospitalier Est, Lyon (France); Zimmer, Luc [Universite Claude Bernard Lyon 1, CNRS INSERM, Lyon Neuroscience Research Center, Lyon (France); Hospices Civils de Lyon, Lyon (France); CERMEP-Imaging Platform, Groupement Hospitalier Est, Lyon (France)

2014-10-21

Brain serotonin 6 receptor (5-HT{sub 6}) is one of the most recently identified serotonin receptors. It is a potent therapeutic target for psychiatric and neurological diseases, e.g. schizophrenia and Alzheimer's disease. Since no specific fluorinated radioligand has yet been successfully used to study this receptor by positron emission tomography (PET) neuroimaging, the objective of the present study was to study the first 5-HT{sub 6} {sup 18}F-labelled radiotracer. 2FNQ1P, inspired by the quinolone core of a previous radiotracer candidate, GSK215083, was selected according its 5-HT{sub 6} affinity and selectivity and was radiolabelled by {sup 18}F nucleophilic substitution. The cerebral distribution of [{sup 18}F]2FNQ1P was studied in vivo in rats, cats and macaque monkeys. The chemical and radiochemical purities of [{sup 18}F]2FNQ1P were >98 %. In rats, in vitro competition with the 5-HT{sub 6} antagonist, SB258585, revealed that the radioligand was displaced dose dependently. Rat microPET studies showed low brain uptake of [{sup 18}F]2FNQ1P, reversed by the P-glycoprotein inhibitor, cyclosporin. On the contrary, PET scans in cats showed good brain penetration and specific striatal binding blocked after pretreatment with unlabelled 2FNQ1P. PET scans in macaque monkeys confirmed high specific binding in both cortical and subcortical regions, specifically decreased by pretreatment with the 5-HT{sub 6} receptor antagonist, SB258585. 2FNQ1P was initially selected because of its suitable characteristics for 5-HT{sub 6} receptor probing in vitro in terms of affinity and specificity. Although in vivo imaging in rats cannot be considered as predictive of the clinical characteristics of the radiotracer, [{sup 18}F]2FNQ1P appeared to be a suitable 5-HT{sub 6} PET tracer in feline and primate models. These preclinical results encourage us to pursue the clinical development of this first fluorinated 5-HT{sub 6} PET radiotracer. (orig.)

Characterisation of the appearance of radioactive metabolites in monkey and human plasma from the 5-HT1A receptor radioligand, [carbonyl-11C]WAY-100635 - explanation of high signal contrast in PET and an aid to biomathematical modelling

International Nuclear Information System (INIS)

Osman, Safiye; Lundkvist, Camilla; Pike, Victor W.; Halldin, Christer; McCarron, Julie A.; Swahn, Carl-Gunnar; Farde, Lars; Ginovart, Nathalie; Luthra, Sajinder K.; Gunn, Roger N.; Bench, Christopher J.; Sargent, Peter A.; Grasby, Paul M.

1998-01-01

N-(2-(4-(2-Methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl) cyclohexanecarboxamide (WAY-100635), labelled in its amido carbonyl group with 11 C (t 1/2 = 20.4 min), is a promising radioligand for the study of brain 5-HT 1A receptors with positron emission tomography (PET). Thus, in PET experiments in six cynomolgus monkeys and seven healthy male volunteers, [carbonyl- 11 C]WAY-100635 was taken up avidly by brain. Radioactivity was retained in regions rich in 5-HT 1A receptors, such as occipital cortex, temporal cortex and raphe nuclei, but cleared rapidly from cerebellum, a region almost devoid of 5-HT 1A receptors. [Carbonyl- 11 C]WAY-100635 provides about 3- and 10-fold higher signal contrast (receptor-specific to nonspecific binding) than [O-methyl- 11 C]WAY-100635 in receptor-rich areas of monkey and human brain, respectively. To elucidate the effect of label position on radioligand behaviour and to aid in the future biomathematical interpretation of the kinetics of regional cerebral radioactivity uptake in terms of receptor-binding parameters, HPLC was used to measure [carbonyl- 11 C]WAY-100635 and its radioactive metabolites in plasma at various times after intravenous injection. Radioactivity cleared rapidly from monkey and human plasma. Parent radioligand represented 19% of the radioactivity in monkey plasma at 47 min and 8% of the radioactivity in human plasma at 40 min. [Carbonyl- 11 C]desmethyl-WAY-100635 was below detectable limits in monkey plasma and at most a very minor radioactive metabolite in human plasma. [ 11 C]Cyclohexanecarboxylic acid was identified as a significant radioactive metabolite. In human plasma this maximally represented 21% of the radioactivity at 10 min after radioligand injection. All other major radioactive metabolites in monkey and human plasma were even more polar. No-carrier-added [carbonyl- 11 C]cyclohexanecarboxylic acid was prepared in the laboratory and after intravenous administration into cynomolgus monkey was

A novel multiplex poliovirus binding inhibition assay applicable for large serosurveillance and vaccine studies, without the use of live poliovirus.

Science.gov (United States)

Schepp, Rutger M; Berbers, Guy A M; Ferreira, José A; Reimerink, Johan H; van der Klis, Fiona R

2017-03-01

Large-scale serosurveillance or vaccine studies for poliovirus using the "gold standard" WHO neutralisation test (NT) are very laborious and time consuming. With the polio eradication at hand and with the removal of live attenuated Sabin strains from the oral poliovirus vaccine (OPV), starting with type 2 (as of April 2016), laboratories will need to conform to much more stringent laboratory biosafety regulations when handling live poliovirus strains. In this study, a poliovirus binding inhibition multiplex immunoassay (polio MIA) using inactivated poliovirus vaccine (IPV-Salk) was developed for simultaneous quantification of serum antibodies directed to all three poliovirus types. Our assay shows a good correlation with the NT and an excellent correlation with the ELISA-based binding inhibition assay (POBI). The assay is highly type-specific and reproducible. Additionally, serum sample throughput increases about fivefold relative to NT and POBI and the amount of serum needed is reduced by more than 90%. In conclusion, the polio MIA can be used as a safe and high throughput application, especially for large-scale surveillance and vaccine studies, reducing laboratory time and serum amounts needed. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Binding assays for the quantitative detection of P. brevis polyether neurotoxins in biological samples and antibodies as therapeutic aids for polyether marine intoxication. Annual report, 1 December 1987-30 November 1988

Energy Technology Data Exchange (ETDEWEB)

Baden, D.G.

1988-12-15

The polyether lipid-soluble toxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) can be detected using two separate types of specific binding reaction. Using tritiated PbTx-3 as a specific probe for binding to voltage-dependent sodium channels in rat brain synaptosomes or to specific polyclonal antibodies, binding equilibria and displacement by unlabeled brevetoxins were compared. Labeled toxin can be displaced in a competitive manner by any of the other 5 naturally-occurring toxins; the quantitative displacement ability of each appears to reflect individual potency in fish bioassay. A comparison of ED50 in Radioimmunoassay and ED50 in synaptosome binding assay indicates that the former assay is useful for detection of toxins which possess the structural backbone of PbTx-3, the immunizing hapten. Thus, the two assays have quantitative applicability; the sodium channel with respect to potency and the antibodies with respect to structure. Microtiter plate assays utilizing each specific brevetoxin binding component and enzyme-linked toxin hapten have been successful and indicate a general applicability of colorimetric prototypes. There, is however, considerable manipulation required to decrease non-specific binding of the hydrophobic toxin-enzyme complex to the plates. Preliminary studies aimed at producing monoclonal antibodies have been explored using brevetoxins linked to keyhole limpet hemocyanin.

Platelet binding and biodistribution of [{sup 99m}Tc]rBitistatin in animal species and humans

Energy Technology Data Exchange (ETDEWEB)

Knight, Linda C. [Department of Radiology, Temple University School of Medicine, Philadelphia, PA 19140 (United States)], E-mail: lknight@temple.edu; Romano, Jan E. [Department of Radiology, Temple University School of Medicine, Philadelphia, PA 19140 (United States); Bright, Lewis T.; Agelan, Alexis [University Laboratory Animal Resources, Temple University School of Medicine, Philadelphia, PA 19140 (United States); Kantor, Steven; Maurer, Alan H. [Department of Radiology, Temple University School of Medicine, Philadelphia, PA 19140 (United States)

2007-10-15

Introduction: {sup 99m}Tc recombinant bitistatin (rBitistatin) is a radioligand for {alpha}{sub IIb}{beta}{sub 3} (glycoproteins IIb/IIIa) receptor on platelets and is being developed as a diagnostic radiopharmaceutical for in vivo imaging of acute thrombi and emboli. Prior to the first administration of [{sup 99m}Tc]rBitistatin to human subjects, its biodistribution and effects on platelets were evaluated in animals. This paper reports findings in animal studies in comparison with initial findings in normal human subjects. Methods: [{sup 99m}Tc]rBitistatin was administered to mice, guinea pigs and dogs to assess time-dependent organ distribution, urinary excretion and blood disappearance rates. Blood samples were analyzed to determine radioligand binding to circulating platelets and the extent of plasma protein binding. The effect of [{sup 99m}Tc]rBitistatin on circulating platelet count was determined. These factors were also determined in normal human subjects who received [{sup 99m}Tc]rBitistatin as part of a Phase I clinical trial. Results: The main organs that accumulated [{sup 99m}Tc]rBitistatin were kidneys, liver and spleen in all animal species and humans. The main organs seen on human images were the kidneys and spleen. Liver uptake was fainter, and soft-tissue background was low. [{sup 99m}Tc]rBitistatin bound to circulating platelets in blood, with a higher percentage of binding to platelets in guinea pigs and dogs compared to that in humans. Plasma protein binding was low and of little consequence in view of platelet binding. The main route of excretion was through the urine. [{sup 99m}Tc]rBitistatin did not affect platelet counts in humans or dogs. Conclusions: [{sup 99m}Tc]rBitistatin, when administered at low doses for imaging, has no adverse effects on platelets and has the qualitative biodistribution predicted by animal studies. [{sup 99m}Tc]rBitistatin was found to bind to circulating platelets in humans, suggesting that it will be able to bind

Advances in the Development of PET Ligands Targeting Histone Deacetylases for the Assessment of Neurodegenerative Diseases

Directory of Open Access Journals (Sweden)

Tetsuro Tago

2018-01-01

Full Text Available Epigenetic alterations of gene expression have emerged as a key factor in several neurodegenerative diseases. In particular, inhibitors targeting histone deacetylases (HDACs, which are enzymes responsible for deacetylation of histones and other proteins, show therapeutic effects in animal neurodegenerative disease models. However, the details of the interaction between changes in HDAC levels in the brain and disease progression remain unknown. In this review, we focus on recent advances in development of radioligands for HDAC imaging in the brain with positron emission tomography (PET. We summarize the results of radiosynthesis and biological evaluation of the HDAC ligands to identify their successful results and challenges. Since 2006, several small molecules that are radiolabeled with a radioisotope such as carbon-11 or fluorine-18 have been developed and evaluated using various assays including in vitro HDAC binding assays and PET imaging in rodents and non-human primates. Although most compounds do not readily cross the blood-brain barrier, adamantane-conjugated radioligands tend to show good brain uptake. Until now, only one HDAC radioligand has been tested clinically in a brain PET study. Further PET imaging studies to clarify age-related and disease-related changes in HDACs in disease models and humans will increase our understanding of the roles of HDACs in neurodegenerative diseases.

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

DEFF Research Database (Denmark)

Barington, Line; Rummel, Pia C; Lückmann, Michael

2016-01-01

and aromatic residues in extracellular loop 2 (ECL2) for ligand binding and activation in the chemokine receptor CCR8. We used IP3 accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action...... in CCR8. We find that the 7 transmembrane (7TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix (TM)III and ECL2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only...

Molecular imaging of σ receptors: synthesis and evaluation of the potent σ1 selective radioligand [18F]fluspidine

International Nuclear Information System (INIS)

Fischer, Steffen; Hiller, Achim; Deuther-Conrad, Winnie; Scheunemann, Matthias; Steinbach, Joerg; Brust, Peter; Wiese, Christian; Grosse Maestrup, Eva; Schepmann, Dirk; Wuensch, Bernhard

2011-01-01

Neuroimaging of σ 1 receptors in the human brain has been proposed for the investigation of the pathophysiology of neurodegenerative and psychiatric diseases. However, there is a lack of suitable 18 F-labelled PET radioligands for that purpose. The selective σ 1 receptor ligand [ 18 F]fluspidine (1'-benzyl-3-(2-[ 18 F]fluoroethyl)-3H-spiro[[2]benzofuran-1,4'-piperidine]) was synthesized by nucleophilic 18 F - substitution of the tosyl precursor. In vitro receptor binding affinity and selectivity were assessed by radioligand competition in tissue homogenate and autoradiographic approaches. In female CD-1 mice, in vivo properties of [ 18 F]fluspidine were evaluated by ex vivo brain section imaging and organ distribution of intravenously administered radiotracer. Target specificity was validated by organ distribution of [ 18 F]fluspidine after treatment with 1 mg/kg i.p. of the σ receptor antagonist haloperidol or the emopamil binding protein (EBP) inhibitor tamoxifen. In vitro metabolic stability and in vivo metabolism were investigated by LC-MS n and radio-HPLC analysis. [ 18 F]Fluspidine was obtained with a radiochemical yield of 35-45%, a radiochemical purity of ≥ 99.6% and a specific activity of 150-350 GBq/μmol (n = 6) within a total synthesis time of 90-120 min. In vitro, fluspidine bound specifically and with high affinity to σ 1 receptors (K i = 0.59 nM). In mice, [ 18 F]fluspidine rapidly accumulated in brain with uptake values of 3.9 and 4.7%ID/g and brain to blood ratios of 7 and 13 at 5 and 30 min after intravenous application of the radiotracer, respectively. By ex vivo autoradiography of brain slices, resemblance between binding site occupancy of [ 18 F]fluspidine and the expression of σ 1 receptors was shown. The radiotracer uptake in the brain as well as in peripheral σ 1 receptor expressing organs was significantly inhibited by haloperidol but not by tamoxifen. Incubation with rat liver microsomes led to a fast biotransformation of

Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter.

Science.gov (United States)

Rogge, George A; Shen, Li-Ling; Kuhar, Michael J

2010-07-16

Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Microbial methodological artifacts in [3H]glutamate receptor binding assays

International Nuclear Information System (INIS)

Yoneda, Y.; Ogita, K.

1989-01-01

Incubation of radiolabeled L-glutamic acid, a putative central excitatory neurotransmitter, in 50 mM Tris-acetate buffer (pH 7.4) at 30 degrees C in the absence of brain synaptic membranes resulted in a significant adsorption of the radioactivity to glass fiber filters routinely employed to trap the bound ligand in receptor binding assays. The adsorption was not only eliminated by the inclusion of L-isomers of structurally related amino acids, but also inhibited by that of most presumed agonists and antagonists for the brain glutamate receptors. This displaceable adsorption was a temperature-dependent nonreversible, and saturable phenomenon. Scatchard analysis of these data revealed that the adsorption consisted of a single component with an apparent dissociation constant of 73 nM. The displaceable adsorption was significantly attenuated by a concurrent incubation with papain, pronase E, and phospholipase C. A significant amount of the radioactivity was detected in the pass-through fraction of the Dowex column following an application of the reaction mixture incubated with purified [ 3 H]glutamate at 30 degree C for 60 min in the absence of membranous proteins added. Complete abolition of the displaceable adsorption resulted from the use of incubation buffer boiled at 100 degrees C as well as filtered through a nitrocellulose membrane filter with a pore size of 0.45 micron immediately before use. These results suggest that the displaceable adsorption may be attributable to the radioactive metabolite of [ 3 H]glutamate by microorganisms contaminating the Tris-acetate buffer. This might in part contribute to some of the controversial results with regard to receptor binding studies on acidic amino acids

11C-PBR28 binding to translocator protein increases with progression of Alzheimer’s disease

OpenAIRE

Kreisl, William C.; Lyoo, Chul Hyoung; Liow, Jeih-San; Wei, Monica; Snow, Joseph; Page, Emily; Jenko, Kimberly J.; Morse, Cheryl L.; Zoghbi, Sami S.; Pike, Victor W.; Turner, R. Scott; Innis, Robert B.

2016-01-01

This longitudinal study sought to determine whether the 18 kDa translocator protein (TSPO), a marker of neuroinflammation, increases over time in Alzheimer’s disease. Positron emission tomography (PET) imaging with the TSPO radioligand 11C-PBR28 imaging was performed at baseline and after a median follow-up of 2.7 years in 14 amyloid-positive patients and eight amyloid-negative controls. Patients had a greater increase in TSPO binding than controls in inferior parietal lobul...

The effect of Zhimu on the turnover of heart β-adrenoceptors in hyperthyroid mice

International Nuclear Information System (INIS)

Lu Zhiliang; Xu Jianguo; Yi Ningyu; Xia Zongqin

1992-01-01

Radioligand binding assay of receptors with 125 I-iodopindolol revealed that Zhimu can significantly inhibit the increase of mouse heart β-adrenoceptors induced by T 3 . The kinetic parameters were calculated from the data of heart β-adrenoceptor binding sites determined at various periods after irreversible blocking of the preexisting β-adrenoceptors. Zhimu can decrease the production rate and degradation rate constant of hyperthyroid mice by 48% and 35% respectively. These results indicate that Zhimu can decrease the production rate more potently than the degradation rate of heart β-adrenoceptors in hyperthyroid mice, and hence can make the number of receptor binding sites approach normal

Serotonin 2A receptor agonist binding in the human brain with [¹¹C]Cimbi-36

DEFF Research Database (Denmark)

Ettrup, Anders; Svarer, Claus; McMahon, Brenda

2016-01-01

INTRODUCTION: [(11)C]Cimbi-36 is a recently developed serotonin 2A (5-HT2A) receptor agonist positron emission tomography (PET) radioligand that has been successfully applied for human neuroimaging. Here, we investigate the test-retest variability of cerebral [(11)C]Cimbi-36 PET and compare [(11)C...... test-retest variability in [(11)C]Cimbi-36 binding measures, and another eight were scanned after a bolus plus constant infusion with [(18)F]altanserin. Regional differences in the brain distribution of [(11)C]Cimbi-36 and [(18)F]altanserin were assessed with a correlation of regional binding measures...... and with voxel-based analysis. RESULTS: Test-retest variability of [(11)C]Cimbi-36 non-displaceable binding potential (BPND) was consistently correlation between regional...

Internalization and cellular processing of cholecystokinin in rat pancreatic acinar cells

International Nuclear Information System (INIS)

Izzo, R.S.; Pellecchia, C.; Praissman, M.

1988-01-01

To evaluate the internalization of cholecystokinin, monoiodinated imidoester of cholecystokinin octapeptide [ 125 I-(IE)-CCK-8] was bound to dispersed pancreatic acinar cells, and surface-bound and internalized radioligand were differentiated by treating with an acidified glycine buffer. The amount of internalized radioligand was four- and sevenfold greater at 24 and 37 degree C than at 4 degree C between 5 and 60 min of association. Specific binding of radioligand to cell surface receptors was not significantly different at these temperatures. Chloroquine, a lysosomotropic agent that blocks intracellular proteolysis, significantly increased the amount of CCK-8 internalized by 18 and 16% at 30 and 60 min of binding, respectively, compared with control. Dithiothreitol (DTT), a sulfhydryl reducing agent, also augmented the amount of CCK-8 radioligand internalized by 25 and 29% at 30 and 60 min, respectively. The effect of chloroquine and DTT on the processing of internalized radioligand was also considered after an initial 60 min of binding of radioligand to acinar cells. After 180 min of processing, the amount of radioligand internalized was significantly greater in the presence of chloroquine compared with controls, whereas the amount of radioligand declined in acinar cells treated with DTT. Internalized and released radioactivity from acinar cells was rebound to pancreatic membrane homogenates to determine the amount of intact radioligand during intracellular processing. Chloroquine significantly increased the amount of intact 125 I-(IE)-CCK-8 radioligand in released and internalized radioactivity while DTT increased the amount of intact radioligand only in internalized samples. This study shows that pancreatic acinar cells rapidly internalize large amounts of CCK-8 and that chloroquine and DTT inhibit intracellular degradation

Evaluation of the novel 5-HT4 receptor PET ligand [11C]SB207145 in the Gottingen minipig

DEFF Research Database (Denmark)

Kornum, B.R.; Lind, N.M.; Gillings, N.

2009-01-01

This study investigates 5-hydroxytryptamine 4 (5-HT(4)) receptor binding in the minipig brain with positron emission tomography (PET), tissue homogenate-binding assays, and autoradiography in vitro. The cerebral uptake and binding of the novel 5-HT(4) receptor radioligand [(11)C]SB207145 in vivo...... was modelled and the outcome compared with postmortem receptor binding. Different models for quantification of [(11)C]SB207145 binding were evaluated: One-tissue and two-tissue compartment kinetic modelling, Logan arterial input, and three different reference tissue models. We report that the pig...... model provides stable and precise estimates of the binding potential in all regions. The binding potentials calculated for striatum, midbrain, and cortex from the PET data were highly correlated with 5-HT(4) receptor concentrations determined in brain homogenates from the same regions, except...

Evaluation of the novel 5-HT4 receptor PET ligand [11C]SB207145 in the Göttingen minipig

DEFF Research Database (Denmark)

Kornum, Birgitte R; Lind, Nanna M; Gillings, Nic

2009-01-01

This study investigates 5-hydroxytryptamine 4 (5-HT(4)) receptor binding in the minipig brain with positron emission tomography (PET), tissue homogenate-binding assays, and autoradiography in vitro. The cerebral uptake and binding of the novel 5-HT(4) receptor radioligand [(11)C]SB207145 in vivo...... was modelled and the outcome compared with postmortem receptor binding. Different models for quantification of [(11)C]SB207145 binding were evaluated: One-tissue and two-tissue compartment kinetic modelling, Logan arterial input, and three different reference tissue models. We report that the pig...... model provides stable and precise estimates of the binding potential in all regions. The binding potentials calculated for striatum, midbrain, and cortex from the PET data were highly correlated with 5-HT(4) receptor concentrations determined in brain homogenates from the same regions, except...

Modulation of [3H]-dopamine binding by cholecystokinin octapeptide (CCK-8)

International Nuclear Information System (INIS)

Murphy, R.B.; Schuster, D.I.

1982-01-01

Cholecystokinin-octapeptide (CCK-8) is a putative neurotransmitter which has been demonstrated previously to occur in midbrain dopamine neurones. We observe that CCK-8 causes changes in both the affinity and density of binding sites for [ 3 H]-dopamine in rat striatal homogenates, in vitro, upon incubation with the peptide at a concentration of 1 micromolar. A dose-response study of the competetion of CCK-8 with [ 3 H]-dopamine binding indicates an IC50 for the peptide of 450 nM; desulfated CCK-8 and the related peptide caerulin are at least 4-fold less active than CCK-8. CCK-8 was also administered to rats in a separate study; the binding of [ 3 H]-dopamine was evaluated to homogenates of striata and olfactory tubercles obtained from these animals, which had been treated with systemic injection at a dose of 20 micrograms/kg, daily, for four days. A decrease in the number of striatal binding sites for the radioligand was observed, with a concomitant increase in the number of binding sites in the olfactory tubercle. These data collectively suggest a possible regulatory role for CCK-8 in the ascending dopamine systems

Magnetic levitation as a platform for competitive protein-ligand binding assays.

Science.gov (United States)

Shapiro, Nathan D; Soh, Siowling; Mirica, Katherine A; Whitesides, George M

2012-07-17

This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (K(d) values within the range from ~10 nM to 100 μM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 min-2 h). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight greater

Exploring 6-(substituted sulfonyl)imidazopyridines as a potential scaffold for the design of 5-HT6 ligands

International Nuclear Information System (INIS)

Heloire, V.M.; Furman, C.; Melnyk, P.; Carato, P.

2013-01-01

Cognitive dysfunction is a characteristic of various forms of dementia such as Alzheimer's disease. We have focused on the 5-HT 6 receptor in order to identify potent ligands. Herein we report the design of a novel series of 6-sulfonylimidazole derivatives substituted with an alkylamino chain at the 2- or 3-position, their synthesis, and their ability to interact with 5-HT 6 receptors as evaluated in radioligand binding assays. (author)

Binding of (/sup 3/H)imipramine to human platelet membranes with compensation for saturable binding to filters and its implication for binding studies with brain membranes

Energy Technology Data Exchange (ETDEWEB)

Phillips, O.M.; Wood, K.M.; Williams, D.C.

1984-08-01

Apparent specific binding of (/sup 3/H)imipramine to human platelet membranes at high concentrations of imipramine showed deviation from that expected of a single binding site, a result consistent with a low-affinity binding site. The deviation was due to displaceable, saturable binding to the glass fibre filters used in the assays. Imipramine, chloripramine, desipramine, and fluoxetine inhibited binding to filters whereas 5-hydroxytryptamine and ethanol were ineffective. Experimental conditions were developed that eliminated filter binding, allowing assay of high- and low-affinity binding to membranes. Failure to correct for filter binding may lead to overestimation of binding parameters, Bmax and KD for high-affinity binding to membranes, and may also be misinterpreted as indicating a low-affinity binding component in both platelet and brain membranes. Low-affinity binding (KD less than 2 microM) of imipramine to human platelet membranes was demonstrated and its significance discussed.

PSMA-Based Radioligand Therapy for Metastatic Castration-Resistant Prostate Cancer: The Bad Berka Experience Since 2013.

Science.gov (United States)

Kulkarni, Harshad R; Singh, Aviral; Schuchardt, Christiane; Niepsch, Karin; Sayeg, Manal; Leshch, Yevgeniy; Wester, Hans-Juergen; Baum, Richard P

2016-10-01

A potential milestone in personalized nuclear medicine is theranostics of metastatic castration-resistant prostate cancer (mCRPC) based on molecular imaging using PET/CT with 68 Ga-labeled prostate-specific membrane antigen (PSMA) ligands and molecular radiotherapy using PSMA-targeted radioligand therapy (PRLT) with 177 Lu-PSMA ligands. 68 Ga-PSMA PET/CT enables accurate detection of mCRPC lesions with high diagnostic sensitivity and specificity and provides quantitative and reproducible data that can be used to select patients for PRLT and therapeutic monitoring. Our comprehensive experience over the last 3 years using different radioligands indicates that PRLT is highly effective for the treatment of mCRPC, even in advanced cases, and potentially lends a significant benefit to overall and progression-free survival. Additionally, significant improvement in clinical symptoms and excellent palliation of pain can be achieved. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Hyperthermostable binding molecules on phage: Assay components for point-of-care diagnostics for active tuberculosis infection.

Science.gov (United States)

Zhao, Ning; Spencer, John; Schmitt, Margaret A; Fisk, John D

2017-03-15

Tuberculosis is the leading cause of death from infectious disease worldwide. The low sensitivity, extended processing time, and high expense of current diagnostics are major challenges to the detection and treatment of tuberculosis. Mycobacterium tuberculosis ornithine transcarbamylase (Mtb OTC, Rv1656) has been identified in the urine of patients with active TB infection and is a promising target for point-of-care diagnostics. Specific binding proteins with low nanomolar affinities for Mtb OTC were selected from a phage display library built upon a hyperthermostable Sso7d scaffold. Phage particles displaying Sso7d variants were utilized to generate a sandwich ELISA-based assay for Mtb OTC. The assay response is linear between 2 ng/mL and 125 ng/mL recombinant Mtb OTC and has a limit of detection of 400 pg/mL recombinant Mtb OTC. The assay employing a phage-based detection reagent is comparable to commercially-available antibody-based biosensors. Importantly, the assay maintains functionality at both neutral and basic pH in presence of salt and urea over the range of concentrations typical for human urine. Phage-based diagnostic systems may feature improved physical stability and cost of production relative to traditional antibody-based reagents, without sacrificing specificity and sensitivity. Copyright © 2017 Elsevier Inc. All rights reserved.

An improved automated synthesis and in vivo evaluation of PET radioligand for serotonin re-uptake sites. [11C]McN5652X

International Nuclear Information System (INIS)

Sasaki, Masahiro; Suhara, Tetsuya; Suzuki, Kazutoshi; Kubodera, Akiko.

1996-01-01

Carbon-11 labeled serotonin (5-HT) re-uptake inhibitor, [ 11 C]McN5 652X ((6S,10bR)-trans-( + )-1,2,3,5,6,10b-hexahydro-6-[4-(methylthio)phenyl]pyrrolo-[2,1-a]-isoquinoline), has recently been reported to be favorable for studying human 5-HT re-uptake site by positron emission tomography (PET) because of its rapid and high specific binding characteristics as radioligands. [ 11 C]McN5652X has been synthesized by S-methylation of the corresponding des-methyl precursor A with [ 11 C]iodomethane. One serious disadvantage of this procedure, however, is the lack of stability of A. The improved method for the synthesis of A has been desired. We have found that the decomposition of A is significantly reduced by adding a protecting agent for SH groups, dithiothreitol (DTT), into the reaction medium immediately after the demethylation of McN5652X. By using this stabilized precursor A, we have developed an automated procedure giving [ 11 C]McN5652X with 98.6±0.4% radiochemical purity in high specific activity (181.3±7.4GBq/μmol). Preclinical evaluation of the produ ct was carried out by injecting the solution of [ 11 C]McN5652X obtained by this procedure into mice. [ 11 C]McN5652X showed the high accumulation into mouse thalamus, striatum and cerebral cortex, organs known to have high level of 5-HT receptor density, after intravenous injection. Human PET studies also showed the high uptakes of this radioligand into the thalamus, striatum and midbrain

Identification of Rift Valley fever virus nucleocapsid protein-RNA binding inhibitors using a high-throughput screening assay.

Science.gov (United States)

Ellenbecker, Mary; Lanchy, Jean-Marc; Lodmell, J Stephen

2012-09-01

Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection, and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential antiviral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug-screening assay and tested 26 424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of Food and Drug Administration-approved drugs, druglike molecules, and natural product extracts, we identified several lead compounds that are promising candidates for medicinal chemistry.

Effects of anesthetics on vesicular monoamine transporter type 2 binding to 18F-FP-(+)-DTBZ: a biodistribution study in rat brain

International Nuclear Information System (INIS)

Chen, Zhengping; Tang, Jie; Liu, Chunyi; Li, Xiaomin; Huang, Hongbo; Xu, Xijie; Yu, Huixin

2016-01-01

Objectives: The in vivo binding analysis of vesicular monoamine transporter type 2 (VMAT2) to radioligand has provided a means of investigating related disorders. Anesthesia is often inevitable when the investigations are performed in animals. In the present study, we tested effects of four commonly-used anesthetics: isoflurane, pentobarbital, chloral hydrate and ketamine, on in vivo VMAT2 binding to 18 F-FP-(+)-DTBZ, a specific VMAT2 radioligand, in rat brain. Methods: The transient equilibrium time window for in vivo binding of 18 F-FP-(+)-DTBZ after a bolus injection was firstly determined. The brain biodistribution studies under anesthetized and awake rats were then performed at the equilibrium time. Standard uptake values (SUVs) of the interest brain regions: the striatum (ST), hippocampus (HP), cortex (CX) and cerebellum (CB) were obtained; and ratios of tissue to cerebellum were calculated. Results: Isoflurane and pentobarbital did not alter distribution of 18 F-FP-(+)-DTBZ in the brain relative to the awake group; neither SUVs nor ratios of ST/CB and HP/CB were altered significantly. Chloral hydrate significantly increased SUVs of all the brain regions, but did not significantly alter ratios of ST/CB and HP/CB. Ketamine significantly increased SUVs of the striatum, hippocampus and cortex, and insignificantly increased the SUV of the cerebellum; consequently, ketamine significantly increased ratios of ST/CB and HP/CB. Conclusions: It is concluded that in vivo VMAT2 binding to 18 F-FP-(+)-DTBZ are not altered by isoflurane and pentobarbital, but altered by chloral hydrate and ketamine. Isoflurane and pentobarbital may be promising anesthetic compounds for investigating in vivo VMAT2 binding. Further studies are warranted to investigate the interactions of anesthetics with VMAT2 binding potential with in vivo PET studies.

Demonstration of pulmonary {beta}{sub 2}-adrenergic receptor binding in vivo with [{sup 18}F]fluoroethyl-fenoterol in a guinea pig model

Energy Technology Data Exchange (ETDEWEB)

Helisch, A.; Schirrmacher, E.; Schirrmacher, R.; Buchholz, H.G.; Bartenstein, P. [University Hospital, Department of Nuclear Medicine, Mainz (Germany); Thews, O.; Dillenburg, W.; Tillmanns, J. [University of Mainz, Institute of Physiology and Pathophysiology, Mainz (Germany); Hoehnemann, S.; Roesch, F. [University of Mainz, Institute of Nuclear Chemistry, Mainz (Germany); Wessler, I. [University of Mainz, Institute of Pharmacology, Mainz (Germany); Buhl, R. [University Hospital, Pulmonary Division, Mainz (Germany)

2005-11-01

The new {beta}{sub 2} radioligand (R,R)(S,S) 5-(2-(2-[4-(2-[{sup 18}F]fluoroethoxy)phenyl]-1-methylethylamino)-1-hydroxyethyl)-benzene-1,3-diol ([{sup 18}F]FE-fenoterol; [{sup 18}F]FEFE), a fluoroethylated derivative of racemic fenoterol, was evaluated in vivo and ex vivo using a guinea pig model. Dynamic PET studies over 60 min with [{sup 18}F]FEFE were performed in nine Hartley guinea pigs in which a baseline (group 1, n=3), a predose (group 2, n=3; 2 mg/kg fenoterol 5 min prior to injection of [{sup 18}F]FEFE) or a displacement study (group 3, n=3; 2 mg/kg fenoterol 5 min post injection of [{sup 18}F]FEFE) was conducted. In all animal groups, the lungs could be visualised and semi-quantified separately by calculating uptake ratios to non-specific binding in the neck area. Premedication with non-radioactive fenoterol and displacement tests showed significant reduction of lung uptake, by 94% and 76%, respectively. These data demonstrate specific binding of the new radioligand to the pulmonary {beta}{sub 2}-receptors in accordance with ex vivo measurements. Therefore, [{sup 18}F]FEFE seems to be suitable for the in vivo visualisation and quantification of the pulmonary {beta}{sub 2}-receptor binding in this animal model. (orig.)

Radioreceptor opioid assay

International Nuclear Information System (INIS)

Miller, R.J.; Chang, K.-J.

1981-01-01

A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

Science.gov (United States)

Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael

2015-12-01

Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

Measurement of circulating 1,25-dihydroxyvitamin D employing radioimmunoassay

International Nuclear Information System (INIS)

Yamamoto, Itsuo; Hamazu, Hisatoshi; Kigami, Yuusuke; Morita, Rikushi

1997-01-01

Measurement of serum 1,25-dihydroxyvitamin D levels is important for diagnosis of various calcium metabolism disorders. Conventional assays for 1,25-dihydroxyvitamin D employed specific 1,25-dihydroxyvitamin D receptor as binding site for the ligand and thus, biologically active 1,25-dihydroxyvitamin D ligand, which is labeled with 3 H, was required. Usage of 3 H made assays cumbersome works. A new assay which uses specific antibody as the binding site and the radioligand labeled with 125 I is now available as a commercial kit. Using these kits, we first studied basically the reproducibility, recovery, cross-reactivity and comparison with conventional assays. All of those results were satisfactory. Secondly, we measured clinically in 111 healthy adults and in patients with various disorders such as renal failure, primary hyperparathyroidism, hypoparathyroidism and sarcoidosis. This newly available kit for measurement of circulating 1,25-dihydroxyvitamin D is proved to be useful in clinical evaluation of calcium metabolic disorders. (author)

Muscarinic receptor subtypes in porcine detrusor: comparison with humans and regulation by bladder augmentation

NARCIS (Netherlands)

Goepel, M.; Gronewald, A.; Krege, S.; Michel, M. C.

1998-01-01

The properties of muscarinic acetylcholine receptors of porcine and human bladder detrusor were compared in radioligand binding studies using [3H]quinuclidinylbenzylate as the radioligand. The receptor affinity for the radioligand and the density of muscarinic receptors was similar in male and

Methodology for benzodiazepine receptor binding assays at physiological temperature. Rapid change in equilibrium with falling temperature

International Nuclear Information System (INIS)

Dawson, R.M.

1986-01-01

Benzodiazepine receptors of rat cerebellum were assayed with [ 3 H]-labeled flunitrazepam at 37 0 C, and assays were terminated by filtration in a cold room according to one of three protocols: keeping each sample at 37 degrees C until ready for filtration, taking the batch of samples (30) into the cold room and filtering sequentially in the order 1-30, and taking the batch of 30 samples into the cold room and filtering sequentially in the order 30-1. the results for each protocol were substantially different from each other, indicating that rapid disruption of equilibrium occurred as the samples cooled in the cold room while waiting to be filtered. Positive or negative cooperativity of binding was apparent, and misleading effects of gamma-aminobutyric acid on the affinity of diazepam were observed, unless each sample was kept at 37 0 C until just prior to filtration

Synthesis and biological evaluation in rat and cat of [18F]12ST05 as a potential 5-HT6 PET radioligand

International Nuclear Information System (INIS)

Tang, Sandrine; Verdurand, Mathieu; Joseph, Benoit; Lemoine, Laetitia; Daoust, Alexia; Billard, Thierry; Fournet, Guy; Le Bars, Didier; Zimmer, Luc

2007-01-01

Introduction: 5-hydroxytryptamine (5-HT) 6 receptors represent one of the more recently discovered serotoninergic receptor family. However, no 5-HT 6 positron emission tomography (PET) radiotracer is currently used in clinical imaging studies. The purpose of this study was to propose the first fluorinated PET radiotracer for this brain receptor. Methods: A new compound presenting in vitro high affinity towards the serotoninergic 5-HT 6 receptor, N-[2-(1-[(4-fluorophenyl)sulfonyl]-1H-indol-4-yloxy)ethyl] -N,N-dimethylamine, was labelled with fluorine 18 via a nitro-/fluoronucleophilic substitution. Biological evaluations included (i) in vitro and ex vivo autoradiographies in rat brain and (ii) a PET scan on anaesthetized cat. Results and Conclusion: Although the radioligand showed excellent brain penetration, it did not reveal any specific binding to the 5-HT 6 receptors indicating that this radiotracer is not suitable for mapping 5-HT 6 receptors using PET

Specific assay measuring binding of /sup 125/I-Gp 120 from HIV to T4/sup +//CD4/sup +/ cells

Energy Technology Data Exchange (ETDEWEB)

Lundin, K.; Nygren, A.; Ramstedt, U.; Gidlund, M.; Wigzell, H.; Arthur, L.O.; Robey, W.G.; Morein, B.

1987-02-26

The HIV (HTLV-III) envelope glycoprotein, Gp120, was isolated from virus-infected tissue culture cells using affinity chromatography. A radioimmunoassay was developed to determine the degree of iodinated Gp120 to target CD4/sup +/ (T4/sup +/) cells. /sup 125/I-Gp120 could be shown to selectively bind to CD4/sup +/ cells only. The Gp120 remained bound to these cells after repeated washes. Monoclonal anti-CD4 antibodies block the binding of Gp120 to CD4/sup +/ cells. Monoclonal antibodies to other cell surface components do not interfere with /sup 125/I-Gp120 binding. All IgG antibodies from HIV seropositive donors tested block /sup 125/I-GP120 binding, though with variable titers. The authors believe that this assay provides further proof for the use of CD4 (T4) as a component of the receptor for HIV. It represents a safe, objective and sensitive method for the analysis of Gp120-CD4 interactions, as well as the potential of antibodies to interfere with this binding. (Auth.). 24 refs.; 2 figs.; 8 tabs.

Novel Radioligands for Cyclic Nucleotide Phosphodiesterase Imaging with Positron Emission Tomography: An Update on Developments Since 2012

Directory of Open Access Journals (Sweden)

Susann Schröder

2016-05-01

Full Text Available Cyclic nucleotide phosphodiesterases (PDEs are a class of intracellular enzymes that inactivate the secondary messenger molecules, cyclic adenosine monophosphate (cAMP and cyclic guanosine monophosphate (cGMP. Thus, PDEs regulate the signaling cascades mediated by these cyclic nucleotides and affect fundamental intracellular processes. Pharmacological inhibition of PDE activity is a promising strategy for treatment of several diseases. However, the role of the different PDEs in related pathologies is not completely clarified yet. PDE-specific radioligands enable non-invasive visualization and quantification of these enzymes by positron emission tomography (PET in vivo and provide an important translational tool for elucidation of the relationship between altered expression of PDEs and pathophysiological effects as well as (pre-clinical evaluation of novel PDE inhibitors developed as therapeutics. Herein we present an overview of novel PDE radioligands for PET published since 2012.

Potent haloperidol derivatives covalently binding to the dopamine D2 receptor.

Science.gov (United States)

Schwalbe, Tobias; Kaindl, Jonas; Hübner, Harald; Gmeiner, Peter

2017-10-01

The dopamine D 2 receptor (D 2 R) is a common drug target for the treatment of a variety of neurological disorders including schizophrenia. Structure based design of subtype selective D 2 R antagonists requires high resolution crystal structures of the receptor and pharmacological tools promoting a better understanding of the protein-ligand interactions. Recently, we reported the development of a chemically activated dopamine derivative (FAUC150) designed to covalently bind the L94C mutant of the dopamine D 2 receptor. Using FAUC150 as a template, we elaborated the design and synthesis of irreversible analogs of the potent antipsychotic drug haloperidol forming covalent D 2 R-ligand complexes. The disulfide- and Michael acceptor-functionalized compounds showed significant receptor affinity and an irreversible binding profile in radioligand depletion experiments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of in vivo selective binding of [{sup 11}C]doxepin to histamine H{sub 1} receptors in five animal species

Energy Technology Data Exchange (ETDEWEB)

Ishiwata, Kiichi E-mail: ishiwata@pet.tmig.or.jp; Kawamura, Kazunori; Wang Weifang; Tsukada, Hideo; Harada, Norihiro; Mochizuki, Hideki; Kimura, Yuichi; Ishii, Kenji; Iwata, Ren; Yanai, Kazuhiko

2004-05-01

The specific binding of [{sup 11}C]doxepin, which has been used as a radioligand for mapping histamine H{sub 1} receptors in human brain by positron emission tomography, was evaluated in five animal species. In mice the [{sup 11}C]doxepin uptake was reduced by treatment with cold doxepin and two H{sub 1} receptor antagonists, but not with H{sub 2}/H{sub 3} antagonists. The specific binding evaluated with treatment with (+)-chlorpheniramine (H{sub 1} antagonist) was in the range of 10-30% in mouse, rat, rabbit, and monkey, but was not detected in guinea pig.

Differences in receptor binding affinity of several phytocannabinoids do not explain their effects on neural cell cultures.

Science.gov (United States)

Rosenthaler, Sarah; Pöhn, Birgit; Kolmanz, Caroline; Huu, Chi Nguyen; Krewenka, Christopher; Huber, Alexandra; Kranner, Barbara; Rausch, Wolf-Dieter; Moldzio, Rudolf

2014-01-01

Phytocannabinoids are potential candidates for neurodegenerative disease treatment. Nonetheless, the exact mode of action of major phytocannabinoids has to be elucidated, but both, receptor and non-receptor mediated effects are discussed. Focusing on the often presumed structure-affinity-relationship, Ki values of phytocannabinoids cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), THC acid (THCA) and THC to human CB1 and CB2 receptors were detected by using competitive inhibition between radioligand [(3)H]CP-55,940 and the phytocannabinoids. The resulting Ki values to CB1 range from 23.5 nM (THCA) to 14711 nM (CBDV), whereas Ki values to CB2 range from 8.5 nM (THC) to 574.2 nM (CBDV). To study the relationship between binding affinity and effects on neurons, we investigated possible CB1 related cytotoxic properties in murine mesencephalic primary cell cultures and N18TG2 neuroblastoma cell line. Most of the phytocannabinoids did not affect the number of dopaminergic neurons in primary cultures, whereas propidium iodide and resazurin formation assays revealed cytotoxic properties of CBN, CBDV and CBG. However, THC showed positive effects on N18TG2 cell viability at a concentration of 10 μM, whereas CBC and THCA also displayed slightly positive activities. These findings are not linked to the receptor binding affinity therewith pointing to another mechanism than a receptor mediated one. [Corrected] Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of a competitive binding assay for cortisol using horse transcortin

International Nuclear Information System (INIS)

Stahl, F.; Hubl, W.; Schnorr, D.; Doerner, G.

1978-01-01

A non-chromatographic competitive binding assay (CBA) using horse transcortin has been employed in the routine measurement of cortisol in plasma, urine and amniotic fluids. Comparing the values with those of a radioimmunoassay (RIA) or a fluorimetric method (FM) an excellent correlation between the three methods both in plasma and urine has been calculated in normal subjects and in patients with various endocrine disorders. In amniotic fluids, however, there were discrepancies between CBA and RIA. Whereas CBA showed no differences, RIA gave significantly higher values in a amniotic fluids of female than of male fetuses. Elevated free plasma cortisol levels observed in patients with prostatic cancer after diethyl stilboestrol diphosphate therapy did not correlate with unconjugated urinary cortisol concentration as measured with CBA and FM. In newborns, a relatively high plasma level found 12 hours after birth was followed by a nadir on the 2nd and 3rd day of life and by an increase until levels of adults on the 5th day of life were reached. (author)

Pi overlapping ring systems contained in a homogeneous assay: a novel homogeneous assay for antigens

Science.gov (United States)

Kidwell, David A.

1993-05-01

A novel immunoassay, Pi overlapping ring systems contained in a homogeneous assay (PORSCHA), is described. This assay relies upon the change in fluorescent spectral properties that pyrene and its derivatives show with varying concentration. Because antibodies and other biomolecules can bind two molecules simultaneously, they can change the local concentration of the molecules that they bind. This concentration change may be detected spectrally as a change in the fluorescence emission wavelength of an appropriately labeled biomolecule. Several tests of PORSCHA have been performed which demonstrate this principle. For example: with streptavidin as the binding biomolecule and a biotin labeled pyrene derivative, the production of the excimer emitting at 470 nm is observed. Without the streptavidin present, only the monomer emitting at 378 and 390 nm is observed. The ratio of monomer to excimer provides the concentration of unlabeled biotin in the sample. Approximately 1 ng/mL of biotin may be detected with this system using a 50 (mu) l sample (2 X 10-16 moles biotin). The principles behind PORSCHA, the results with the streptavidin/biotin system are discussed and extensions of the PORSCHA concept to antibodies as the binding partner and DNA in homogeneous assays are suggested.

Single prostacyclin receptor of gel-filtered platelets provides a correlation with antiaggregatory potency of PGI2 mimics

International Nuclear Information System (INIS)

Eggerman, T.L.; Hartzell, C.J.; Selfe, S.; Andersen, N.H.

1987-01-01

Gel-filtered human platelets (GFP) display only a single binding site for [ 3 H]-PGI2: KD = 61nM, 234 fmol/10(8) platelets (1410 sites/platelet). Platelet-rich plasma (PRP) displays the same receptor density but the KD value increases to 123 nM due to protein binding of PGI2 which lowers its effective concentration. The [ 3 H]-PGI2/GFP binding assay has been used to evaluate the molecular basis of aggregation inhibition for prostacyclin analogs and mimics, three PGE type structures, and PGD2. Antiaggregatory IC50s and radioligand binding IC50s correlate for PGE2, E1, and six PGI2 analogs. PGD2, and to a lesser extent 6-oxo-PGE1, display greater antiaggregatory potency than expected based on PGI2-binding site affinity data

Identification of Tight-Binding Plasmepsin II and Falcipain 2 Inhibitors in Aqueous Extracts of Marine Invertebrates by the Combination of Enzymatic and Interaction-Based Assays

Science.gov (United States)

Salas-Sarduy, Emir; Guerra, Yasel; Covaleda Cortés, Giovanni; Avilés, Francesc Xavier; Chávez Planes, María A.

2017-01-01

Natural products from marine origin constitute a very promising and underexplored source of interesting compounds for modern biotechnological and pharmaceutical industries. However, their evaluation is quite challenging and requires specifically designed assays to reliably identify the compounds of interest in a highly heterogeneous and interfering context. In the present study, we describe a general strategy for the confident identification of tight-binding protease inhibitors in the aqueous extracts of 62 Cuban marine invertebrates, using Plasmodium falciparum hemoglobinases Plasmepsin II and Falcipain 2 as model enzymes. To this end, we first developed a screening strategy that combined enzymatic with interaction-based assays and then validated screening conditions using five reference extracts. Interferences were evaluated and minimized. The results from the massive screening of such extracts, the validation of several hits by a variety of interaction-based assays and the purification and functional characterization of PhPI, a multifunctional and reversible tight-binding inhibitor for Plasmepsin II and Falcipain 2 from the gorgonian Plexaura homomalla, are presented. PMID:28430158

Development of [11C]/[3H]THK-5351 – A potential novel carbon-11 tau imaging PET radioligand

International Nuclear Information System (INIS)

Stepanov, Vladimir; Svedberg, Marie; Jia, Zhisheng; Krasikova, Raisa; Lemoine, Laetitia; Okamura, Nobujuki; Furumoto, Shozo; Mitsios, Nicholas; Mulder, Jan; Långström, Bengt; Nordberg, Agneta; Halldin, Christer

2017-01-01

Introduction: Due to the rise in the number of patients with dementia the imperative for finding new diagnostic and treatment options becomes ever more pressing. While significant progress has been made in PET imaging of Aβ aggregates both in vitro and in vivo, options for imaging tau protein aggregates selectively are still limited. Based on the work previously published by researchers from the Tohoku University, Japan, that resulted in the development of [ 18 F]THK-5351, we have undertaken an effort to develop a carbon-11 version of the identical structure - [ 11 C]THK-5351. In parallel, THK-5351 was also labeled with tritium ([ 3 H]THK-5351) for use in in vitro autoradiography (ARG). Methods: The carbon-11 labeling was performed starting with di-protected enantiomeric pure precursor - tert-butyl 5-(6-((2S)-3-fluoro-2-(tetrahydro-2H-pyran-2-yloxy)propoxy)quinolin-2-yl) pyridin-2-yl carbamate, which was reacted with [ 11 C]MeI, using DMF as the solvent and NaH as base, followed by deprotection with trifluoroacetic acid/water mixture, resulting in enantiomerically pure carbon-11 radioligand, [ 11 C]THK-5351 - (S)-1-fluoro-3-(2-(6-([ 11 C]methylamino)pyridin-3-yl)quinolin-6-yloxy) propan-2-ol. Tritium labeling and purification of [ 3 H]THK-5351 were undertaken using similar approach, resulting in [ 3 H]THK-5351 with RCP >99.8% and specific radioactivity of 1.3 GBq/μmol. Results: [ 11 C]THK-5351 was produced in good yield (1900 ± 355 MBq), specific radioactivity (SRA) (361 ± 119 GBq/μmol at EOS + 20 min) and radiochemical purity (RCP) (>99.8%), with enantiomeric purity of 98.7%. [ 3 H]THK-5351 was evaluated for ARG of tau binding in post-mortem human brain tissue using cortical sections from one AD patient and one control subject. [ 3 H]THK-5351 binding density was higher in the AD patient compared to the control subject, the binding was displaced by unlabeled THK-5351 confirming specific [ 3 H]THK-5351 binding.

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

Science.gov (United States)

Polireddy, Kishore; Khan, Mohiuddin Md Taimur; Chavan, Hemantkumar; Young, Susan; Ma, Xiaochao; Waller, Anna; Garcia, Matthew; Perez, Dominique; Chavez, Stephanie; Strouse, Jacob J; Haynes, Mark K; Bologa, Cristian G; Oprea, Tudor I; Tegos, George P; Sklar, Larry A; Krishnamurthy, Partha

2012-01-01

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that [3H]dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

International Nuclear Information System (INIS)

Leff, S.E.; Creese, I.

1985-01-01

The interactions of dopaminergic agonists and antagonists with 3 H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of [ 3 H]dopamine and [ 3 H]apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/[ 3 H]dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific [ 3 H]dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and [ 3 H]flupentixol-binding activities. The affinities of agonists to inhibit D3 specific [ 3 H]dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/[ 3 H]flupentixol competition curves. Both D3 specific [ 3 H] dopamine binding and the high affinity agonist-binding component of dopamine/[ 3 H]flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays.

Science.gov (United States)

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-12-01

To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques-an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (kappa > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (kappa = 0.63). The assay kit marketed as Varelisa allows accurate detection of LKM1.

PET imaging evaluation of [{sup 18}F]DBT-10, a novel radioligand specific to α{sub 7} nicotinic acetylcholine receptors, in nonhuman primates

Energy Technology Data Exchange (ETDEWEB)

Hillmer, Ansel T.; Zheng, Ming-Qiang; Li, Songye; Lin, Shu-fei; Holden, Daniel; Labaree, David; Ropchan, Jim; Carson, Richard E.; Huang, Yiyun [Yale University, PET Center, 801 Howard Ave, PO Box 208048, New Haven, CT (United States); Scheunemann, Matthias; Teodoro, Rodrigo; Deuther-Conrad, Winnie; Brust, Peter [Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Leipzig (Germany)

2016-03-15

Positron emission tomography (PET) radioligands specific to α{sub 7} nicotinic acetylcholine receptors (nAChRs) afford in vivo imaging of this receptor for neuropathologies such as Alzheimer's disease, schizophrenia, and substance abuse. This work aims to characterize the kinetic properties of an α{sub 7}-nAChR-specific radioligand, 7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-2-[{sup 18}F]-fluorodibenzo[b,d]thiophene 5,5-dioxide ([{sup 18}F]DBT-10), in nonhuman primates. [{sup 18}F]DBT-10 was produced via nucleophilic substitution of the nitro-precursor. Four Macaca mulatta subjects were imaged with [{sup 18}F]DBT-10 PET, with measurement of [{sup 18}F]DBT-10 parent concentrations and metabolism in arterial plasma. Baseline PET scans were acquired for all subjects. Following one scan, ex vivo analysis of brain tissue was performed to inspect for radiolabeled metabolites in brain. Three blocking scans with 0.69 and 1.24 mg/kg of the α{sub 7}-nAChR-specific ligand ASEM were also acquired to assess dose-dependent blockade of [{sup 18}F]DBT-10 binding. Kinetic analysis of PET data was performed using the metabolite-corrected input function to calculate the parent fraction corrected total distribution volume (V{sub T}/f{sub P}). [{sup 18}F]DBT-10 was produced within 90 min at high specific activities of 428 ± 436 GBq/μmol at end of synthesis. Metabolism of [{sup 18}F]DBT-10 varied across subjects, stabilizing by 120 min post-injection at parent fractions of 15-55 %. Uptake of [{sup 18}F]DBT-10 in brain occurred rapidly, reaching peak standardized uptake values (SUVs) of 2.9-3.7 within 30 min. The plasma-free fraction was 18.8 ± 3.4 %. No evidence for radiolabeled [{sup 18}F]DBT-10 metabolites was found in ex vivo brain tissue samples. Kinetic analysis of PET data was best described by the two-tissue compartment model. Estimated V{sub T}/f{sub P} values were 193-376 ml/cm{sup 3} across regions, with regional rank order of thalamus > frontal cortex > striatum

Characterization of (/sup 3/H)paroxetine binding in rat brain

Energy Technology Data Exchange (ETDEWEB)

Marcusson, J.O.; Bergstroem, M.E.; Eriksson, K.; Ross, S.B.

1988-06-01

The binding of the 5-hydroxytryptamine (5-HT, serotonin) uptake inhibitor (3H)paroxetine to rat cortical homogenates has been characterized. The effect of tissue concentration was examined and, with 0.75 mg wet weight tissue/ml in a total volume of 1,600 microliter, the binding was optimized with an apparent dissociation constant (KD) of 0.03-0.05 nM. Competition experiments with 5-HT, citalopram, norzimeldine, and desipramine revealed a high (90%) proportion of displaceable binding that fitted a single-site binding model. Fluoxetine and imipramine revealed, in addition to a high-affinity (nanomolar) site, also a low-affinity (micromolar) site representing approximately 10% of the displaceable binding. The specificity of the (3H)paroxetine binding was emphasized by the fact that 5-HT was the only active neurotransmitter bound and that the serotonin S1 and S2 antagonist methysergide was without effect on the binding. Both 5-HT- and fluoxetine-sensitive (3H)paroxetine binding was completely abolished after protease treatment, suggesting that the binding site is of protein nature. Saturation studies with 5-HT (100 microM) sensitive (3H)paroxetine binding were also consistent with a single-site binding model, and the binding was competitively inhibited by 5-HT and imipramine. The number of binding sites (Bmax) for 5-HT-sensitive (3H)paroxetine and (3H)imipramine binding was the same, indicating that the radioligands bind to the same sites. Lesion experiments with p-chloroamphetamine resulted in a binding in frontal and parietal cortices becoming undetectable and a greater than 60% reduction in the striatum and hypothalamus, indicating a selective localization on 5-HT terminals. Together these findings suggest that (3H)paroxetine specifically and selectively labels the substrate recognition site for 5-HT uptake in rat brain.

[123I]FP-CIT binding in rat brain after acute and sub-chronic administration of dopaminergic medication

International Nuclear Information System (INIS)

Lavalaye, J.; Knol, R.J.J.; Bruin, K. de; Reneman, L.; Booij, J.; Janssen, A.G.M.

2000-01-01

The recently developed radioligand [ 123 I]FP-CIT is suitable for clinical single-photon emission tomography (SPET) imaging of the dopamine (DA) transporter in vivo. To date it has remained unclear whether dopaminergic medication influences the striatal [ 123 I]FP-CIT binding. The purpose of this study was to investigate the influence of this medication on [ 123 I]FP-CIT binding in the brain. We used an animal model in which we administered dopaminomimetics, antipsychotics and an antidepressant. In vivo [ 123 I]FP-CIT binding to the DA and serotonin transporters was evaluated after sub-chronic and acute administration of the drugs. The administered medication induced small changes in striatal [ 123 I]FP-CIT binding which were not statistically significant. As expected, the DA reuptake blocker GBR 12,909 induced a significant decrease in [ 123 I]FP-CIT binding. [ 123 I]FP-CIT binding in the serotonin-rich hypothalamus was decreased only after acute administration of fluvoxamine. The results of this study suggest that dopaminergic medication will not affect the results of DA transporter SPET imaging with [ 123 I]FP-CIT. (orig.)

Candidate PET radioligands for cannabinoid CB1 receptors: [18F]AM5144 and related pyrazole compounds

International Nuclear Information System (INIS)

Li Zizhong; Gifford, Andrew; Liu Qian; Thotapally, Rajesh; Ding Yushin; Makriyannis, Alexandros; Gatley, S. John

2005-01-01

Introduction: The mammalian brain contains abundant G protein-coupled cannabinoid CB 1 receptors that respond to Δ 9 -tetrahydrocannabinol, the active ingredient of cannabis. The availability of a positron emission tomography (PET) radioligand would facilitate studies of the addictive and medicinal properties of compounds that bind to this receptor. Among the known classes of ligands for CB 1 receptors, the pyrazoles are attractive targets for radiopharmaceutical development because they are antagonists and are generally less lipophilic than the other classes. Methods: A convenient high-yield synthesis of N-(4-[ 18 F]fluorophenyl)-5-(4-bromophenyl)-1-(2,4-dichlorophenyl)- 1H-pyrazole-3-carboxamide (AM5144) was devised by coupling the appropriate pyrazole-3-carboxyl chloride compound with 4-[ 18 F]fluoroaniline. The labeled precursor was synthesized from 1-[ 18 F]fluoro-4-nitrobenzene in 60% radiochemical yield for 10 min using an improved procedure involving sodium borohydride reduction with cobalt chloride catalysis. The product was purified by HPLC to give a specific activity >400 mCi/μmol and a radiochemical purity >95%, and a PET study was conducted in a baboon. Results: Although the regional uptake of AM5144 in baboon brain was consistent with binding to cannabinoid CB 1 receptors, absolute uptake at 1 receptor ligands is not surprising because of their high lipophilicity as compared with most brain PET radiotracers. However, for nine pyrazole compounds for which rodent data are available, brain uptake and calculated logP values are not correlated. Thus, high logP values should not preclude evaluation of radiotracers for targets such as the CB 1 receptor that may require very lipophilic ligands

A generic template for automated bioanalytical ligand-binding assays using modular robotic scripts in support of discovery biotherapeutic programs.

Science.gov (United States)

Duo, Jia; Dong, Huijin; DeSilva, Binodh; Zhang, Yan J

2013-07-01

Sample dilution and reagent pipetting are time-consuming steps in ligand-binding assays (LBAs). Traditional automation-assisted LBAs use assay-specific scripts that require labor-intensive script writing and user training. Five major script modules were developed on Tecan Freedom EVO liquid handling software to facilitate the automated sample preparation and LBA procedure: sample dilution, sample minimum required dilution, standard/QC minimum required dilution, standard/QC/sample addition, and reagent addition. The modular design of automation scripts allowed the users to assemble an automated assay with minimal script modification. The application of the template was demonstrated in three LBAs to support discovery biotherapeutic programs. The results demonstrated that the modular scripts provided the flexibility in adapting to various LBA formats and the significant time saving in script writing and scientist training. Data generated by the automated process were comparable to those by manual process while the bioanalytical productivity was significantly improved using the modular robotic scripts.

Synthesis and biological evaluation of I-125/I-123-labelled analogues of citalopram and escitalopram as potential radioligands for imaging of the serotonin transporter

DEFF Research Database (Denmark)

Madsen, Jacob; Elfving, Betina; Frokjaer, Vibe G.

2011-01-01

Two novel radioligands for the serotonin transporter (SERT), [I-125]{3-[5-iodo-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]-propyl}-dimethylamine ([I-125]-2) and S-[I-125]{3-[5-iodo-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]-propyl}-dimethylamine ([I-125]-(S)-2) were synthesized in a ...... of the radioligand in imaging cortical SERT distribution in vivo. These data suggest that the iodine-labelled derivatives of citalopram and escitalopram are not superior to another SPECT tracer for the SERT, namely [I-123] ADAM....

Heterogeneous binding of sigma radioligands in the rat brain and liver; Possible relationship to subforms of cytochrome P-450

Energy Technology Data Exchange (ETDEWEB)

Ross, S B [Research Laboratories, Astra Research Centre AB, Soedertaejle (Sweden)

1991-01-01

The binding of four sigma receptor ligands, {sup 3}H-(+)-N-allyl-N-normetazocine ({sup 3}H-(+)-SKF 10,047), {sup 3}H-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ({sup 3}H-(+)-3-PPP), {sup 3}H-haloperidol and {sup 3}H-N,N'-di(o-totyl)guanidine ({sup 3}H-DTG), and the cytochrome P450IID6 ligand and dopamine uptake inhibitor {sup 3}H-1-(2-(diphenylmethoxy)ethyl)-4-(3-phenylpropyl)piperazine ({sup 3}H-GBR 12935) to membranal preparations of rat liver or whole rat brain was examined regarding kinetical properties and inhibition by various compounds with affinity for sigma binding sites or cytochrome P-450. In rat brain the density of binding sites was increased in order (+)-SKF 10,047<(+)-3-PPPbinding sites were also indicated by the low Hill coefficients found for most of the compounds studied. It was found that the cytochrome P-450 inhibitor proadifen (SKF 525A), like haloperidol, was a potent inhibitor of the binding of {sup 3}H-(+)-SKF 10,047, {sup 3}H-(+)-3-PPP and {sup 3}H-haloperidol to the liver and brain preparations, less active in inhibiting the binding of {sup 3}H-DTG and least effective on the binding of {sup 3}H-GBR 12935. Another cytochrome P-450 inhibitor, L-lobeline, was particularly potent in inhibiting the binding of {sup 3}H-DTG but was also quite potent inhibitor of the binding of the other sigma ligands. It was less potent in inhibiting the binding of {sup 3}H-GBR 12935. The binding of the latter ligand was potently inhibited by the analogous compound GBR 12909 but of the other compounds examined only L-lobeline, proadifen, haloperidol, DTG and (+)-3-PPP had IC50 values below 10 {mu}M. (Abstract Truncated)

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

Science.gov (United States)

Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

2017-01-01

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Effects of tamsulosin metabolites at alpha-1 adrenoceptor subtypes

NARCIS (Netherlands)

Taguchi, K.; Saitoh, M.; Sato, S.; Asano, M.; Michel, M. C.

1997-01-01

We have investigated the affinity and selectivity of tamsulosin and its metabolites, M1, M2, M3, M4 and AM1, at the tissue and the cloned alpha-1 adrenoceptor subtypes in the radioligand binding and the functional studies. In the radioligand binding studies, the compounds competed for [3H]prazosin

Early postnatal diagnosis of hereditary spherocytosis by combining light microscopy, acidified glycerol lysis test and eosin-5'-maleimide binding assay.

Science.gov (United States)

Andres, Oliver; Eber, Stefan; Speer, Christian P

2015-12-01

Exact diagnosis of hereditary spherocytosis (HS) is widely considered unreliable around birth. However, early postnatal diagnosis at the beginning of congenital hemolysis may be essential for managing neonatal anemia and hemolytic icterus, identifying those at high risk for severe hyperbilirubinemia, irreversible kernicterus, or sudden need for red cell transfusion. We analyzed 37 blood samples from neonates or infants up to six weeks of life that had been collected in-house or shipped to our laboratory due to suspected red cell membrane disorder. By combining assessment of red cell morphology, acidified glycerol lysis test (AGLT), and eosin-5'-maleimide (EMA) binding assay, we were able to clearly exclude HS in 22 and confirm HS in 10 patients, of which one had undergone red cell transfusion prior to blood sampling. Assessment of red cell morphology and normal test results allowed diagnosis of infantile pyknocytosis or Heinz body anemia in three neonates. Re-evaluation of five patients with inconsistent results of AGLT and EMA binding led to confirmation of HS in two cases. Automated analysis of hematologic parameters revealed elevated proportion of hyperdense cells to be a highly significant indicator for HS in neonatal infants. We showed that assessment of red cell morphology in combination with AGLT and EMA binding assay is a reliable basis for confirming or rejecting suspected diagnosis of HS even in neonates. Our data underline the necessity for blood sampling and laboratory exploration in suspected red cell membrane or enzyme defects at the earliest occasion.

Evaluation of PET Radioligands for the neuronal nicotinic acetylcholine receptor

International Nuclear Information System (INIS)

Schoenbaechler, R.; Westera, G.; Nan-Horng Lin

2002-01-01

Full text: A-186253.1, a compound made by Abbott laboratories, was labelled with carbon-11 and evaluated as a PET ligand for the neuronal nicotinic acetylcholine receptor (nAChR). The compound was labelled with C-11 by methylation with 11C-MeI of the desmethyl precursor A-183828.1. The affinity of A-186253.1 for the α4β2 and the α7 subtype of the nAChR was determined in displacement studies. PET-studies were performed in rats and pigs Inhibitory constants (K i ) versus cytsine were 461 ± 99 pM for A-186253.1 and versus α-Bungarotoxin >100 μM. which means a very high selectivity for the α4β2-receptor (>227,000). Highest uptake of [ 11 C]-A-186253.1 was observed in the thalamus where an increase in radiotracer uptake was seen until 45 min p.i.. Thereafter, the radiotracer concentration remained constant until the end of the scan indicating slow washout of [ 11 C]-A-186253.1. Application of cold A-186253.1 (0.5 mg/kg) 40 min p.i. resulted in a decrease in radiotracer concentration in the thalamus and the cortex indicating displacement of [ 11 C]-A-186253.1. Blockade studies with cytisine (0.5 mg/kg), a selective ligand for the α4β2 nicotinic receptor, showed just a slight reduction of the radioligand uptake in the thalamus and in the cortex whereas the blockade with cold A-186253.1 (1 mg/kg) resulted in a 50 % reduction. These results suggest, that 50 % of the [ 11 C]-A-186253.1 in the brain corresponds to specifically bound radioligand, but not to the α4β2 subtype of the nicotinic receptor. (author)

DNA-Aptamers Binding Aminoglycoside Antibiotics

Directory of Open Access Journals (Sweden)

Nadia Nikolaus

2014-02-01

Full Text Available Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

Directory of Open Access Journals (Sweden)

Kishore Polireddy

Full Text Available ABCB6 is a member of the adenosine triphosphate (ATP-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS, can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

Clinical value of determination of TSH-binding inhibiting immunoglobulins (TBII) by a radioreceptor assay

International Nuclear Information System (INIS)

Heberling, H.J.; Bierwolf, B.; Lohmann, D.

1986-01-01

The clinical value of a commercial kit for determination of TBII was evaluated. 50 patients with untreated Graves' disease, 21 patients with Graves' disease before and during medical therapy, 18 patients after finishing medical therapy and 10 patients after surgical treatment were examined. Besides these, 41 patients with other thyroid diseases and 36 patients without any thyroid disorder were included. In 47 (94%) of 50 patients with untreated Graves' disease TBII were detectable in serum using a TSH standard curve. Binding activities exceeding 10 U/l TSH equivalents were regarded as positive. In other thyroid diseases TBII were negative with the exception of 3 of 22 patient with autonomously functioning thyroid nodules. After 12 months of antithyroid drug treatment of 19 patients the incidence of positive antibody findings was 26%. During follow-up after medical therapy (1-9 years) 7 of 18 patients had increased TBII in correlation with clinical and functional findings. The determination of TBII by TRAK assay proved to be a sensitive and specific method. The assay can be used to differentiate between hyperthyroidism of autoimmune or non-immunogenic origin. Thus the method seems to be helpful for the follow-up under medical treatment of patients with Graves' disease. (author)

Effect of reagins and allergen extracts on radioallergosorbent assays for mite allergen

International Nuclear Information System (INIS)

Tovey, E.R.; Vandenberg, R.A.

1978-01-01

The reproducibility of the radioallergosorbent (RAST) inhibition and direct binding assays with mite allergen were investigated in the presence of heterogeneous extracts and non-mite sensitive atopic sera. Both contain components similar to potential contaminants which would occur in the assay of mite allergen and dust allergen extracts. The standardized inhibition and direct binding assays employed had a day to day (n = 4) coefficient of variation [(s.d. x 100)/mean] of 15% and 24% respectively. The inhibition assay for mite allergen was reproducible in the presence of protein concentrations of added plant, fungal, arthropod and animal extracts in excess of the protein concentrations that occur under the operational mite assay conditions. The mite inhibition assay was also reproducible in the presence of non-mite allergen extracts, with and without additional sera containing IgE specific for the non0mite allergens. The binding of a constant quantity of mite allergen to the activated solid phase in the direct binding assay was reproducible in the presence of added bovine serum albumin, and of a fungal or arthropod extract, representing the heterogeneous components of an allergen extract at the concentrations of total protein known to occur in the direct binding assay of mite extracts. (author)

Comparative Evaluation of the Translocator Protein Radioligands 11C-DPA-713, 18F-DPA- 714, and 11C-PK11195 in a Rat Model of Acute Neuro-inflammation

International Nuclear Information System (INIS)

Chauveau, F.; Van Camp, N.; Dolle, F.; Kuhnast, B.; Hinnen, F.; Damont, A.; Boutin, H.; Tavitian, B.; Chauveau, F.; Van Camp, N.; Boutin, H; Tavitian, B.; Chauveau, F.; Boutin, H.; James, M.; Kassiou, M.; Kassiou, M.

2009-01-01

Overexpression of the translocator protein, TSPO (18 kDa), formerly known as the peripheral benzodiazepine receptor, is a hallmark of activation of cells of monocytic lineage (micro-glia and macrophages) during neuro-inflammation. Radiolabeling of TSPO ligands enables the detection of neuro-inflammatory lesions by PET. Two new radioligands, 11 C-labeled N, N-diethyl-2-[2-(4- methoxy-phenyl)-5, 7-dimethylpyrazolo[1, 5-a]pyrimidin-3-yl] acetamide (DPA-713) and 18 F-labeled N, N-diethyl-2-(2-(4-(2- fluoroethoxy)phenyl)-5, 7-dimethylpyrazolo[1, 5-a]pyrimidin-3-yl) acetamide (DPA-714), both belonging to the pyrazolopyrimidine class, were compared in vivo and in vitro using a rodent model of neuro-inflammation. Methods: 11 C-DPA-713 and 18 F-DPA-714, as well as the classic radioligand 11 C-labeled (R)-N-methyl- N-(1-methylpropyl)-1-(2-chlorophenyl)isoquinoline-3-carboxamide (PK11195), were used in the same rat model, in which intra-striatal injection of (R, S)-a-amino-3-hydroxy-5-methyl-4-isoxazolopropionique gave rise to a strong neuro-inflammatory response. Comparative endpoints included in vitro autoradiography and in vivo imaging on a dedicated small-animal PET scanner under identical conditions. Results: 11 C-DPA-713 and 18 F-DPA-714 could specifically localize the neuro-inflammatory site with a similar signal-to-noise ratio in vitro. In vivo, 18 F-DPA-714 performed better than 11 C-DPA-713 and 11 C-PK11195, with the highest ratio of ipsilateral to contralateral uptake and the highest binding potential. Conclusion: 18 F-DPA-714 appears to be an attractive alternative to 11 C-PK11195 because of its increased bioavailability in brain tissue and its reduced nonspecific binding. Moreover, its labeling with 18 F, the preferred PET isotope for radiopharmaceutical chemistry, favors its dissemination and wide clinical use. 18 F-DPA-714 will be further evaluated in longitudinal studies of neuro-inflammatory conditions such as are encountered in stroke or neuro

The production of KIR-Fc fusion proteins and their use in a multiplex HLA class I binding assay.

Science.gov (United States)

Hilton, Hugo G; Moesta, Achim K; Guethlein, Lisbeth A; Blokhuis, Jeroen; Parham, Peter; Norman, Paul J

2015-10-01

Soluble recombinant proteins that comprise the extracellular part of a surface expressed receptor attached to the Fc region of an IgG antibody have facilitated the determination of ligand specificity for an array of immune system receptors. Among such receptors is the family of killer cell immunoglobulin-like receptors (KIR) that recognize HLA class I ligands. These receptors, expressed on natural killer (NK) cells and T cells, play important roles in both immune defense and placental development in early pregnancy. Here we describe a method for the production of two domain KIR-Fc fusion proteins using baculovirus infected insect cells. This method is more scalable than traditional mammalian cell expression systems and produces efficiently folded proteins that carry posttranslational modifications found in native KIR. We also describe a multiplex binding assay using the Luminex platform that determines the avidity and specificity of two domain KIR-Fc for a panel of microbeads, each coated with one of 97 HLA class I allotypes. This assay is simple to perform, and represents a major improvement over the assays used previously, which were limited in the number of KIR and HLA class I combinations that could be assayed at any one time. The results obtained from this assay can be used to predict the response of NK cell and T cells when their KIR recognize HLA class I. Copyright © 2015 Elsevier B.V. All rights reserved.

Biodistribution and dosimetry of iodine-123-labelled Z-MIVE: an oestrogen receptor radioligand for breast cancer imaging

NARCIS (Netherlands)

Rijks, L. J.; Busemann Sokole, E.; Stabin, M. G.; de Bruin, K.; Janssen, A. G.; van Royen, E. A.

1998-01-01

This study reports on the distribution and radiation dosimetry of iodine-123-labelled cis-11beta-methoxy-17alpha-iodovinyloestradiol (Z-[123I]MIVE), a promising radioligand for imaging of oestrogen receptors (ERs) in human breast cancer. Whole-body scans were performed up to 24 h after intravenous

Radiosynthesis and in vivo evaluation of [{sup 11}C]MP-10 as a positron emission tomography radioligand for phosphodiesterase 10A

Energy Technology Data Exchange (ETDEWEB)

Plisson, Christophe, E-mail: Christophe.2.plisson@gsk.com [GlaxoSmithKline, Clinical Imaging Centre Hammersmith Hospital, London, W12 0NN (United Kingdom); Salinas, Cristian [GlaxoSmithKline, Clinical Imaging Centre Hammersmith Hospital, London, W12 0NN (United Kingdom); Weinzimmer, David; Labaree, David; Lin, Shu-Fei; Ding, Yu-Shin [Yale University PET Center, Yale University School of Medicine, PO Box 208048 New Haven, CT (United States); Jakobsen, Steen [Aarhus PET Centre, Aarhus Sygehus, Norrebrogade 44, DK-8000 Aarhus C (Denmark); Smith, Paul W. [GlaxoSmithKline, Clinical Imaging Centre Hammersmith Hospital, London, W12 0NN (United Kingdom); Eiji, Kawanishi [Medicinal Chemistry Research Laboratories II, Research Division, Mitsubishi Tanabe Pharma Corporation, Saitama 335-8505 (Japan); Carson, Richard E. [Yale University PET Center, Yale University School of Medicine, PO Box 208048 New Haven, CT (United States); Gunn, Roger N.; Rabiner, Eugenii A. [GlaxoSmithKline, Clinical Imaging Centre Hammersmith Hospital, London, W12 0NN (United Kingdom)

2011-08-15

Introduction: The aim of this study was to evaluate a newly reported positron emission tomography (PET) radioligand [{sup 11}C]MP-10, a potent and selective inhibitor of the central phosphodiesterase 10A enzyme (PDE10A) in vivo, using PET. Methods: A procedure was developed for labeling MP-10 with carbon-11. [{sup 11}C]MP-10 was evaluated in vivo both in the pig and baboon brain. Results: Alkylation of the corresponding desmethyl compound with [{sup 11}C]methyl iodide produced [{sup 11}C]MP-10 with good radiochemical yield and specific activity. PET studies in the pig showed that [{sup 11}C]MP-10 rapidly entered the brain reaching peak tissue concentration at 1-2 min postadministration, followed by washout from the tissue. Administration of a selective PDE10A inhibitor reduced the binding in all brain regions to the levels of the cerebellum, demonstrating the saturability and selectivity of [{sup 11}C]MP-10 binding. In the nonhuman primate, the brain tissue kinetics of [{sup 11}C]MP-10 were slower, reaching peak tissue concentrations at 30-60 min postadministration. In both species, the observed rank order of regional brain signal was striatum>diencephalon>cortical regions=cerebellum, consistent with the known distribution and concentration of PDE10A. [{sup 11}C]MP-10 brain kinetics were well described by a two-tissue compartment model, and estimates of total volume of distribution (V{sub T}) were obtained. Blocking studies with unlabeled MP-10 revealed the suitability of the cerebellum as a reference tissue and enabled the estimation of regional binding potential (BP{sub ND}) as the outcome measure of specific binding. Quantification of [{sup 11}C]MP-10 binding using the simplified reference tissue model with cerebellar input function produced BP{sub ND} estimates consistent with those obtained by the two-tissue compartment model. Conclusion: We demonstrated that [{sup 11}C]MP-10 possesses good characteristics for the in vivo quantification of the PDE10A in the

Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

Science.gov (United States)

Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

2015-02-21

This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions.

Science.gov (United States)

Srikant, C B; Dahan, A; Craig, C

1990-02-04

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2

Molecular imaging of {alpha}7 nicotinic acetylcholine receptors: design and evaluation of the potent radioligand [{sup 18}F]NS10743

Energy Technology Data Exchange (ETDEWEB)

Deuther-Conrad, Winnie; Fischer, Steffen; Hiller, Achim; Brust, Peter [Institute of Interdisciplinary Isotope Research, Leipzig (Germany); Oestergaard Nielsen, Elsebet; Brunicardi Timmermann, Daniel; Peters, Dan [NeuroSearch A/S, Ballerup (Denmark); Steinbach, Joerg [Institute of Interdisciplinary Isotope Research, Leipzig (Germany); Forschungszentrum Dresden-Rossendorf, Institute of Radiopharmacy, Dresden (Germany); Sabri, Osama [Universitaet Leipzig, Department of Nuclear Medicine, Leipzig (Germany)

2009-05-15

The outstanding diversity of cellular properties mediated by neuronal and nonneuronal {alpha}7 nicotinic acetylcholine receptors ({alpha}7 nAChR) points to the diagnostic potential of quantitative nuclear molecular imaging of {alpha}7 nAChR in neurology and oncology. It was our goal to radiolabel the {alpha}7 nAChR agonist 4-[5-(4-fluoro-phenyl)-[1,3,4]oxadiazol-2-yl]-1,4-diaza-bicyclo[3.2.2]nonane (NS10743) and to assess the selectivity of [{sup 18}F]NS10743 binding site occupancy in animal experiments. [{sup 18}F]NS10743 was synthesized by nucleophilic substitution of the nitro precursor. In vitro receptor affinity and selectivity were assessed by radioligand competition and autoradiography. The radiotracer properties were evaluated in female CD-1 mice by brain autoradiography and organ distribution. Target specificity was validated after treatment with SSR180711 (10 mg/kg, intraperitoneal), and metabolic stability was investigated using radio-HPLC. The specific activity of [{sup 18}F]NS10743 exceeded 150 GBq/{mu}mol at a radiochemical purity >99%. In vitro, NS10743 and [{sup 18}F]NS10743 showed high affinity and specificity towards {alpha}7 nAChR. The brain permeation of [{sup 18}F]NS10743 was fast and sufficient with values of 4.83 and 1.60% injected dose per gram and brain to plasma ratios of 3.83 and 2.05 at 5 and 60 min after radiotracer administration. Brain autoradiography and organ distribution showed target-specific accumulation of [{sup 18}F]NS10743 in brain substructures and various {alpha}7 nAChR-expressing organs. The radiotracer showed a high metabolic stability in vivo with a single polar radiometabolite, which did not cross the blood-brain barrier. The good in vitro and in vivo features of [{sup 18}F]NS10743 make this radioligand a promising candidate for quantitative in vivo imaging of {alpha}7 nAChR expression and encourage further investigations. (orig.)

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

Directory of Open Access Journals (Sweden)

M.H. Sonobe

2007-01-01

Full Text Available A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test. In this study, the serum titers (values between 1.0 and 19.5 IU measured by a modified TOBI test (Modi-TOBI test and toxin neutralization assays were correlated (P < 0.0001. Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001 and for diphtheria (r² = 0.93, P < 0.0001 between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

Metabolism and quantification of [18F]DPA-714, a new TSPO positron emission tomography radioligand

International Nuclear Information System (INIS)

Peyronneau, Marie-Anne; Saba, Wadad; Goutal, Sebastien; Damont, Annelaure; Dolle, Frederic; Bottlaender, Michel; Valette, Heric; Kassiou, Michael

2013-01-01

[ 18 F]DPA-714 [N,N-diethyl-2-(2-(4-(2[ 18 F]-fluoroethoxy)phenyl) 5,7-dimethyl-pyrazolo[1,5a]pyrimidin-3-yl)acetamide] is a new radioligand currently used for imaging the 18-kDa translocator protein in animal models of neuro-inflammation and recently in humans. The biodistribution by positron emission tomography (PET) in baboons and the in vitro and in vivo metabolism of [ 18 F]DPA-714 were investigated in rats, baboons, and humans. Whole-body PET experiments showed a high uptake of radioactivity in the kidneys, heart, liver, and gallbladder. The liver was a major route of elimination of [ 18 F]DPA-714, and urine was a route of excretion for radio-metabolites. In rat and baboon plasma, high-performance liquid chromatography (HPLC) metabolic profiles showed three major radio-metabolites accounting for 85% and 89% of total radioactivity at 120 minutes after injection, respectively. Rat microsomal incubations and analyses by liquid chromatography-mass spectrometry (LC-MS) identified seven metabolites, characterized as O-de-ethyl, hydroxyl, and N-de-ethyl derivatives of nonradioactive DPA-714, two of them having the same retention times than those detected in rat and baboon plasma. The third plasma radio-metabolite was suggested to be a carboxylic acid compound that accounted for 15% of the rat brain radioactivity. O-de-ethylation led to a nonradioactive compound and [ 18 F] fluoroacetic acid. Human CYP3A4 and CYP2D6 were shown to be involved in the oxidation of the radioligand. Finally an easy, rapid, and accurate method-indispensable for PET quantitative clinical studies - for quantifying [ 18 F]DPA-714 by solid-phase extraction was developed. In vivo, an extensive metabolism of [ 18 F]DPA-714 was observed in rats and baboons, identified as [ 18 F]de-ethyl, [ 18 F]hydroxyl, and [ 18 F]carboxylic acid derivatives of [ 18 F]DPA-714. The main route of excretion of the unchanged radioligand in baboons was hepatobiliary while that of radio-metabolites was the urinary

Molecular imaging of {sigma} receptors: synthesis and evaluation of the potent {sigma}{sub 1} selective radioligand [{sup 18}F]fluspidine

Energy Technology Data Exchange (ETDEWEB)

Fischer, Steffen; Hiller, Achim; Deuther-Conrad, Winnie; Scheunemann, Matthias; Steinbach, Joerg; Brust, Peter [Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, Research Site Leipzig, Interdisciplinary Isotope Research, Leipzig (Germany); Wiese, Christian; Grosse Maestrup, Eva; Schepmann, Dirk; Wuensch, Bernhard [Institut fuer Pharmazeutische und Medizinische Chemie der Westfaelischen Wilhelms-Universitaet Muenster, Muenster (Germany)

2011-03-15

Neuroimaging of {sigma}{sub 1} receptors in the human brain has been proposed for the investigation of the pathophysiology of neurodegenerative and psychiatric diseases. However, there is a lack of suitable {sup 18}F-labelled PET radioligands for that purpose. The selective {sigma}{sub 1} receptor ligand [{sup 18}F]fluspidine (1'-benzyl-3-(2-[{sup 18}F]fluoroethyl)-3H-spiro[[2]benzofuran-1,4'-piperidine]) was synthesized by nucleophilic {sup 18}F{sup -} substitution of the tosyl precursor. In vitro receptor binding affinity and selectivity were assessed by radioligand competition in tissue homogenate and autoradiographic approaches. In female CD-1 mice, in vivo properties of [{sup 18}F]fluspidine were evaluated by ex vivo brain section imaging and organ distribution of intravenously administered radiotracer. Target specificity was validated by organ distribution of [{sup 18}F]fluspidine after treatment with 1 mg/kg i.p. of the {sigma} receptor antagonist haloperidol or the emopamil binding protein (EBP) inhibitor tamoxifen. In vitro metabolic stability and in vivo metabolism were investigated by LC-MS{sup n} and radio-HPLC analysis. [{sup 18}F]Fluspidine was obtained with a radiochemical yield of 35-45%, a radiochemical purity of {>=} 99.6% and a specific activity of 150-350 GBq/{mu}mol (n = 6) within a total synthesis time of 90-120 min. In vitro, fluspidine bound specifically and with high affinity to {sigma}{sub 1} receptors (K{sub i} = 0.59 nM). In mice, [{sup 18}F]fluspidine rapidly accumulated in brain with uptake values of 3.9 and 4.7%ID/g and brain to blood ratios of 7 and 13 at 5 and 30 min after intravenous application of the radiotracer, respectively. By ex vivo autoradiography of brain slices, resemblance between binding site occupancy of [{sup 18}F]fluspidine and the expression of {sigma}{sub 1} receptors was shown. The radiotracer uptake in the brain as well as in peripheral {sigma}{sub 1} receptor expressing organs was significantly

Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

Directory of Open Access Journals (Sweden)

Yaron Orenstein

Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

Pet's research at the SHFJ, Cea, one example: development and validation of a radioligand for the study of the cerebral dopaminergic system

International Nuclear Information System (INIS)

Ribeiro, M.J.

2005-01-01

The aim of this work is the evaluation of biodistribution and radiation dosimetry of a cocaine analog, the N-(3-iodo-prop-2E-enyl)-2beta-carbo-methoxy-3beta-(4-methyl-phenyl) nor-tropane (PE2I), labeled with carbon 11 ([ 11 C]PE2I). The [ 11 C]PE2I is a selective radioligand for imaging neuronal dopamine transporter (DAT) with positron emission tomography (PET). The DAT is a membrane-bound pre synaptically located protein that regulates the concentration of dopamine at nerve terminals. DAT radioligands are often used to evaluate the progression of Parkinson's disease or the efficiency of neuro-protective therapeutics and, typically, these studies required several successive PET scans. (author)

The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

Science.gov (United States)

Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

2017-12-05

Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of ["1"1C]Lu AE92686 as a PET radioligand for phosphodiesterase 10A in the nonhuman primate brain

International Nuclear Information System (INIS)

Yang, Kai-Chun; Stepanov, Vladimir; Amini, Nahid; Martinsson, Stefan; Takano, Akihiro; Halldin, Christer; Nielsen, Jacob; Bundgaard, Christoffer; Bang-Andersen, Benny; Grimwood, Sarah; Farde, Lars; Finnema, Sjoerd J.

2017-01-01

["1"1C]Lu AE92686 is a positron emission tomography (PET) radioligand that has recently been validated for examining phosphodiesterase 10A (PDE10A) in the human striatum. ["1"1C]Lu AE92686 has high affinity for PDE10A (IC_5_0 = 0.39 nM) and may also be suitable for examination of the substantia nigra, a region with low density of PDE10A. Here, we report characterization of regional ["1"1C]Lu AE92686 binding to PDE10A in the nonhuman primate (NHP) brain. A total of 11 PET measurements, seven baseline and four following pretreatment with unlabeled Lu AE92686 or the structurally unrelated PDE10A inhibitor MP-10, were performed in five NHPs using a high resolution research tomograph (HRRT). ["1"1C]Lu AE92686 binding was quantified using a radiometabolite-corrected arterial input function and compartmental and graphical modeling approaches. Regional time-activity curves were best described with the two-tissue compartment model (2TCM). However, the distribution volume (V_T) values for all regions were obtained by the Logan plot analysis, as reliable cerebellar V_T values could not be derived by the 2TCM. For cerebellum, a proposed reference region, V_T values increased by ∝30 % with increasing PET measurement duration from 63 to 123 min, while V_T values in target regions remained stable. Both pretreatment drugs significantly decreased ["1"1C]Lu AE92686 binding in target regions, while no significant effect on cerebellum was observed. Binding potential (BP_N_D) values, derived with the simplified reference tissue model (SRTM), were 13-17 in putamen and 3-5 in substantia nigra and correlated well to values from the Logan plot analysis. The method proposed for quantification of ["1"1C]Lu AE92686 binding in applied studies in NHP is based on 63 min PET data and SRTM with cerebellum as a reference region. The study supports that ["1"1C]Lu AE92686 can be used for PET examinations of PDE10A binding also in substantia nigra. (orig.)

Characterization of [{sup 11}C]Lu AE92686 as a PET radioligand for phosphodiesterase 10A in the nonhuman primate brain

Energy Technology Data Exchange (ETDEWEB)

Yang, Kai-Chun; Stepanov, Vladimir; Amini, Nahid; Martinsson, Stefan; Takano, Akihiro; Halldin, Christer [Karolinska Institutet, Karolinska University Hospital, Department of Clinical Neuroscience, Center for Psychiatric Research, Stockholm (Sweden); Nielsen, Jacob [H. Lundbeck A/S, Synaptic Transmission, Valby (Denmark); Bundgaard, Christoffer; Bang-Andersen, Benny [H. Lundbeck A/S, Discovery Chemistry and DMPK, Valby (Denmark); Grimwood, Sarah [Pfizer Inc., Neuroscience and Pain Research Unit, Cambridge, MA (United States); Farde, Lars [Karolinska Institutet, Karolinska University Hospital, Department of Clinical Neuroscience, Center for Psychiatric Research, Stockholm (Sweden); AstraZeneca PET Science Center at Karolinska Institutet, Personalized Health Care and Biomarkers, Stockholm (Sweden); Finnema, Sjoerd J. [Karolinska Institutet, Karolinska University Hospital, Department of Clinical Neuroscience, Center for Psychiatric Research, Stockholm (Sweden); Yale University, Department of Radiology and Biomedical Imaging, New Haven, CT (United States)

2017-02-15

[{sup 11}C]Lu AE92686 is a positron emission tomography (PET) radioligand that has recently been validated for examining phosphodiesterase 10A (PDE10A) in the human striatum. [{sup 11}C]Lu AE92686 has high affinity for PDE10A (IC{sub 50} = 0.39 nM) and may also be suitable for examination of the substantia nigra, a region with low density of PDE10A. Here, we report characterization of regional [{sup 11}C]Lu AE92686 binding to PDE10A in the nonhuman primate (NHP) brain. A total of 11 PET measurements, seven baseline and four following pretreatment with unlabeled Lu AE92686 or the structurally unrelated PDE10A inhibitor MP-10, were performed in five NHPs using a high resolution research tomograph (HRRT). [{sup 11}C]Lu AE92686 binding was quantified using a radiometabolite-corrected arterial input function and compartmental and graphical modeling approaches. Regional time-activity curves were best described with the two-tissue compartment model (2TCM). However, the distribution volume (V{sub T}) values for all regions were obtained by the Logan plot analysis, as reliable cerebellar V{sub T} values could not be derived by the 2TCM. For cerebellum, a proposed reference region, V{sub T} values increased by ∝30 % with increasing PET measurement duration from 63 to 123 min, while V{sub T} values in target regions remained stable. Both pretreatment drugs significantly decreased [{sup 11}C]Lu AE92686 binding in target regions, while no significant effect on cerebellum was observed. Binding potential (BP{sub ND}) values, derived with the simplified reference tissue model (SRTM), were 13-17 in putamen and 3-5 in substantia nigra and correlated well to values from the Logan plot analysis. The method proposed for quantification of [{sup 11}C]Lu AE92686 binding in applied studies in NHP is based on 63 min PET data and SRTM with cerebellum as a reference region. The study supports that [{sup 11}C]Lu AE92686 can be used for PET examinations of PDE10A binding also in substantia

Ultrasensitive human thyrotropin (h TSH) immunoradiometric assay (IRMA) set up, through identification and minimization of non specific bindings

International Nuclear Information System (INIS)

Peroni, C.N.

1994-01-01

An IRMA of h TSH, based on magnetic solid phase separation, was studied especially for what concerns its non specific bindings. These were identified as a product of the interaction between an altered form of radioiodinated anti-h TSH monoclonal antibody ( 125 I-m AB) and the uncoupled magnetizable cellulose particle (matrix). Apparently this form of 125 I-m AB is a type of aggregate that can be partly resolved from the main peak on Sephadex G-200 and further minimized via a single pre-incubation with the same matrix. Solid phase saturation with milk proteins, tracer storage at 4 0 C and serum addition during incubation were also found particularly effective is preventing its formation. These findings were used in order to reproducibly decrease non specific bindings to values 60 /B O ) up to values of 300-500. This way we obtained h TSH radio assays with functional sensitivities of about 0.05 m IU/L and analytical sensitivities of the order of 0.02 m IU/L, which classify them at least as among the best second generation assays and that are excellent indeed for magnetic IRMA s. A more optimistic sensitivity calculation, based on Rodbard's definition, provided values down to 0.008 m IU/L. Such sensitivities, moreover, were obtained in a very reproducible way and all over the useful tracer life. (author). 83 refs, 13 figs, 25 tabs

Immunoradiometric assay for cytomegalovirus-specific IgG antibodies; Assay development and evaluation in blood transfusion practice

Energy Technology Data Exchange (ETDEWEB)

Klapper, P.E.; Cleator, G.M.; Prinja-Wolks, D.; Morris, D.J. (Medical School, Manchester (United Kingdom). Department of Medical microbiology, Virology Unit); Morell, G. (Regional Blood Transfusion Centre, manchester (United Kingdom))

1990-03-01

An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 (human cytomegalovirus (CMV)) has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specifity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT). Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate. (author). 23 refs.; 1 tab.

A model for evaluating steroids acting at the hypothalamus-pituitary axis using radioimmunoassay and related procedures

International Nuclear Information System (INIS)

Spona, J.; Bieglmayer, Ch.; Schroeder, R.; Poeckl, E.

1978-01-01

The relative affinity constants for binding of estrone (E 1 ), estriol (E 3 ), 17β-estradiol(E 2 ) and 17α-ethinyl-17β-estradiol(EE 2 ) to cytosol estrogen-receptors of rat hypothalamus and pituitary were estimated by a radioligand-receptor assay procedure. The relative affinity constants in the hypothalamic system were 6.5x10 -10 M for E 2 , 1x10 -9 M for EE 2 and 2x10 -8 M for E 1 and E 3 . The affinity constants were 1x10 -9 M for E 2 and E 3 and 7x10 -9 M for E 1 and E 3 when pituitary cytosol samples were used. Some discrepancies between biological activity and affinity for the estrogen-receptor were noted. These may be due to differences in the metabolism and cellular uptake of the estrogens. The radioligand-receptor assay procedure may be useful in evaluating the action of estrogens and anti-estrogens acting at the hypothalamic and pituitary level. Sedimentation patterns of cytosol samples labelled with the estrogens used in this study revealed, upon ultracentrifugation, protein moieties sedimenting in the 8 S region. The potency of progesterone and D-Norgestrel to modulate the release of LH and FSH stimulated by luteinizing hormone-releasing hormone (LH-RH) in castrated female rats was found to correlate well with the biological activity of the progestogens. It is concluded that the radioligand-receptor assay procedure for estrogens and the in-vivo model for the evaluation of the central action of progestogens may be valuable tools for testing new steroids to be used in oral contraceptives. (author)

Synthesis and biological evaluation in rat and cat of [{sup 18}F]12ST05 as a potential 5-HT{sub 6} PET radioligand

Energy Technology Data Exchange (ETDEWEB)

Tang, Sandrine [Institut de Chimie et Biochimie Moleculaires et Supramoleculaires, UMR CNRS 5246, Universite Lyon 1, Universite de Lyon, Lyon, 69677 (Cermep) (France); Verdurand, Mathieu [Laboratoire de Neuropharmacologie, FRE CNRS 3006, Universite Lyon 1, Universite de Lyon, Lyon, 69373 (FRE CNRS 3006) (France); CERMEP-Imagerie du Vivant, PET Department, Lyon, 69622 (ICBMS) (France); Joseph, Benoit [Institut de Chimie et Biochimie Moleculaires et Supramoleculaires, UMR CNRS 5246, Universite Lyon 1, Universite de Lyon, Lyon, 69677 (Cermep) (France); Lemoine, Laetitia [Laboratoire de Neuropharmacologie, FRE CNRS 3006, Universite Lyon 1, Universite de Lyon, Lyon, 69373 (FRE CNRS 3006) (France); CERMEP-Imagerie du Vivant, PET Department, Lyon, 69622 (ICBMS) (France); Daoust, Alexia [CERMEP-Imagerie du Vivant, PET Department, Lyon, 69622 (ICBMS) (France); Billard, Thierry; Fournet, Guy [Institut de Chimie et Biochimie Moleculaires et Supramoleculaires, UMR CNRS 5246, Universite Lyon 1, Universite de Lyon, Lyon, 69677 (Cermep) (France); Le Bars, Didier [Institut de Chimie et Biochimie Moleculaires et Supramoleculaires, UMR CNRS 5246, Universite Lyon 1, Universite de Lyon, Lyon, 69677 (Cermep) (France); CERMEP-Imagerie du Vivant, PET Department, Lyon, 69622 (ICBMS) (France); Zimmer, Luc [Laboratoire de Neuropharmacologie, FRE CNRS 3006, Universite Lyon 1, Universite de Lyon, Lyon, 69373 (FRE CNRS 3006) (France); CERMEP-Imagerie du Vivant, PET Department, Lyon, 69622 (ICBMS) (France)], E-mail: zimmer@univ-lyon1.fr

2007-11-15

Introduction: 5-hydroxytryptamine (5-HT){sub 6} receptors represent one of the more recently discovered serotoninergic receptor family. However, no 5-HT{sub 6} positron emission tomography (PET) radiotracer is currently used in clinical imaging studies. The purpose of this study was to propose the first fluorinated PET radiotracer for this brain receptor. Methods: A new compound presenting in vitro high affinity towards the serotoninergic 5-HT{sub 6} receptor, N-[2-(1-[(4-fluorophenyl)sulfonyl]-1H-indol-4-yloxy)ethyl] -N,N-dimethylamine, was labelled with fluorine 18 via a nitro-/fluoronucleophilic substitution. Biological evaluations included (i) in vitro and ex vivo autoradiographies in rat brain and (ii) a PET scan on anaesthetized cat. Results and Conclusion: Although the radioligand showed excellent brain penetration, it did not reveal any specific binding to the 5-HT{sub 6} receptors indicating that this radiotracer is not suitable for mapping 5-HT{sub 6} receptors using PET.

Immune chromatography: a quantitative radioimmunological assay

International Nuclear Information System (INIS)

Davis, J.W.; Demetriades, M.; Bowen, J.M.

1984-01-01

Immune chromatography, a radioimmunological binding assay, employs paper chromatography to separate immune complexes from free antigen and antibodies. During chromatography free antigen and antibodies become distributed throughout the paper, while immune complexes remain near the bottoms of the strips. The chromatographic differences can be made quantitative by using either iodinated antigens or antibodies. Under these conditions nanogram quantities of antigen can be detected or antibodies in sera diluted several 1000-fold. The immune chromatography assay can also be performed as an indirect assay, since the paper strips are cut from nitrocellulose paper. In this case the immune components are absorbed by the paper during chromatography. Antigen is then detected with an iodinated second antibody. The indirect immune chromatography assay is particularly useful for identifying different sera that react with the same antigen. Reaction with the first serum before chromatography reduces the amount of antigen available to the second serum following chromatography. In addition to characterizing the immune chromatography procedure, we discuss the possible applications of chromatography assays for the quantitation of other types of molecular binding interactions. (Auth.)

Radioimmunoassay of thyroid hormones

International Nuclear Information System (INIS)

Bartalena, L.; Mariotti, S.; Pinchera, A.

1987-01-01

For many years, methods based on iodine content determination have represented the only techniques available for the estimation of total thyroid hormone concentrations in serum. Subsequently, simple, sensitive, and specific radioligand assays for thyroid hormones have replaced these chemical methods. For the purpose of this chapter, iodometric techniques are only briefly summarized for their historical importance, whereas attention is focused on radioligand assays

Production and assay of forskolin antibodies

International Nuclear Information System (INIS)

Ho, L.T.; Ho, R.J.

1986-01-01

Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using 3 H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added 3 H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC 50 was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound

Equol is more active than soy isoflavone itself to compete for binding to thromboxane A(2) receptor in human platelets.

Science.gov (United States)

Muñoz, Yenny; Garrido, Argelia; Valladares, Luis

2009-03-01

Several dietary intervention studies examining the health effect of soy isoflavones allude to the importance of equol in establishing the cardiovascular response to soy protein. Although, the specific mechanism by which this action occurs has not been established. The aim of this study was to investigate the inhibitory effect of soy-isoflavones and the metabolite of daidzein, equol, on agonist-induced platelet responses dependent on thromboxane A(2) (TxA(2)) receptor. Competitive radioligand binding assay was used to screen for affinity of these compounds to the TxA(2) receptor. The effect of equol on platelet activation, evaluate through of release of the ATP, by analogs of TxA(2) was analyzed. The effect of equol on platelet aggregation was investigated with ADP, U46619 (a TxA(2) mimic) and the calcium ionophore A23187. The data showed that aglycone isoflavones and equol bind to TxA(2) receptor in the micromol/L range, whereas their glucoside derivates had very low binding activity for this receptor. Under equilibrium conditions, the following order of the relative affinity in inhibiting [(3)H]-SQ29585 binding was: equol>genistein>daidzein>glycitein>genistin, daidzin, glycitin. Equol interaction was reversible and competitive for labeled-SQ29548 with not apparent decrease in the number of TxA(2) binding sites. In addition, from platelet activation studies, equol effectively inhibited ATP secretion elicited by the TxA(2) analog U46619. On the other hand, equol inhibited the platelet aggregation induced by U46619 and A23187, while it failed to inhibit that induced by ADP. The aglycone isoflavones from soy, and particularly equol, have been found to have biological effects attributable to thromboxane A(2) receptor antagonism. These findings may help elucidate how dietary isoflavone modulate platelet function and explain why soy-rich foods are claimed to have beneficial effects in the prevention of thrombotic events.

Mechanism of Coomassie Brilliant Blue G-250 binding to cetyltrimethylammonium bromide: an interference with the Bradford assay.

Science.gov (United States)

Aminian, Mahdi; Nabatchian, Fariba; Vaisi-Raygani, Asad; Torabi, Mojgan

2013-03-15

The Bradford protein assay is a popular method because of its rapidity, sensitivity, and relative specificity. This method is subject to some interference by nonprotein compounds. In this study, we describe the interference of cetyltrimethylammonium bromide (CTAB) with the Bradford assay. This interference is based on the interaction of Coomassie Brilliant Blue G-250 (CBB) with this cationic detergent. This study suggests that both electrostatic and hydrophobic interactions are involved in the interaction of CTAB and CBB. The anionic and neutral forms of CBB bind to CTAB by electrostatic attraction, which accelerates hydrophobic interactions of these CBB forms and the hydrophobic tail of CTAB. Consequently, the hydrophobic regions of the dominant free cationic form of CBB dye compete for the tail of CTAB with two other forms of the dye and gradually displace the primary hydrophobic interactions and rearrange the primary CBB-CTAB complex. This interaction of CTAB and CBB dye produces a primary 650-nm-absorbing complex that then gradually rearranges to a complex that shows an absorbance shoulder at 800-950 nm. This study conclusively shows a strong response of CBB to CTAB that causes a time-dependent and nearly additive interference with the Bradford assay. This study also may promote an application of CBB for CTAB quantification. Copyright © 2012 Elsevier Inc. All rights reserved.

Developmental changes in the role of a pertussis toxin sensitive guanine nucleotide binding protein in the rat cardiac alpha1-adrenergic system

International Nuclear Information System (INIS)

Han, H.M.

1989-01-01

During development, the cardiac alpha 1 -adrenergic chronotropic response changes from positive in the neonate to negative in the adult. This thesis examined the possibility of a developmental change in coupling of a PT-sensitive G-protein to the alpha 1 -adrenergic receptor. Radioligand binding experiments performed with the iodinated alpha 1 -selective radioligand [ 125 I]-I-2-[β-(4-hydroxphenyl)ethylaminomethyl]tetralone ([ 125 I]-IBE 2254) demonstrated that the alpha 1 -adrenergic receptor is coupled to a G-protein in both neonatal and adult rat hearts. However, in the neonate the alpha 1 -adrenergic receptor is coupled to a PT-insensitive G-protein, whereas in the adult the alpha 1 -adrenergic receptor is coupled to both a PT-insensitive and a PT-sensitive G-protein. Consistent with the results from binding experiments, PT did not have any effect on the alpha 1 -mediated positive chronotropic response in the neonate, whereas in the adult the alpha 1 -mediated negative chronotropic response was completely converted to a positive one after PT-treatment. This thesis also examined the possibility of an alteration in coupling of the alpha 1 -adrenergic receptor to its effector under certain circumstances such as high potassium (K + ) depolarization in nerve-muscle (NM) co-cultures, a system which has been previously shown to be a convenient in vitro model to study the mature inhibitory alpha 1 -response

Analysis of angiotensin II binding to human platelets: Differences in young and old subjects

International Nuclear Information System (INIS)

Siebers, M.J.; Goodfriend, T.L.; Ball, D.; Elliott, M.E.

1990-01-01

We examined the binding of radiolabeled angiotensin II (AII) to human platelets to characterize the apparent increase in AII receptors observed in older subjects. At 22 degrees C, the amount of radioactivity associated with platelets from older subjects increased continuously for more than 2 hours. The same amount of radioactivity was displaced by addition of unlabeled AII at 30 min and 60 min. In the presence of phenylarsine oxide, in the cold, or when labeled antagonist was the ligand, binding came to equilibrium by 30 min. High pressure liquid chromatography demonstrated that 125 I-AII was the major radioactive compound in the supernatant and platelets after incubation, but the platelets also contained radiolabeled AII fragments. Thus, some degradation accompanied interaction of AII and platelets. Phenylarsine oxide did not prevent degradation of bound AII, suggesting that degradation precedes internalization. On average, maximum binding was greater in older subjects whether platelets were incubated with 125 I-AII alone, with 125 I-AII and phenylarsine oxide to prevent internalization, or when the competitive inhibitor 125 I-sar1,ile8-AII was the radioligand. Variability of binding among subjects also increased with age. Thus, platelets bind, degrade, and internalize AII, and the three processes occur to a greater extent in platelets from some, but not all older subjects

125I-iomazenil - benzodiazepine receptor binding and serum corticosterone level during psychological stress in a rat model

International Nuclear Information System (INIS)

Fukumitsu, Nobuyoshi; Ogi, Shigeyuki; Uchiyama, Mayuki; Mori, Yutaka

2004-01-01

To test the hypothesis that benzodiazepine receptor density decreases in response to stress, we correlated 125 I-iomazenil ( 125 I-IMZ) binding with serum corticosterone levels in a rat model. Wistar male rats were divided into four groups; control group (CON, 10 rats), no physical or psychological stress; and one-, three-, and five-day stress groups of 12 rats each (1-DAY, 3-DAY, and 5-DAY, respectively), receiving psychological stress for the given number of days. Psychological stress were given to rats with a communication box. The standardized uptake value (SUV) of 125 I-iomazenil of the 3-DAY and 5-DAY showed that 125 I-iomazenil - benzodiazepine receptor binding was significantly reduced in the cortices, accumbens nuclei, amygdala and caudate putamen (p 125 I-IMZ is a useful radioligand to reflect received stress and its binding in the cortices, accumbens nuclei, amygdala and caudate putamen is strongly affected by psychological stress

Elevation of corticosteroid-binding globulin in Obese strain (OS) chickens: possible implications for the disturbed immunoregulation and the development of spontaneous autoimmune thyroiditis

International Nuclear Information System (INIS)

Faessler, R.; Schauenstein, K.; Kroemer, G.; Schwarz, S.; Wick, G.

1986-01-01

Basal plasma levels of corticosterone and corticosteroid-binding globulin (CBG) have been investigated in Obese strain (OS) chickens afflicted with spontaneous autoimmune thyroiditis (SAT). Corticosterone was determined radioimmunologically, and CBG by using a highly sensitive radioligand saturation assay. OS chickens displayed total corticosterone levels not different from healthy normal White Leghorn (NWL) chickens. CBG, however, was found to be twice as high in OS chickens as compared with their healthy counterparts, irrespective of sex or age. This quantitative difference in the CBG level is not compensated for by either altered affinity or specificity of the molecule. Furthermore, no differences were found in the response of OS and NWL lymphocytes to the suppressive effect of glucocorticoids in vitro. It was therefore assumed that OS animals are deficient in free, hormonally active corticosterone. An additional indication for such a diminished glucocorticoid tonus was that in vivo treatment of OS chickens with glucocorticoid hormones, thus increasing the free and active hormone fraction, normalizes the T cell hyperreactivity and significantly reduces thyroid infiltration. Possible pathophysiological implications of a diminished glucocorticoid tonus for spontaneous autoimmunity, as well as possible explanations for the beneficial effects of glucocorticoid treatment on the development of SAT, are discussed

Assay of oestrogen

International Nuclear Information System (INIS)

Edwards, J.C.

1981-01-01

A particular problem with the direct radioimmunoassay of unconjugated oestriol in pregnancy is caused by the increased amount of steroid-binding proteins present in pregnancy serum and plasma. The steroid-binding proteins react with oestriol and 125 I-labelled oestriol during the assay procedure and the steroid-protein bound 125 I-labelled oestriol is precipitated along with the antibody-bound 125 I-labelled oestriol by the ammonium sulphate solution separation system. A novel method is described whereby progesterone (1-20 μg/ml) is used to block the action of steroid-binding proteins in pregnancy serum and plasma samples, thus minimizing interference in a direct radioimmunoassay for unconjugated oestriol using a specific anti-oestriol serum. (U.K.)

(125I)LSD labels 5-TCsub(IC) recognition sites in pig choriod plexus membranes. Comparison with (3H)mesulergine and (3H)5-HT binding

International Nuclear Information System (INIS)

Hoyer, D.; Srivatsa, S.; Pazos, A.; Engel, G.; Palacios, J.M.

1986-01-01

The mammalian choroid plexus is enriched in a newly described serotonin recognition site, the binding characteristics of ( 125 I)LSD, ( 3 H)mesulergine and ( 3 H)serotonin to pig choroid plexus membranes were compared. These ligands labelled with high affinity a similar number of sites. The binding profiles of the sites labelled with these radioligands are indistinguishable as illustrated by highly significant correlation parameters. These sites are very similar to those labelled by Nsub(I)-methyl-2-( 125 I)LSD in pig and rat choroid plexus membranes. The data demonstrate that these ligands label 5-HTsub(IC) recognition sites in the pig and rat choroid plexus membranes. (author)

Brain serotonin 4 receptor binding is inversely associated with verbal memory recall.

Science.gov (United States)

Stenbæk, Dea S; Fisher, Patrick M; Ozenne, Brice; Andersen, Emil; Hjordt, Liv V; McMahon, Brenda; Hasselbalch, Steen G; Frokjaer, Vibe G; Knudsen, Gitte M

2017-04-01

We have previously identified an inverse relationship between cerebral serotonin 4 receptor (5-HT 4 R) binding and nonaffective episodic memory in healthy individuals. Here, we investigate in a novel sample if the association is related to affective components of memory, by examining the association between cerebral 5-HT 4 R binding and affective verbal memory recall. Twenty-four healthy volunteers were scanned with the 5-HT 4 R radioligand [ 11 C]SB207145 and positron emission tomography, and were tested with the Verbal Affective Memory Test-24. The association between 5-HT 4 R binding and affective verbal memory was evaluated using a linear latent variable structural equation model. We observed a significant inverse association across all regions between 5-HT 4 R binding and affective verbal memory performances for positive ( p  = 5.5 × 10 -4 ) and neutral ( p  = .004) word recall, and an inverse but nonsignificant association for negative ( p  = .07) word recall. Differences in the associations with 5-HT 4 R binding between word categories (i.e., positive, negative, and neutral) did not reach statistical significance. Our findings replicate our previous observation of a negative association between 5-HT 4 R binding and memory performance in an independent cohort and provide novel evidence linking 5-HT 4 R binding, as a biomarker for synaptic 5-HT levels, to the mnestic processing of positive and neutral word stimuli in healthy humans.

Beta 2-adrenergic receptors on eosinophils. Binding and functional studies

International Nuclear Information System (INIS)

Yukawa, T.; Ukena, D.; Kroegel, C.; Chanez, P.; Dent, G.; Chung, K.F.; Barnes, P.J.

1990-01-01

We have studied the binding characteristics and functional effects of beta-adrenoceptors on human and guinea pig eosinophils. We determined the binding of the beta-antagonist radioligand [125I]pindolol (IPIN) to intact eosinophils obtained from the peritoneal cavity of guinea pigs and from blood of patients with eosinophilia. Specific binding was saturable, and Scatchard analysis showed a single binding site with a dissociation constant (Kd) of 24.6 pM and maximal number of binding sites (Bmax) of 7,166 per cell. ICI 118,551, a beta 2-selective antagonist, inhibited IPIN binding with a Ki value of 0.28 nM and was approximately 5,000-fold more effective than the beta 1-selective antagonist, atenolol. Isoproterenol increased cAMP levels about 5.5-fold above basal levels (EC50 = 25 microM); albuterol, a beta 2-agonist, behaved as a partial agonist with a maximal stimulation of 80%. Binding to human eosinophils gave similar results with a Kd of 25.3 pM and a Bmax corresponding to 4,333 sites per cell. Incubation of both human and guinea pig eosinophils with opsonized zymosan (2 mg/ml) or with phorbol myristate acetate (PMA) (10(-8) and 10(-6) M) resulted in superoxide anion generation and the release of eosinophil peroxidase; albuterol (10(-7) to 10(-5) M) had no inhibitory effect on the release of these products. Thus, eosinophils from patients with eosinophilia and from the peritoneal cavity of guinea pigs possess beta-receptors of the beta 2-subtype that are coupled to adenylate cyclase; however, these receptors do not modulate oxidative metabolism or degranulation. The possible therapeutic consequences of these observations to asthma are discussed

Binding of [125I]iodipine to parathyroid cell membranes: Evidence of a dihydropyridine-sensitive calcium channel

International Nuclear Information System (INIS)

Jones, J.I.; Fitzpatrick, L.A.

1990-01-01

The parathyroid cell is unusual, in that an increase in extracellular calcium concentrations inhibits PTH release. Calcium channels are glycoproteins that span cell membranes and allow entry of extracellular calcium into cells. We have demonstrated that the calcium channel agonist (+)202-791, which opens calcium channels, inhibits PTH release and that the antagonist (-)202-791, which closes calcium channels, stimulates PTH release. To identify the calcium channels responsible for these effects, we used a radioligand that specifically binds to calcium channels. Bovine parathyroid cell membranes were prepared and incubated under reduced lighting with [125I] iodipine (SA, 2000 Ci/mmol), which recognizes 1,4-dihydropyridine-sensitive calcium channels. Bound ligand was separated from free ligand by rapid filtration through Whatman GF/B filters. Nonspecific binding was measured by the inclusion of nifedipine at 10 microM. Specific binding represented approximately 40% of the total binding. The optimal temperature for [125I] iodipine binding was 4 C, and binding reached equilibrium by 30 min. The equilibrium dissociation constant (Kd) was approximately 550 pM, and the maximum number of binding sites was 780 fmol/mg protein. Both the calcium channel agonist (+)202-791 and antagonist (-)202-791 competitively inhibited [125I] iodipine binding, with 50% inhibition concentrations of 20 and 300 nM, respectively. These data indicate the presence of dihydropyridine-sensitive calcium channels on parathyroid cell membranes

MEL-N16: A Series of Novel Endomorphin Analogs with Good Analgesic Activity and a Favorable Side Effect Profile.

Science.gov (United States)

Liu, Xin; Zhao, Long; Wang, Yuan; Zhou, Jingjing; Wang, Dan; Zhang, Yixin; Zhang, Xianghui; Wang, Zhaojuan; Yang, Dongxu; Mou, Lingyun; Wang, Rui

2017-10-18

Opioid peptides are neuromodulators that bind to opioid receptors and reduce pain sensitivity. Endomorphins are among the most active endogenous opioid peptides, and they have good affinity and selectivity toward the μ opioid receptor. However, their clinical usage is hindered by their inability to cross the blood-brain barrier and their poor in vivo activity after peripheral injection. In order to overcome these defects, we have designed and synthesized a series of novel endomorphin analogs with multiple site modifications. Radioligand binding, cAMP accumulation, and β-arrestin-2 recruitment assays were employed to determine the activity of synthesized endomorphin analogs toward opioid receptors. The blood-brain barrier permeability and antinociceptive effect of these analogs were determined in several rodent models of acute and persistent pain. In addition, the side effects of the analogs were examined. The radioligand binding assay and functional activity examination indicated that the MEL-N16 series of compounds were more active agonists against μ opioid receptor than were the parent peptides. Notably, the analogs displayed biased downstream signaling toward G-protein pathways over β-arrestin-2 recruitment. The analogs showed highly potent antinociceptive effects in the tested nociceptive models. In comparison with endomorphins, the synthesized analogs were better able to penetrate the blood-brain barrier and exerted their pain regulatory activity in the central nervous system after peripheral injection. These analogs also have lower tendency to cause side effects than morphine does at similar or equal antinociceptive doses. The MEL-N16 compounds have highly potent and efficacious analgesic effects in various pain models with a favorable side effect profile.

A Promising PET Tracer for Imaging of α7 Nicotinic Acetylcholine Receptors in the Brain: Design, Synthesis, and in Vivo Evaluation of a Dibenzothiophene-Based Radioligand

Directory of Open Access Journals (Sweden)

Rodrigo Teodoro

2015-10-01

Full Text Available Changes in the expression of α7 nicotinic acetylcholine receptors (α7 nAChRs in the human brain are widely assumed to be associated with neurological and neurooncological processes. Investigation of these receptors in vivo depends on the availability of imaging agents such as radioactively labelled ligands applicable in positron emission tomography (PET. We report on a series of new ligands for α7 nAChRs designed by the combination of dibenzothiophene dioxide as a novel hydrogen bond acceptor functionality with diazabicyclononane as an established cationic center. To assess the structure-activity relationship (SAR of this new basic structure, we further modified the cationic center systematically by introduction of three different piperazine-based scaffolds. Based on in vitro binding affinity and selectivity, assessed by radioligand displacement studies at different rat and human nAChR subtypes and at the structurally related human 5-HT3 receptor, we selected the compound 7-(1,4-diazabicyclo[3.2.2]nonan-4-yl-2-fluorodibenzo-[b,d]thiophene 5,5-dioxide (10a for radiolabeling and further evaluation in vivo. Radiosynthesis of [18F]10a was optimized and transferred to an automated module. Dynamic PET imaging studies with [18F]10a in piglets and a monkey demonstrated high uptake of radioactivity in the brain, followed by washout and target-region specific accumulation under baseline conditions. Kinetic analysis of [18F]10a in pig was performed using a two-tissue compartment model with arterial-derived input function. Our initial evaluation revealed that the dibenzothiophene-based PET radioligand [18F]10a ([18F]DBT-10 has high potential to provide clinically relevant information about the expression and availability of α7 nAChR in the brain.

Evaluation of a new free-thyroxin assay

International Nuclear Information System (INIS)

Welby, M.L.; Guthrie, L.; Reilly, C.P.

1981-01-01

The Amerlex Free Thyroxin (T 4 ) Radioimmunoassay Kit (Amersham International Ltd.) is a new direct equilibrium radioimmunoassay for free T 4 based on an antiserum with very high affinity for T 4 , and a unique 125 l-labeled T 4 analog as tracer. It is a very simple single-tube radioimmunoassay, making use of Amerlex particles to separate antibody-bound from free species. Interassay precision (CV) is 3.7% at 13 pmol/L and 2.3% at 30 pmol/L; within-assay precision is 4.2% at 21 pmol/L. The reference interval is 11-22 pmol/L. The assay did not misclassify any patients tested who had untreated myxedema or untreated thyrotoxicosis. The free T 4 assay excelled both the free T 4 index and the T 4 /T 4 -binding globulin ratio in correcting for increased thyroxin-binding globulin from pregnancy, and it was better than the index but not better than the ratio in correcting for increased thyroxin-binding globulin in users of oral contraceptives

Radioreceptor assay for somatomedin A

Energy Technology Data Exchange (ETDEWEB)

Takano, K [Tokyo Women' s Medical Coll. (Japan)

1975-04-01

Measurement method of somatomedian A by radioreceptor assay using the human placenta membrane was described and discussed. Binding rate of /sup 125/I-somatomedin A to its receptors was studied under various conditions of time and temperature of the incubation, and pH of the system. The influence of somatomedin A, porcine insulin, and porcine calcitonin, on /sup 125/I-somatomedin A bound receptors was studied, and these hormones showed the competitive binding to somatomedin A receptors in some level. The specificity, recovery rate, and clinical applications of somatomedin A were also discussed. Radioreceptor assay for somatomedine A provided easier, faster, and more accurate measurements than conventional bioassay. This technique would be very useful to study somatomedin A receptor and functions of insulin.

[[sup 3]H]imidacloprid: synthesis of a candidate radioligand for the nicotinic acetylcholine receptor

Energy Technology Data Exchange (ETDEWEB)

Latli, B.; Casida, J.E. (California Univ., Berkeley, CA (United States). Dept. of Entomological Sciences)

1992-08-01

Imidacloprid is an exceptionally potent insecticide known from physiological studies to act at the nicotinic acetylcholine receptor. To prepare [[sup 3]H]imidacloprid as a candidate radioligand, 6-chloronicotinoyl chloride was reduced with NaB[sup 2]H[sub 4] (in model studies) or NaB[sup 3]H[sub 4] in absolute ethanol to 2-chloro-5-pyridinylmethanol which was transformed to 2-chloro-5-chloromethylpyridine on refluxing with thionyl chloride. Coupling with 4,5-dihydro-N-nitro-1H-imidazol-2-amine then gave [[sup 2]H[sub 2

A pigeon crop sac radioreceptor assay for prolactin

International Nuclear Information System (INIS)

Forsyth, I.A.; Buntin, J.D.; Nicoll, C.S.

1978-01-01

Ovine prolactin, labelled with 125 I by either lactoperoxidase or a mild chloramine T method, was bound to receptors from the pigeon crop sac mucosa cells of prolactin-injected pigeons. Binding was demonstrated in a crude homogenate of mucosal cells removed from the crop by scraping and in a subcellular fraction in which 5'- nucleotidase activity was enhanced two- to three-fold. The binding was specific, dependent on time, temperature and the concentration of receptors and had a dissociation constant of 7 x 10 -10 mol/l. The binding capacity of the crop tissue was 71 fmol/mg membrane protein. Nine purified preparations of prolactin from four species were assayed by local pigeon crop sac bioassay and by radioreceptor assay. The two methods were highly correlated (r = 0.934). The regression equation was radioreceptor assay = 1.22 bioassay - 0.18 indicating a 1:1 correspondence between the two methods for prolactin purified from sheep, rat, horse and pig anterior pituitary glands. (author)

Radiosynthesis and in vivo evaluation of novel radioligands for PET imaging of cerebral 5-HT7 receptors

DEFF Research Database (Denmark)

Hansen, Hanne D; Herth, Matthias M; Ettrup, Anders

2014-01-01

in the living brain. Here, we present the radiosynthesis and in vivo evaluation of Cimbi-712 (3-{4-[4-(4-methylphenyl)piperazine-1-yl]butyl}p-1,3-dihydro-2H-indol-2-one) and Cimbi-717 (3-{4-[4-(3-methoxyphenyl)piperazine-1-yl]butyl}-1,3-dihydro-2H-indol-2-one) as selective 5-HT7R PET radioligands in the pig...

Development of 99mTc labelled somatostatin analogues with high affinity for somatostatin receptors

International Nuclear Information System (INIS)

Maina, T.; Nock, B.; Nicolopoulou, A.; Tsipra, C.; Poppe, M.; Chiotellis, E.

2001-01-01

A recent development in oncology involves the use of metabolically stabilized peptide hormone analogues labelled with metallic radionuclides for the diagnosis or therapy of malignant disease. This approach was successfully applied for the first time in the visualization of somatostatin positive tumours and their metastases with 111 In DTPA-octreotide. In an effort to obtain a 99m Tc somatostatin receptor affine radioligand we describe herein the synthesis, radiochemistry and preliminary biological evaluation of two novel 99m Tc labelled somatostatin analogues, N 4 -TOC and N 4 -RC-160. In these compounds a tetraamine bifunctional unit was covalently attached to the N-terminal (D)Phe 1 of the peptide chain using Boc-protection strategies. The peptide conjugates were purified by high performance liquid chromatography (HPLC) and characterized by UV/Vis and ES-MS spectroscopies. As revealed by HPLC, 99m Tc labelling was quantitative under mild conditions, leading to a single 99m Tc species in high specific activities. Affinity of 99m Tc N 4 -TOC for the somatostatin receptor, as determined by in vitro binding assays in rat brain cortex membranes, was found unaffected by the presence of the bulky metal chelate. The binding properties of 99m Tc N 4 -RC-160 could not be determined by this assay due to an extremely high non-specific binding of this radioligand, and will be shortly investigated by other methods. Tissue distribution in healthy mice revealed that 99m Tc N 4 -TOC is clearing mainly through the kidneys and the urinary tract whereas 99m Tc N 4 -RC-160 shows a high accumulation in the liver as a result of its lipophilicity. Analysis of urine samples by HPLC showed that 99m Tc N 4 -TOC is excreted integer from the body of mice, while 99m Tc N 4 -RC-160 is totally transformed to an unidentified hydrophilic metabolite in vivo. The location of this metabolism is currently investigated. In vivo blocking experiments using animals pre-treated with 50 μg octreotide

Characterization of the radioactive metabolites of the 5-HT1A receptor radioligand, [O-methyl-C-11]WAY-100635, in monkey and human plasma by HPLC : Comparison of the behaviour of an identified radioactive metabolite with parent radioligand in monkey using PET

NARCIS (Netherlands)

Osman, S; Lundkvist, C; Pike, VW; Halldin, C; McCarron, JA; Swahn, CG; Ginovart, N; Luthra, SK; Bench, CJ; Grasby, PM; Wikstrom, H; Barf, T; Cliffe, IA; Fletcher, A; Farde, L

N-(2-(4-(2-Methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl)cyclohexanecarboxamide (WAY-100635), labelled in the O-methyl group with carbon-11 (t(1/2) = 20.4 min), is a promising radioligand for application with positron emission tomography (PET) to the study of 5-HT1A receptors in living human

Visual evaluation of kinetic characteristics of PET probe for neuroreceptors using a two-phase graphic plot analysis.

Science.gov (United States)

Ito, Hiroshi; Ikoma, Yoko; Seki, Chie; Kimura, Yasuyuki; Kawaguchi, Hiroshi; Takuwa, Hiroyuki; Ichise, Masanori; Suhara, Tetsuya; Kanno, Iwao

2017-05-01

Objectives In PET studies for neuroreceptors, tracer kinetics are described by the two-tissue compartment model (2-TCM), and binding parameters, including the total distribution volume (V T ), non-displaceable distribution volume (V ND ), and binding potential (BP ND ), can be determined from model parameters estimated by kinetic analysis. The stability of binding parameter estimates depends on the kinetic characteristics of radioligands. To describe these kinetic characteristics, we previously developed a two-phase graphic plot analysis in which V ND and V T can be estimated from the x-intercept of regression lines for early and delayed phases, respectively. In this study, we applied this graphic plot analysis to visual evaluation of the kinetic characteristics of radioligands for neuroreceptors, and investigated a relationship between the shape of these graphic plots and the stability of binding parameters estimated by the kinetic analysis with 2-TCM in simulated brain tissue time-activity curves (TACs) with various binding parameters. Methods 90-min TACs were generated with the arterial input function and assumed kinetic parameters according to 2-TCM. Graphic plot analysis was applied to these simulated TACs, and the curvature of the plot for each TAC was evaluated visually. TACs with several noise levels were also generated with various kinetic parameters, and the bias and variation of binding parameters estimated by kinetic analysis were calculated in each TAC. These bias and variation were compared with the shape of graphic plots. Results The graphic plots showed larger curvature for TACs with higher specific binding and slower dissociation of specific binding. The quartile deviations of V ND and BP ND determined by kinetic analysis were smaller for radioligands with slow dissociation. Conclusions The larger curvature of graphic plots for radioligands with slow dissociation might indicate a stable determination of V ND and BP ND by kinetic analysis. For

In vitro and in vivo characterisation of nor-β-CIT: a potential radioligand for visualisation of the serotonin transporter in the brain

International Nuclear Information System (INIS)

Bergstroem, K.A.; Halldin, C.; Hall, H.; Lundkvist, C.; Ginovart, N.; Swahn, C.G.; Farde, L.

1997-01-01

Radiolabelled 2β-carbomethoxy-3β-(4-iodophenyl)tropane (β-CIT) has been used in clinical studies for the imaging of dopamine and serotonin transporters with single-photon emission tomography (SPET). 2β-Carbomethoxy-3β-(4-iodophenyl)nortropane (nor-β-CIT) is a des-methyl analogue of β-CIT, which in vitro has tenfold higher affinity (IC 50 =0.36 nM) to the serotonin transporter than β-CIT (IC 50 =4.2 nM). Nor-β-CIT may thus be a useful radioligand for imaging of the serotonin transporter. In the present study iodine-125 and carbon-11 labelled nor-β-CIT were prepared for in vitro autoradiographic studies on post-mortem human brain cryosections and for in vivo positron emission tomography (PET) studies in Cynomolgus monkeys. Whole hemisphere autoradiography with [ 125 I[nor-β-CIT demonstrated high binding in the striatum, the thalamus and cortical regions of the human brain. Addition of a high concentration (1 μM) of citalopram inhibited binding in the thalamus and the neocortex, but not in the striatum. In PET studies with [ 11 C[nor-β-CIT there was rapid uptake of radioactivity in the monkey brain (6% of injected dose at 15 min) and high accumulation of radioactivity in the striatum, thalamus and neocortex. Thalamus to cerebellum and cortex to cerebellum ratios were 2.5 and 1.8 at 60 min, respectively. The ratios obtained with [ 11 C[nor-β-CIT were 20%-40% higher than those previously obtained with [ 11 C[β-CIT. Radioactivity in the thalamus and the neocortex but not in the striatum was displaceable with citalopram (5 mg/kg). In conclusion, nor-β-CIT binds to the serotonin transporter in the primate brain in vitro and in vivo and has potential for PET and SPET imaging of the serotonin transporter in human brain. (orig.). With 4 figs

Conception, synthesis and evaluation of fluorescent probes and PET radioligands for the oxytocin and vasopressin receptors

International Nuclear Information System (INIS)

Karpenko, Iuliia

2014-01-01

In order to better understand the role of OTR and AVPR in ASD, to reveal new features in its pharmacology and signaling and to establish high-throughput screening method on wild-type G protein-coupled receptors, we developed imaging probes for the oxytocin-vasopressin receptors family, namely radiotracers for positron emission tomography and optical probes for fluorescence detection and imaging. The fluorescent ligands have been used to establish TR-FRET binding assay for OTR and to initiate the development the screening assay for the wild-type oxytocin receptor. The PET radiotracers will be shortly tested in mice and monkeys to evaluate their potency in detecting the central oxytocin receptors. (author)

Development of a radioiodinated ligand for characterising α1-adrenoceptors

International Nuclear Information System (INIS)

Adams, A.; Jarrott, B.

1982-01-01

Two α-adrenoceptor antagonists, phentolamine and 2-(β-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone (BE 2254) which are phenolic derivatives were radioiodinated after chloramine-T oxidation of Na 125 I and the labelled material isolated by chromatography. 125 I-Phentolamine does not bind selectively to α-adrenoceptors in guinea pig brain whereas the 125 I-BE 2254 derivative binds rapidly, reversibly and with high affinity to these receptors with a K/sub d/ of 230 pM. At low concentrations of 125 I-BE 2254 ( 1 subclass of adrenoceptors. Binding measurements to kidney and smooth muscle membrane preparations indicate that 125 I-BE 2254 may also be a useful tool in the study of α-adrenoceptors in peripheral tissues. The high specific activity of 125 I-BE 2254 permits the use of minimal quantities of membrane material for receptor assay and ligand displacement measurements, e.g. 250 μg per assay tube, and this provides a significant advantage over the use of existing radioligands such as 3 H-prazosin which requires approx. 40 times as much tissue

[3H]naloxone as an opioid receptor label: Analysis of binding site heterogeneity and use for determination of opioid affinities of casomorphin analogues

International Nuclear Information System (INIS)

Schnittler, M.; Repke, H.; Liebmann, C.; Schrader, U.; Schulze, H.P.; Neubert, K.

1990-01-01

The nonselective antagonist [ 3 H]naloxone was used to identify opioid receptors in rat brain membranes. The multiple naloxone binding sites were related to different opioid receptors by means of selective opiod ligands as well as various β-casomorphin analogues. Analysis of binding site heterogeneity was performed using several computer curve fitting methods. The results indicate that structurally modified casomorphin peptides are able to discriminate between μ 1 and μ 2 binding sites. The affinities to the μ sites obtained with [ 3 H]naloxone as label are in a good agreement with those from experiments with the μ selective radioligand [ 3 H]DAGO. The μ 1 site affinities of these casomorphin derivatives are well correlated with their antinociceptive potencies. This finding suggests the mediation of the analgesic activity via the high-affinity μ 1 subtype. (author)

Visualization of specific binding sites of benzodiazepine in human brain

International Nuclear Information System (INIS)

Shinotoh, H.; Yamasaki, T.; Inoue, O.; Itoh, T.; Suzuki, K.; Hashimoto, K.; Tateno, Y.; Ikehira, H.

1986-01-01

Using 11C-labeled Ro15-1788 and positron emission tomography, studies of benzodiazepine binding sites in the human brain were performed on four normal volunteers. Rapid and high accumulation of 11C activity was observed in the brain after i.v. injection of [11C]Ro15-1788, the maximum of which was within 12 min. Initial distribution of 11C activity in the brain was similar to the distribution of the normal cerebral blood flow. Ten minutes after injection, however, a high uptake of 11C activity was observed in the cerebral cortex and moderate uptake was seen in the cerebellar cortex, the basal ganglia, and the thalamus. The accumulation of 11C activity was low in the brain stem. This distribution of 11C activity was approximately parallel to the known distribution of benzodiazepine receptors. Saturation experiments were performed on four volunteers with oral administration of 0.3-1.8 mg/kg of cold Ro15-1788 prior to injection. Initial distribution of 11C activity following injection peaked within 2 min and then the accumulation of 11C activity decreased rapidly and remarkably throughout the brain. The results indicated that [11C] Ro15-1788 associates and dissociates to specific and nonspecific binding sites rapidly and has a high ratio of specific receptor binding to nonspecific binding in vivo. Carbon-11 Ro15-1788 is a suitable radioligand for the study of benzodiazepine receptors in vivo in humans

Labelling of pneumococcal penicillin-binding proteins with [3H]propionyl-ampicillin. A rapid method for monitoring penicillin-binding activity

International Nuclear Information System (INIS)

Hakenbeck, R.; Kohiyama, M.

1982-01-01

Penicillin-binding proteins (PBPs) are membrane components ubiquitous to all bacteria examined so far. Some of them are present in only a few copies per cell. The conventional method of visualizing these proteins consists in binding of radioactive penicillin to the fractions containing PBPs followed by SDS-PAGE and finally fluorography. Although this procedure is laborious, it is necessary for the determination of the identity as well as for the quantification of each PBP. On the other hand, when penicillin-binding conditions are to be examined or binding activity has to be followed through fractionation and purification of PBPs, no fast monitoring device for these proteins has been available. The authors developed a rapid and easy assay for penicillin-binding activity with a filter-binding technique using [ 3 H]propionyl ampicillin ( 3 H-PA) of high specific activity. As little 2μg of crude membranes obtained from the highly penicillin-sensitive, β-lactamase-negative organism Streptococcus pneumoniae, are sufficient to detect binding activity. In this paper they describe optimum conditions for the assay of PBPs and show that this binding activity correlates with the presence of native penicillin-binding proteins. (Auth.)

A cell-based MHC stabilization assay for the detection of peptide binding to the canine classical class I molecule, DLA-88.

Science.gov (United States)

Ross, Peter; Holmes, Jennifer C; Gojanovich, Gregory S; Hess, Paul R

2012-12-15

Identifying immunodominant CTL epitopes is essential for studying CD8+ T-cell responses in populations, but remains difficult, as peptides within antigens typically are too numerous for all to be synthesized and screened. Instead, to facilitate discovery, in silico scanning of proteins for sequences that match the motif, or binding preferences, of the restricting MHC class I allele - the largest determinant of immunodominance - can be used to predict likely candidates. The high false positive rate with this analysis ideally requires binding confirmation, which is obtained routinely by an assay using cell lines such as RMA-S that have defective transporter associated with antigen processing (TAP) machinery, and consequently, few surface class I molecules. The stabilization and resultant increased life-span of peptide-MHC complexes on the cell surface by the addition of true binders validates their identity. To determine whether a similar assay could be developed for dogs, we transfected a prevalent class I allele, DLA-88*50801, into RMA-S. In the BARC3 clone, the recombinant heavy chain was associated with murine β2-microglobulin, and importantly, could differentiate motif-matched and -mismatched peptides by surface MHC stabilization. This work demonstrates the potential to use RMA-S cells transfected with canine alleles as a tool for CTL epitope discovery in this species. Copyright © 2012 Elsevier B.V. All rights reserved.

The electrophoretic mobility shift assay (EMSA)

OpenAIRE

sprotocols

2015-01-01

The electrophoretic mobility shift assay (EMSA), also known as “gel shift assay”, is used to examine the binding parameters and relative affinities of protein and DNA interactions. We produced recombinant CCA1 protein and tested its binding affinity for the promoter fragments that contain CBS (AAAAATCT) or evening element (EE, AAAATATCT) (1) using a modified procedure adopted from published protocols (2,3).

Preliminary studies of 99mTc-memantine derivatives for NMDA receptor imaging

International Nuclear Information System (INIS)

Zhou Xingqin; Zhang Jiankang; Yan Chenglong; Cao Guoxian; Zhang Rongjun; Cai Gangming; Jiang Mengjun; Wang Songpei

2012-01-01

Introduction: Novel technetium-labeled ligands, 99m Tc-NCAM and 99m Tc-NHAM were developed from the N-methyl-D-aspartate (NMDA) receptor agonist memantine as a lead compound by coupling with N 2 S 2 . This study evaluated the binding affinity and specificity of the ligands for the NMDA receptor. Methods: Ligand biodistribution and uptake specificity in the brain were investigated in mice. Binding affinity and specificity were determined by radioligand receptor binding assay. Three antagonists were used for competitive binding analysis. In addition, uptake of the complexes into SH-SY5Y nerve cells was evaluated. Results: The radiochemical purity of 99m Tc-labeled ligands was more than 95%. Analysis of brain regional uptake showed higher concentration in the frontal lobe and specific uptake in the hippocampus. 99m Tc-NCAM reached a higher target to nontarget ratio than 99m Tc-NHAM. The results indicated that 99m Tc-NCAM bound to a single site on the NMDA receptor with a K d of 701.21 nmol/l and a B max of 62.47 nmol/mg. Specific inhibitors of the NMDA receptor, ketamine and dizocilpine, but not the dopamine D 2 and 5HT 1A receptor partial agonist aripiprazole, inhibited specific binding of 99m Tc-NCAM to the NMDA receptor. Cell physiology experiments showed that NCAM can increase the viability of SH-SY5Y cells after glutamate-induced injury. Conclusions: The new radioligand 99m Tc-NCAM has good affinity for and specific binding to the NMDA receptor, and easily crosses the blood–brain barrier; suggesting that it might be a potentially useful tracer for NMDA receptor expression.

Radioligand imaging of α4β2* nicotinic acetylcholine receptors in Alzheimer’s disease and Parkinson’s disease

International Nuclear Information System (INIS)

Meyer, P. M.; Tiepolt, S.; Barthel, H.; Hesse, S.; Sabri, O.

2014-01-01

The α4β2 * nicotinic acetylcholine receptors (α4β2 * -nAChR) are highly abundant in the human brain. As neuromodulators they play an important role in cognitive functions such as memory, learning and attention as well as mood and motor function. Post mortem studies suggest that abnormalities of α4β2 * -nAChRs are closely linked to histopathological hallmarks of Alzheimer’s disease (AD), such as amyloid aggregates/oligomers and tangle pathology and of Parkinson’s disease (PD) such as Lewy body pathology and the nigrostriatal dopaminergic deficit. In this review we summarize and discuss nicotinic receptor imaging findings of 2-[18F]FA-85380 PET, [ 11 C]nicotine PET and 5-[ 123 I]IA-85380 SPECT studies investigating α4β2 * -nAChR binding in vivo and their relationship to mental dysfunction in the brain of patients with AD and patients out of the spectrum of Lewy body disorders such as PD and Lewy body dementia (DLB). Furthermore, recent developments of novel α4β2*-nAChR-specific PET radioligands, such as (-)[ 18 F]Flubatine or [ 18 F]AZAN are summarized. We conclude that α4β2*-nAChR-specific PET might become a biomarker for early diagnostics and drug developments in patients with AD, DLB and PD, even at early or prodromal stages.

(E)-[125I]-5-AOIBV: a SPECT radioligand for the vesicular acetylcholine transporter

International Nuclear Information System (INIS)

Emond, Patrick; Mavel, Sylvie; Zea-Ponce, Yolanda; Kassiou, Michael; Garreau, Lucette; Bodard, Sylvie; Drossard, Marie-Laure; Chalon, Sylvie; Guilloteau, Denis

2007-01-01

The premise that, over the course of Alzheimer's disease (AD), changes in the levels of the vesicular acetylcholine transporter (VAChT) occur in parallel with changes to other cholinergic marker proteins provides the basis for the applicability of benzovesamicol derivatives as radioligands for AD studies by single photon emission computed tomography or positron emission tomography. We report the synthesis of enantiopure benzovesamicol derivatives: (R,R) or (S,S)-(E)-2-hydroxy-5-(3-iodoprop-2-en-1-oxy)-3- (4-phenylpiperidino)tetralin [(R,R)-AOIBV: K d =0.45 nM or (S,S)-5-AOIBV: K d =4.3 nM] and their corresponding tributyltin precursors for radioiodination. (R,R or S,S)-5-AOIBV was labeled with iodine-125 from their corresponding n-tributyltin precursors. Both compounds were obtained with radiochemical and optical purity greater than 97% and in radiochemical yields ranging 34-36%. To determine if these compounds could provide an advantage when compared to [ 125 I]-iodo benzovesamicol (IBVM), IBVM was also labeled and used as the reference compound in all ex vivo experiments. Ex vivo biodistribution experiments in rats revealed that [ 125 I]-(R,R)-5-AOIBV displayed the most suitable pharmacological profile as the radioactivity distribution corresponded well with the known VAChT brain density. Moreover, pre-injection of vesamicol prevented the uptake of [ 125 I]-(R,R)-5-AOIBV in striatum, cortex and hippocampus, demonstrating selectivity for the VAChT. However, even if time activity curves of [ 125 I]-(R,R)-5-AOIBV confirmed that this compound could be used to visualize the VAChT in vivo, at each point of the kinetic study, [ 125 I]-(R,R)-5-AOIBV showed a lower specific binding compared to [ 125 I]-IBVM. These results made [ 125 I]-( R,R)-5-AOIBV inferior to [ 125 I]-IBVM for the VAChT exploration in vivo

Serum insulin-like growth factor-I in 1030 healthy children, adolescents, and adults

DEFF Research Database (Denmark)

Juul, A; Bang, P; Hertel, Niels

1994-01-01

Serum levels of insulin-like growth factor-I (IGF-I) increase with age and pubertal development. The large variation in circulating IGF-I levels in adolescence makes it difficult to use the IGF-I value of a single child in the assessment of his growth status. In addition, the interference of IGF......-binding proteins in many IGF-I assays contributes to this problem. We measured IGF-I in acid-ethanol-extracted serum from 1030 healthy children, adolescents, and adults, employing a RIA that reduces interference of IGF-binding proteins by using monoiodinated Tyr31-[125I]des-(1-3)IGF-I as radioligand. Mean serum...... volume. Multiple regression analysis revealed that serum IGF-I levels predicted height velocity in the following year (r = 0.33; P

The effect of heart-and kidney-Benefiting Chinese Herbal Medicine on the function of cholinergic M-and Gamma amino-butyric acid (GABA) receptor in brain tissues of analogue dementia rats

International Nuclear Information System (INIS)

Mo Qizhong; Gong Bin; Fang Jun

1993-01-01

3 H-QNB and 3 H-GABA were used as radioactive ligand for M-and GABA receptors respectively. M-and GABA receptors were assayed by radioligand binding assay (RBA) in cerebral cortex, hippocampus and cerebellum of analogue dementia rats. It was found that R t of M receptor was decreased in cerebral cortex and hippocampus and R t of GABA receptor was decreased in cerebellum of analogue dementia rats. The dissociation constant (K D ) of M-receptor was decreased significantly in cerebral cortex and K D value of (GABA) receptor was decreased in cerebellum of analogue dementia rats. The decreased R t of M-and GABA receptor in brain tissue of analogue dementia rats was raised by Heart- and Kidney-Benefiting Chinese Herbs as well as hydergin

Clinical applications of HPLC-competitive protein binding assay for serum 25-hydroxyvitamin D

International Nuclear Information System (INIS)

Yang Shouli

1989-01-01

This report describes the clinical applications of HPLC-competitive protein binding assay for serum 25(OH) Vit D in 156 cases. Serum 25(OH) Vit D of normal human was 33.1 +- 14.8 nmol/L (13.2 +- 5.9 ng/ml), while for various diseases are as follows: rickets, 18.0 +- 9.0 nmol/L (7.2 +- 3.6 ng/ml, n = 36, P 0.1); pneumonia of children, 33.8 +- 14.8 nmol/L (13.5 +- 5.9 ng/ml, n = 10, P > 0.5); cirrhosis, 13.8 +- 11.3 nmol/L (5.5 +- 4.5 ng/ml, n = 9, P 0.2); administration of anticonvulsant (1 to 15 years), 19.0 +- 6.5 nmol/L (7.6 +- 2.6 ng/ml, n = 19, P 6 months), 15.3 +- 5.0 nmol/L (6.1 +- 2.0 ng/ml, n = 6, P 0.5); pregnant women, 39.8 +- 16.5 nmol/L (15.9 +- 6.6 ng/ml, n = 7, P > 0.1). We found it is a useful reference value for most of the above diseased state especially for differentiation between rickets and hypervitaminosis

Brain serotonin and dopamine transporter bindings in adults with high-functioning autism.

Science.gov (United States)

Nakamura, Kazuhiko; Sekine, Yoshimoto; Ouchi, Yasuomi; Tsujii, Masatsugu; Yoshikawa, Etsuji; Futatsubashi, Masami; Tsuchiya, Kenji J; Sugihara, Genichi; Iwata, Yasuhide; Suzuki, Katsuaki; Matsuzaki, Hideo; Suda, Shiro; Sugiyama, Toshiro; Takei, Nori; Mori, Norio

2010-01-01

Autism is a neurodevelopmental disorder that is characterized by repetitive and/or obsessive interests and behavior and by deficits in sociability and communication. Although its neurobiological underpinnings are postulated to lie in abnormalities of the serotoninergic and dopaminergic systems, the details remain unknown. To determine the occurrence of changes in the binding of serotonin and dopamine transporters, which are highly selective markers for their respective neuronal systems. Using positron emission tomography, we measured the binding of brain serotonin and dopamine transporters in each individual with the radioligands carbon 11 ((11)C)-labeled trans-1,2,3,5,6,10-beta-hexahydro-6-[4-(methylthio)phenyl]pyrrolo-[2,1-a]isoquinoline ([(11)C](+)McN-5652) and 2beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane ([(11)C]WIN-35,428), respectively. Statistical parametric mapping was used for between-subject analysis and within-subject correlation analysis with respect to clinical variables. Participants recruited from the community. Twenty men (age range, 18-26 years; mean [SD] IQ, 99.3 [18.1]) with autism and 20 age- and IQ-matched control subjects. Serotonin transporter binding was significantly lower throughout the brain in autistic individuals compared with controls (P dopamine transporter binding was significantly higher in the orbitofrontal cortex of the autistic group (P dopamine transporter binding was significantly inversely correlated with serotonin transporter binding (r = -0.61; P = .004). The brains of autistic individuals have abnormalities in both serotonin transporter and dopamine transporter binding. The present findings indicate that the gross abnormalities in these neurotransmitter systems may underpin the neurophysiologic mechanism of autism. Our sample was not characteristic or representative of a typical sample of adults with autism in the community.

Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

Science.gov (United States)

Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

2013-01-01

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

Vulnerability of positron emission tomography radiotracers to endogenous competition

International Nuclear Information System (INIS)

Laruelle, M.; Huang, Y.

2001-01-01

PET and SPECT neuro receptor imaging techniques combined with pharmacological challenges have been introduce to measure acute fluctuations of synaptic dopamine (DA) concentrations in the living human brain. Changes in the in vivo binding of radioligands following manipulation of transmitter levels are generally believed to be driven by binding competition between the radioligand and neurotransmitter. This imaging modality has been very successful in the study of DA transmission at D2 receptors. Yet, the extension of this technique to the study of other neurotransmitter systems has proven difficult. This paper reviews recent evidence suggesting that simple binding competition might not be the only phenomenon regulating transmitter-radioligand interactions in vivo, and examines emerging data indicating that receptor trafficking might also be involved. A better understanding of the mechanisms underlying these interactions should facilitate the development of PET and SPECT radiotracers suitable for the reporting of synaptic transmitter levels

Vulnerability of positron emission tomography radiotracers to endogenous competition

Energy Technology Data Exchange (ETDEWEB)

Laruelle, M.; Huang, Y. [Columbia University College of Physicians and Surgeons and State Psychiatric Institute, Dept. of Psychiatry and Radiology, New York, NY (United States)

2001-06-01

PET and SPECT neuro receptor imaging techniques combined with pharmacological challenges have been introduce to measure acute fluctuations of synaptic dopamine (DA) concentrations in the living human brain. Changes in the in vivo binding of radioligands following manipulation of transmitter levels are generally believed to be driven by binding competition between the radioligand and neurotransmitter. This imaging modality has been very successful in the study of DA transmission at D2 receptors. Yet, the extension of this technique to the study of other neurotransmitter systems has proven difficult. This paper reviews recent evidence suggesting that simple binding competition might not be the only phenomenon regulating transmitter-radioligand interactions in vivo, and examines emerging data indicating that receptor trafficking might also be involved. A better understanding of the mechanisms underlying these interactions should facilitate the development of PET and SPECT radiotracers suitable for the reporting of synaptic transmitter levels.

Immunoradiometric assay for cytomegalovirus-specific IgG antibodies

International Nuclear Information System (INIS)

Klapper, P.E.; Cleator, G.M.; Prinja-Wolks, D.; Morris, D.J.

1990-01-01

An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 [human cytomegalovirus (CMV)] has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specifity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT). Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate. (author). 23 refs.; 1 tab

Evaluation of an Immunochromatographic Assay for Rapid Detection of Penicillin-Binding Protein 2a in Human and Animal Staphylococcus intermedius Group, Staphylococcus lugdunensis, and Staphylococcus schleiferi Clinical Isolates.

Science.gov (United States)

Arnold, A R; Burnham, C-A D; Ford, B A; Lawhon, S D; McAllister, S K; Lonsway, D; Albrecht, V; Jerris, R C; Rasheed, J K; Limbago, B; Burd, E M; Westblade, L F

2016-03-01

The performance of a rapid penicillin-binding protein 2a (PBP2a) detection assay, the Alere PBP2a culture colony test, was evaluated for identification of PBP2a-mediated beta-lactam resistance in human and animal clinical isolates of Staphylococcus intermedius group, Staphylococcus lugdunensis, and Staphylococcus schleiferi. The assay was sensitive and specific, with all PBP2a-negative and PBP2a-positive strains testing negative and positive, respectively. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Assay of vitamin B12

International Nuclear Information System (INIS)

Tovey, K.C.; Carrick, D.T.

1982-01-01

A radioassay is described for vitamin B12 which involves denaturing serum protein binding proteins with alkali. In the denaturation step a dithiopolyol and cyanide are used and in the intrinsic factor assay step a vitamin B12 analogue such as cobinamide is used to bind with any remaining serum proteins. The invention also includes a kit in which the dithiopolyol is provided in admixture with the alkali. The dithiopolyol may be dithiothreitol or dithioerythritol. (author)

Effects of anesthesia and species on the uptake or binding of radioligands in vivo in the Göttingen minipig

DEFF Research Database (Denmark)

Alstrup, Aage Kristian Olsen; Landau, Anne M.; Holden, James E.

2013-01-01

Progress in neuroscience research often involves animals, as no adequate alternatives exist to animal models of living systems. However, both the physiological characteristics of the species used and the effects of anesthesia raise questions of common concern. Here, we demonstrate the confounding...... influences of these effects on tracer binding in positron emission tomography (PET). We determined the effects of two routinely used anesthetics (isoflurane and propofol) on the binding of two tracers of monoamine function, [ C 11 ]SCH23390, a tracer of the dopamine D1 and D5 receptors, and the alpha2...

Effects of ligand priming and multiple-dose injection on tissue uptake of 111In-pentetreotide in rats

International Nuclear Information System (INIS)

Breeman, Wout A. P.; Jong, Marion de; Bernard, Bert F.; Bakker, Willem H.; Rolleman, Edgar J.; Kwekkeboom, Kik J.; Visser, Theo J.; Krenning, Eric P.

1997-01-01