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PubMed1996

Synthesis, in vitro validation and in vivo pharmacokinetics of [125I]N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl) ethylamine: a high-affinity ligand for imaging sigma receptor positive tumors.

N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl)ethylamine, IPEMP, and the corresponding bromo derivative, BrPEMP, have been synthesized and characterized. Both BrPEMP and IPEMP were evaluated for sigma-1 and sigma-2 subtype receptor affinities and found to possess very high affinities for both receptor subtypes. The precursor for radioiodination n-tributylstannylphenylethylpiperidinylethylamine was prepared from its bromo derivative by palladium-catalyzed stannylation reaction. Radioiodinated 4-[125I]PEMP was readily prepared in high yields and high specific activity by oxidative iododestannylation reaction using chloramine-T as oxidizing agent. Sites labeled by 4-[125I]PEMP in guinea pig brain membranes showed high affinity for BD1008, haloperidol, and (+)-pentazocine (Ki = 5.06 +/- 0.40, 32.6 +/- 2.75, and 48.1 +/- 8.60 nM, respectively), which is consistent with sigma receptor pharmacology. Competition binding studies of 4-[125I]PEMP in melanoma (A375) and MCF-7 breast cancer cells showed a high affinity, dose-dependent inhibition of binding with known sigma ligand N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl) ethylamine, BD1008 (Ki = 5, 11 nM, respectively), supporting the labeling of sigma sites in these cells. Haloperidol, however showed a weaker (Ki = 100-200 nM) affinity for the sites labeled by 4-[125I]PEMP in these cells. Biodistribution studies of 4-[125I]PEMP in rats showed a fast clearance of this radiopharmaceutical from blood, liver, lung, and other organs. A co-injection of 4-IPEMP with 4-[125I]PEMP resulted in 37%, 69%, and 35% decrease in activity in liver, kidney, and brain (organs possessing sigma receptors), respectively at 1-h postinjection. These results suggest that 4-[125I]PEMP is a promising radiopharmaceutical for pursuing further studies in animal models with tumors.

Animals↗
PubMed1994

Oncostatin M (OSM) inhibits the differentiation of pluripotent embryonic stem cells in vitro.

Oncostatin M (OSM) is a cytokine which shares a common gene structure and amino acid sequence similarity with leukemia inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6), suggesting evolution from a common ancestral gene. These four cytokines share several biological activities including the ability to induce the differentiation of the murine M1 myeloid leukemic cell line. To further define the functional similarities within this family, we have investigated whether OSM can substitute for LIF in the maintenance in vitro of the undifferentiated state of pluripotent embryonic stem (ES) cells. In this study, we demonstrate that human recombinant OSM is similar to LIF in its ability to inhibit the differentiation of MBL-5 murine ES cells cultured in vitro. The level of differentiation was determined by morphological criteria and by the continued expression of the embryonic stem cell-specific surface antigen defined by the ECMA-7 monoclonal antibody. Competition binding studies demonstrate that OSM binds to the LIF receptor on MBL-5 ES cells. Our results implicate OSM as a developmental regulatory factor for embryonic stem cells in vivo.

Animals↗
PubMed2005

A surface plasmon resonance-based assay for small molecule inhibitors of human cyclophilin A.

A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr approximately 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K(dCsA)) binding to the immobilized His-CypA was 23+/-6 nM, with on and off rates of 0.53+/-0.1 microM(-1) s(-1) and 1.2+/-0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.

Binding, Competitive↗
PubMed2004

Aryl hydrocarbon receptor response to indigoids in vitro and in vivo.

Indigo and indirubin have been reported to be present at low levels in human urine. The possibility that indigoids are physiological ligands of the aryl hydrocarbon receptor (AhR) has been suggested by initial studies in yeast, where indirubin was found to be 50 times more potent than 2,3,7,8-tetrachlorodibenzo[p]dioxin (TCDD), and indigo was found to be equipotent. To demonstrate that these indigoids are bona fide agonists in mammalian systems, we employed a number of in vitro and in vivo measures of AhR agonist potency. In a hepatoma cell reporter system, indigo yielded an EC50 of approximately 5x10(-6)M (indirubin 3' -oxime EC50 approximately 5x10(-7)M, indirubin EC50 approximately 1x10(-7)M). A comparison of these EC50 values with that of 2,3,7,8-tetrachlorodibenzofuran (TCDBF) ( approximately 3x10(-9)M) indicated that these compounds are less potent than classic halogenated-dibenzofurans or -dibenzo-p-dioxins. Competitive binding assays for AhR occupancy showed similar IC50 values for indirubin and TCDBF ( approximately 2x10(-9) and 5x10(-9)M), with the IC50 values of indigo and indirubin 3' -oxime being approximately 10-fold higher. When rats were treated with these indigoids in the range of 1.5-50mg/kg, induction of hepatic cytochrome P450 1A1 was detected. Differences in the rank-order of potency observed in vivo and in vitro could, in part, be explained by metabolism. Although their biological potencies are not as high as has been previously suggested, collectively the results show that these indole-derived pigments are agonists of AhR in vivo. The in vivo results suggest that solubility, distribution, and metabolism influence the response to the compounds.

Animals↗
PubMed2005

Unusual pseudosubstrate specificity of a novel 3,5-dimethoxyphenol O-methyltransferase cloned from Ruta graveolens L.

A cDNA was cloned from Ruta graveolens cells encoding a novel O-methyltransferase (OMT) with high similarity to orcinol or chavicol/eugenol OMTs, but containing a serine-rich N-terminus and a 13 amino acid insertion between motifs IV and V. Expression in Escherichia coli revealed S-adenosyl-l-methionine-dependent OMT activity with methoxylated phenols only with an apparent Km of 20.4 for the prime substrate 3,5-dimethoxyphenol. The enzyme forms a homodimer of 84 kDa, and the activity was insignificantly affected by 2.0 mM Ca2+ or Mg2+, whereas Fe2+, Co2+, Zn2+, Cu2+ or Hg2+ were inhibitory (78-100%). Dithiothreitol (DTT) suppressed the OMT activity. This effect was examined further, and, in the presence of Zn2+ as a potential thiol methyltransferase (TMT) cofactor, the recombinant OMT methylated DTT to DTT-monomethylthioether. Sets of kinetic OMT experiments with 3,5-dimethoxyphenol at various Zn2+/DTT concentrations revealed the competitive binding of DTT with an apparent Ki of 52.0 microM. Thus, the OMT exhibited TMT activity with almost equivalent affinity to the thiol pseudosubstrate which is structurally unrelated to methoxyphenols.

Amino Acid Sequence↗
PubMed2006

Influence of prosthetic radioiodination on the chemical and biological behavior of chemotactic peptides labeled at high specific activity.

The influence of radioiodination made through prosthetic group N-succinimidyl-3-[131I]iodo-benzoate ([131I]SIB) on the behavior of small peptides was investigated using as model the chemotactic hexapeptide Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys. No carrier added labeled peptide was isolated by reverse-phase HPLC (RP-HPLC) with coupling efficiencies up to 59-75%. Biodistribution in normal and infected C57 mice showed mainly a hepatobiliary clearance, a very low thyroid uptake and the highest uptake at the infection site was within 1h of injection. Superoxide production and competitive binding assays studies in human polymorphonuclear leukocytes showed a preserved biological activity and high-affinity specific binding. However, the results indicated that the changes observed in the receptor-binding properties with an IC50 almost twice than the unlabeled peptide and the increasing in the hepatobiliary excretion could be the consequence of the increased lipophicity observed due to the presence of the prosthetic group together with a strong influence of the radioisotope per se.

Animals↗
PubMed2004

Mechanistic basis for estrogenic effects in fathead minnow (Pimephales promelas) following exposure to the androgen 17alpha-methyltestosterone: conversion of 17alpha-methyltestosterone to 17alpha-methylestradiol.

Exposure of adult fathead minnows (Pimephales promelas) to the androgen 17alpha-methyltestosterone (MT) produces both androgenic and estrogenic effects, manifested as nuptial tubercle formation in females, and vitellogenin production in males and females, respectively. The present study was conducted to determine if the unanticipated estrogenic effects are produced by conversion of MT via aromatase activity to 17alpha-methylestradiol (ME2). Aromatase activity at the end of a 7-day waterborne MT exposure (20, 200microg/l) was significantly decreased in ovarian microsomes and brain homogenates from exposed fish, to about 30-50% of control activity. Although aromatase activity was decreased by 7 days, it is possible that the conversion of MT to ME2 occurred soon after initial exposure. In support of this, ME2 was detected in plasma samples of the fish following the 7-day exposure, confirming their ability convert the androgen MT to the estrogen ME2. The concentration of ME2 in plasma was within the range of plasma 17ss-estradiol (E2) found in control female fathead minnows (4-5ng/ml). These results, in conjunction with competitive binding assays that indicate ME2 binds to the fathead minnow estrogen receptor with a relative binding affinity of 68.3% of E2, support the hypothesis that aromatization of MT to ME2 contributes to the estrogenic effects in fathead minnows following exposure to this androgen.

Analysis of Variance↗
PubMed2005

Colorectal cancer metastases affect the biochemical characteristics of the human liver beta-adrenoceptor-G-protein-adenylate cyclase system.

The sympathetic-catecholamine system is involved in the regulation of hepatic metabolic pathways mainly through cAMP-linked beta2-adrenoceptors (beta2-ARs) in humans and to a lesser extent through cAMP-independent mechanisms, but no information is available about the possible biochemical changes of beta2-ARs and their signalling pathways in human colorectal cancer (CRC) and colorectal cancer hepatic metastases (CRCHM). Changes in density and distribution of beta-ARs as well as in post-receptor signalling components were studied in membranes of human liver with CRCHM, and for comparison, in membranes of nonadjacent, non-metastatic human liver (NA-NM) obtained from 13 patients, using binding and competition binding studies. Studies were also carried out using normal and cancerous human colon tissues. In CRCHM, the density of beta-ARs (B(max)) was significantly reduced, compared to NA-NM liver tissues (40.09+/-2.83 vs. 23.09+/-3.24 fmol/mg protein; P<0.001). A similar decrease in the beta-AR density was observed in the colon with primary colorectal cancer compared to healthy colon (37.6+/-2.2 vs. 23.8+/-3.5 fmol/mg protein), whereas the affinity of ICYP binding to the receptor remained unaffected. Desensitized beta-ARs were uncoupled from stimulatory G-protein (G(S)), as total density of beta-adrenoceptors in the high affinity state was significantly reduced. Concomitantly, CRCHM elicited decrease in the catalytic adenylate cyclase (AC) activity (cAMP formation) in response to isoproterenol plus GTP or forskolin or NaF. In NA-NM and CRCHM liver, the inhibition-concentration curves of ICI 118.551 showed the presence of a homogeneous population of the beta2-AR subtypes. Neither the binding patterns nor the inhibition constant (K(i)) of ICI 118.551 were altered in CRCHM. In CRCHM, the hepatic beta-AR-G-protein(s)-AC signalling system was markedly impaired, thus, these changes may well influence beta-AR-mediated functions in both organs.

Adenylyl Cyclases↗
PubMed2004

Discovery of novel peptide/receptor interactions: identification of PHM-27 as a potent agonist of the human calcitonin receptor.

Many naturally occurring peptides exhibit a high degree of promiscuity across G-protein coupled receptor subtypes. The degree to which this phenomenon occurs, and its physiological significance is not well characterized. In addition, many 'orphan' peptides exist for which there are no known receptors. Therefore, to identify novel interactions between biologically active peptides and G-protein coupled receptors, a library of nearly 200 peptides was screened against the human calcitonin (hCTr), human Parathyroid Hormone (PTH1R), human Corticotropin Releasing Factor (CRF1), and the human Glucagon-like peptide (GLP1) receptors using a cell-based functional assay (Receptor Selection and Amplification Technology). Functional profiling revealed that the 'orphan peptide' PHM-27 selectively activated the hCTr; no activity was observed at the PTH1, CRF1, or GLP1 receptors. PHM-27 was a potent agonist at the hCTr, with similar efficacy as human calcitonin, and a potency of 11 nM. These results were confirmed in cyclic AMP assays. Responses to calcitonin and PHM-27 could be suppressed by the antagonist salmon calcitonin (8-32). In competition binding studies, salmon calcitonin (8-32), calcitonin, and PHM-27 were each able to inhibit (125)I-calcitonin from cell membranes containing transiently expressed hCTr. These results indicate that the orphan peptide PHM-27 is a potent agonist at the hCTr.

3T3 Cells↗
PubMed2005

Cys2,7EtalphaCGRP is a potent agonist for CGRP1 receptors in SK-N-MC cells.

The present study reveals that cystein2,7 ethyl-amidealphaCGRP (Cys2,7EtalphaCGRP), an advertised calcitonin gene-related peptide 2 (CGRP2) receptor subtype-selective agonist, is also a potent agonist for the calcitonin gene-related peptide 1 (CGRP1) receptors natively expressed in the SK-N-MC human neuroblastoma cell line. Cys2,7EtalphaCGRP and alpha calcitonin gene-related peptide (alphaCGRP) promote cyclic AMP accumulation in intact SK-N-MC cells to the same extent with EC50 of 1.6+/-0.2 and 0.4+/-0.08 nM, respectively. The antagonist alpha calcitonin gene-related peptide-8-37 (alphaCGRP-(8-37)) produces a concentration-dependent rightward shift of the alphaCGRP- and Cys2,7EtalphaCGRP concentration-response curves with KB-values (71+/-33 and 47+/-21 nM, respectively). The competitive antagonism by alphaCGRP-(8-37) and the similar KB-values suggests that alphaCGRP and Cys2,7EtalphaCGRP stimulate the same receptor. In competition binding studies with [125I]-alphaCGRP on SK-N-MC cell membranes, Cys2,7EtalphaCGRP and alphaCGRP-(8-37) display high affinity for the majority of the binding sites with Ki-values of 0.030+/-0.013 and 0.60+/-0.013 nM, respectively. The present findings are at odds with the proclaimed utilization of Cys2,7EtalphaCGRP as a CGRP2 receptor-selective pharmacological tool. Differences between the agonistic profile of this ligand in this and other experimental systems might be species--or even cell type--dependent.

Binding Sites↗
PubMed2005

Ribonucleic guanidine demonstrates an unexpected marked preference for complementary DNA rather than RNA.

Replacement of the phosphodiester linkages of DNA and RNA by guanidinium linkages provides DNG and RNG. We report here the order of stability of mixed duplexes (RNG-U5.DNA-A5>>RNA-U5.RNA-A5>RNG-U5.RNA-A5>RNA-U5.DNA-A5>DNA-T5.DNA-A5). The considerable stability of RNG.DNA compared to RNG.RNA is shown to be due to the rigid backbone of RNG existing only in B-form and therefore lowering its affinity for A-RNA. RNG oligomers are putative antigene agents which are specific for DNA and would have minimal competitive binding to ncRNA.

Binding, Competitive↗
PubMed2005

Estrogenic effects of environmental chemicals: an interspecies comparison.

The development of various in vitro screening methods has led to identification of novel estrogenic chemicals of natural and anthropogenic origin. In this study, the (anti)estrogenic potential of several environmental chemicals were compared in an array of in vitro test systems comprising: (i) competitive binding to estrogen receptors derived from the human breast cancer cell line MCF-7 (hER) and rainbow trout (Oncorhynchus mykiss) (rtER), (ii) a proliferation assay with MCF-7 cells (E-SCREEN), and iii) induction of vitellogenin (rtVtg) in isolated rainbow trout hepatocytes. The results showed substantial differences in assay sensitivity for potent estrogens like 17beta-estradiol, diethylstilbestrol and zearalenone (ranking order of sensitivity: E-SCREEN > hER approximately rtER approximately rtVtg). Chemicals like 4-n-nonylphenol and bisphenol A had higher relative binding affinity to the hER, whereas 4-t-butylphenol and 4-n-butylphenol showed highest affinity to the rtER. Zearalenone and the novel estrogen 4-t-butylhexanol displayed a considerable higher relative potency in the E-SCREEN than the rtVtg assay, whereas alkylphenols and the novel estrogen mimic 4-t-butyl-nitrobenzene were most potent in fish cells. Correlation analysis of data from the test systems suggest that interspecies differences is largely due to inter-assay variation of the ER-dependent cellular responses, whereas binding to the ER are fairly similar in the two species tested.

Animals↗
PubMedDate not supplied

Characterization of monoclonal antibodies directed against the canine distemper virus nucleocapsid protein.

We have established four monoclonal antibodies (MAbs) against the nucleocapsid protein (NP) of canine distemper virus (CDV). A competitive binding assay has revealed that the MAbs are directed against two antigenic domains. An immunofluorescence assay using a series of deletion clones of the NP and an immunoprecipitation assay using the NP have revealed that two of the MAbs recognize the C-terminal region of the NP while the other two recognize the tertiary structure of the N-terminal domain. These MAbs reacted with all eight strains of CDV used in this study, but showed different reactivities against measles virus and rinderpest virus.

Animals↗
PubMed2006

Combined testing of antibody titer and affinity improves insulin autoantibody measurement: Diabetes Antibody Standardization Program.

The aim of the workshop was to assess whether four laboratories could reproducibly measure insulin autoantibody (IAA) affinity in coded sera from non-diabetic relatives of patients with type 1 diabetes, newly diagnosed patients, and healthy blood donors, and whether combining affinity with autoantibody titer could improve concordance and performance of IAA assays. IAA affinity was measured by competitive binding using constant amounts of Tyr14A [125I] human insulin and increasing quantities of unlabeled human insulin. There was high concordance between laboratories in distinguishing high, moderate, and low affinity IAA, although IAA binding to insulin varied with assay format. Multiple islet autoantibody-positive and patient sera were identified by high affinity IAA regardless of laboratory-designated IAA status. Combining affinity and titer significantly improved sensitivity, specificity, and concordance of IAA measurement. This workshop has demonstrated that different laboratories are able to reproduce IAA affinity results and that considering IAA affinity is likely to improve the diagnostic performance of IAA assays.

Adolescent↗
PubMed2005

Synthesis and evaluation of 7-amino-2-(2(3)-furyl)-5-phenylethylamino-oxazolo[5,4-d]pyrimidines as potential A2A adenosine receptor antagonists for positron emission tomography (PET).

The brain A2A adenosine receptor (A2AAR) participates with the dopamine D2 receptor in the control of movement and also might influence behavior. Because PET is an important tool for studying the roles of receptors in disease, a ligand for imaging the brain A2AAR is desirable. This report describes the synthesis and A2AAR antagonist activities of a panel of phenyl-substituted 7-amino-2-(2-furyl)-5-phenylethylamino-oxazolo[5,4-d]pyrimidines, 11aa-af, and their 3-furyl congeners, 11ba-bd. In competitive binding studies all compounds displaced [3H]CGS21680 from the A2AAR with Ki values of 14-33 nM with selectivity for the A2AAR over the A1AR of 5- to 94-fold. Autoradiography of brain sections showed a high level of unspecific binding that obscured specific binding. Thus, these compounds are not promising PET ligands.

Adenosine A2 Receptor Antagonists↗
PubMed2004

Norchloro-fluoro-homoepibatidine: specificity to neuronal nicotinic acetylcholine receptor subtypes in vitro.

The subtype-specificity of newly synthesised epibatidine-related compounds, norchloro-fluoro-homoepibatidine (NCFHEB) and derivatives, to neuronal nicotinic acetylcholine receptors (nAChR) has been investigated. NCFHEBs were assayed in competitive binding assays to (+/-)-[(3)H]epibatidine-labelled rat thalamic nAChRs and human alpha4beta2, alpha3beta4, and alpha7 nAChRs, expressed in stably transfected HEK-293 and SH-SY5Y cells. The binding affinity of (+)-NCFHEB (K(i): 0.064 nM) and (-)-NCFHEB (K(i): 0.112 nM) to human alpha4beta2 nAChR is in the same order of magnitude as that of epibatidine (K(i): 0.014 nM). However, because the affinity of both NCFHEB-enantiomers to human alpha3beta4 nAChR is up to 65 times lower than that of epibatidine, the alpha4beta2 subtype-specificity of NCFHEB is increased up to 1,400% compared to epibatidine.

Animals↗
PubMed2006

Effects of alginate inclusion on the vector properties of chitosan-based nanoparticles.

Chitosan nanoparticles have shown considerable promise as gene vectors but do not mediate transfection with satisfactory efficiency. To improve upon the transfection efficiency of chitosan, we approached the development of alginate-chitosan nanoparticles with the goals of maintaining low toxicity and biocompatibility. Through ionic gelation, particles were formed with a mean Z-average diameter of 157 nm and a zeta potential of +32 mV. Competition binding assays indicated that the presence of alginate reduces the strength of interaction between chitosan and DNA, contributing to improved transfection. Cell viability assays indicated that nanoparticles exhibit the same low toxicity as chitosan, and significantly reduced toxicity compared to a commercial liposome formulation. As well, complexation with nanoparticles maintained DNA integrity and protected it from nuclease degradation better than chitosan alone. Alginate-chitosan nanoparticles were able to mediate transfection of 293T cells four times that achieved by chitosan nanoparticles; at 48 h, the transfection efficiency was as high as with Lipofectamine, with significantly reduced cytotoxicity. Overall, alginate inclusion improved the vector properties of chitosan-based nanoparticles, demonstrating superior transfection ability while maintaining biocompatibility and low toxicity.

Alginates↗
PubMed2004

Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications.

Two sets of expression vectors were constructed that permitted the efficient expression of single-chain Fv fragments (scFvs) fused N-terminally to an enhanced mutant of the green fluorescent protein GFP+ or the red fluorescent protein DsRed in insect and mammalian cells. The vectors allowed rapid cloning of scFv fragments and secretion of the fusion proteins in a native conformation. Fluorescent scFv fusion proteins directed against a series of cluster of differentiation (CD) antigens were efficiently secreted by transiently transfected mammalian cells and insect cells infected with baculoviral expression constructs. Yields of the secreted proteins varied from 100 microg/l to 3 mg/l. The purified proteins were functionally active in flow cytometry, immunofluorescent microscopy, and competition binding experiments performed to delineate the epitopes recognized by different monoclonal antibodies against the same polypeptide. The use of two different scFv fragments fused with red and green fluorescent proteins and reacting with T- and B-cell lineage markers (CD7 and CD19), respectively, allowed a simplified quantitation of both subsets in two-color flow cytometry experiments with mixed populations of T- and B-lymphoid cells. Due to the lack of Fc domains in the scFv proteins, the fluorescent fusion proteins showed more than 20-fold reduced background fluorescence compared with whole antibodies of the same specificity in experiments with effector cells expressing the high affinity FcgammaRI receptor CD64. Thus, for a number of analytical applications, fluorescent scFv fusion proteins offer advantages over the use of complete primary antibodies and chemically labeled fluorescent secondary antibodies.

Animals↗