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At least 919 records · Page 51Linked to original sources
PubMed2001

Cloning and functional pharmacology of two corticotropin-releasing factor receptors from a teleost fish.

Although it is well established that fish possess corticotropin-releasing factor (CRF) and a CRF-like peptide, urotensin I, comparatively little is known about the pharmacology of their cognate receptors. Here we report the isolation and functional expression of two complementary DNAs (cDNAs), from the chum salmon Oncorhynchus keta, which encode orthologues of the mammalian and amphibian CRF type 1 (CRF(1)) and type 2 (CRF(2)) receptors. Radioligand competition binding experiments have revealed that the salmon CRF(1) and CRF(2) receptors bind urotensin I with approximately 8-fold higher affinity than rat/human CRF. These two peptides together with two related CRF-like peptides, namely, sauvagine and urocortin, were also tested in cAMP assays; for cells expressing the salmon CRF(1) receptor, EC(50) values for the stimulation of cAMP production were between 4.5+/-1.8 and 15.3+/-3.1 nM. For the salmon CRF(2) receptor, the corresponding values were: rat/human CRF, 9.4+/-0.4 nM; urotensin I, 21.2+/-2.1 nM; sauvagine, 0.7+/-0.1 nM; and urocortin, 2.2+/-0.7 nM. We have also functionally coupled the O. keta CRF(1) receptor, in Xenopus laevis oocytes, to the endogenous Ca(2+)-activated chloride conductance by co-expression with the G-protein alpha subunit, G(alpha16). The EC(50) value for channel activation by rat/human CRF (11.2+/-2.6 nM) agrees well with that obtained in cAMP assays (15.3+/-3.1 nM). We conclude that although sauvagine is 13- and 30-fold more potent than rat/human CRF and urotensin I, respectively, in activating the salmon CRF(2) receptor, neither receptor appears able to discriminate between the native ligands CRF and urotensin I.

Amino Acid Sequence↗
PubMed1997

14 beta-Chlorocinnamoylamino derivatives of metopon: long-term mu-opioid receptor antagonists.

The affinity, selectivity and antinociceptive properties of 5 beta-methyl-14 beta-(p-chlorocinnamoylamino)-7,8-dihydromorphinone (MET-Cl-CAMO) and N-cyclopropyl-methyl-5 beta-methyl-14 beta-(p-chlorocinnamoylamino)-7, 8-dihydronormorphinone (N-CPM-MET-Cl-CAMO) for the multiple opioid receptors were characterized. In competition binding assays using bovine striatal membranes, both compounds inhibited the binding of 0.25 nM [3H][D-Ala2, (Me)-Phe4,Gly(ol)5]enkephalin (DAMGO) with IC 50 values of less than 2 nM. Preincubation of membranes with MET-CI-CAMO and N-CPM-MET-Cl-CAMO produced a concentration-dependent, wash-resistant inhibition of mu-opioid receptor binding. Saturation binding experiments with N-CPM-MET-Cl-CAMO showed a reduction in the number of mu-opioid binding sites without a change in affinity. In the mouse 55 degrees C warm-water tail-flick assay, neither MET-Cl-CAMO nor N-CPM-MET-Cl-CAMO at doses up to 100 nmol produced antinociception after intracerebroventricular administration, but morphine-induced antinociception was antagonized in a time- and dose-dependent manner by both compounds. The antagonism produced by 1 nmol of either MET-Cl-CAMO or N-CPM-MET-Cl-CAMO reached a maximal effect after 24 h, and lasted up to 48 h. Analgesia mediated by delta- or kappa-opioids was not altered by either compound. In summary, the data suggest that MET-Cl-CAMO and N-CPM-MET-Cl-CAMO are long-term, mu-opioid receptor antagonists, devoid of agonist properties in the mouse tail-flick assay, and that N-CPM-MET-Cl-CAMO may produce its antagonistic effects by binding irreversibly to the mu-opioid receptor.

Analgesics↗
PubMed1997

Noradrenaline and adrenaline are high affinity agonists at dopamine D4 receptors.

The activity of monoamine neurotransmitters was examined at dopamine D4 receptors. In competition binding with [3H]spiperone, noradrenaline and adrenaline exhibited a high affinity binding component (KH = 12.1 nM and 5.0 nM, respectively), similar to that of dopamine (KH = 2.6 nM), whereas serotonin (5-hydroxytryptamine, 5-HT) and histamine had low affinity (Ki > 1000 nM). Noradrenaline and adrenaline acted as agonists at dopamine D4 receptors, stimulating receptor-mediated [35S]guanylyl-gamma-thiotriphosphate ([35S]GTP gamma S) binding (EC50 = 7.8 and 5.8 microM, respectively, versus 0.1 microM for dopamine). The dopamine D4 receptor-selective ligand, 3-(4-[4-chlorophenyl]piperazin-1-yl)methyl-1H-pyrrolo[2,3b]-pyridi ne (L 745,870) and the dopaminergic antagonists, spiperone, haloperidol and clozapine, inhibited noradrenaline-stimulated [35S]GTP gamma S binding whereas alpha 1-, alpha 2- and beta-adrenoceptor antagonists did not. These results indicate that dopamine D4 receptors are activated by noradrenaline and adrenaline, although at 50-100-fold higher concentrations than dopamine.

Animals↗
PubMed1997

Bot IT2: a new scorpion toxin to study receptor site on insect sodium channels.

The insect-specific Bothus occitanus tunetanus IT2 toxin is distinguishable from other scorpion toxins by its amino acid sequence and effects on sodium conductance. The present study reveals that Bot IT2 possesses in cockroach neuronal membranes a single class of high affinity (Kd = 0.3 +/- 0.1 nM) and low capacity (Bmax = 2.4 +/- 0.5 pmol/mg) binding sites. Competitive binding experiments with several known sodium channel neurotoxins reveal that the Bot IT2 binding site is in close proximity to the other toxins.

Animals↗
PubMed1985

Preliminary results with the antiprogestational compound RU-486 (mifepristone) for interruption of early pregnancy.

RU-486 (mifepristone) (Roussel-Uclaf, Paris, France) is an antiprogesterone that acts through a competitive binding at the site of the progesterone and cortisol receptor. It induced a complete expulsion in 29 (83%) of 35 patients with an unwanted pregnancy with up to 55 days of amenorrhea. A complete expulsion occurred in 17 (89%) of the 19 nulliparous women, and no serious side effects were seen. However, our results were less successful in ine patients with a more advanced pregnancy (between 8 and 10 weeks). In this group, only three complete expulsions were seen.

Abortion, Induced↗
PubMed1997

Optimization of post-column chemiluminescent detection for low-molecular-mass conjugates of acridinium esters.

In this study, various factors are examined that affect the post-column chemiluminescence detection of low-molecular-mass compounds labelled with acridinium esters, using 9-phenyl acridinium ester (PAE) as the initial model. Reaction conditions examined included the effect of hydroxide or hydrogen peroxide concentration, ionic strength and surfactant content on the degree and length of light production by the acridinium ester label. Based on these results, a system was developed for the post-column detection of acridinium ester conjugates in HPLC. The final post-column reactor had optimum conditions similar to those for a benchtop luminometer but gave light production over slightly longer periods of time and had a broader range of reagent concentrations that gave a maximum response. The detection limit of this system for PAE was 7 x 10(-19) mol per injection, with linear and dynamic ranges that extended up to 3 x 10(-15) and 3 x 10(-13) mol, respectively. Some preliminary work was conducted examining the use of this system in chromatography-based competitive binding immunoassays with a thyroxine-acridinium ester conjugate being used as the label. The estimated limit of detection was 2.5 x 10(-17) mol (or roughly 10(-12) M for a 25 microliters sample) based on the retained fraction of the conjugate.

Acridines↗
PubMed1999

Analysis of urinary drugs of abuse by a multianalyte capillary electrophoretic immunoassay.

This paper characterizes a novel multianalyte competitive binding, electrokinetic capillary-based immunoassay for urinary methadone, opiates, benzoylecgonine (cocaine metabolite) and amphetamines. After incubation of 25 microliters urine with the reactants for several minutes in the presence of an internal standard, a small aliquot of the mixture is applied onto a fused-silica capillary and the unbound fluorescein labelled drug tracers are monitored by capillary electrophoresis with on-column laser induced fluorescence detection. The multianalyte assay is shown to be rapid, simple, quantitative, capable of recognizing urinary drug concentrations > or = 30 ng/ml and suitable for screening of patient urines. Data are demonstrated to compare well with those obtained by routine screening methods based on enzyme multiplied immunoassay techniques and fluorescence polarization immunoassays. The electrokinetic capillary assay has been validated via analysis of external quality control urines and confirmation analysis of patient urines using GC-MS.

Amphetamines↗
PubMed1977

Immunochemical studies of infectious mononucleosis. VI. a radioimmunoassay for the detection of infectious mononucleosis heterophile antibody and antigen.

The coated-tube method of solid-phase radioimmunoassay has been adapted to the detection of heterophile antibodies and antigens of infectious mononucleosis. Disposable plastic hemagglutination trays were coated with purified glycoprotein from horse erythrocytes and the subsequent uptake of antibody from test sera was detected by radio-iodinated horse erythrocyte glycoprotein. In a preliminary survey of sera from patients with infectious mononucleosis and sera from controls, the assay proved highly sensitive and specific. The test system was also useful in a competitive binding assay for immunochemical studies of glycoproteins from other heterophile antigen-positive species.

Animals↗
PubMed1997

Human breast carcinoma cells synthesize a protein immunorelated to platelet glycoprotein-Ib alpha with different functional properties.

Although tumor cell-induced platelet aggregation is thought to mediate an early step in the metastatic process, little is known about tumor adhesive receptors responsible for the initial platelet-tumor attachments. Because our preliminary work demonstrated that a platelet-immunorelated glycoprotein Ib alpha (GPIb alpha) receptor expressed by the human breast carcinoma cell line MCF-7 participates in tumor-induced platelet aggregation, we examined the synthesis and functional characteristics of this MCF-7-immunorelated GPIb alpha. When 35S-cysteine-labeled, digitonin-lysed MCF-7 cells were immunoprecipitated with platelet-specific monoclonal antibodies (mAbs) to GPIb alpha, major radioactive bands were observed. Northern blots showed MCF-7 transcripts for GPIb alpha under both high- and low-stringency hybridization conditions. In the presence of purified human iodine 125-labeled von Willebrand factor (125I-labeled vWf) with or without the addition of ristocetin, unlabeled vWf was observed to competitively bind to fixed MCF-7 cells (50% inhibitory concentration = 10 microg/ml, dissociation constant = approximately 3.8 +/- 1.9 nmol/L, 2.7 x 106 + 445,000 binding sites/cell) in which non-GPIb alpha vWf binding sites were blocked. 125I-vWf binding to blocked MCF-7 cells could be selectively and completely inhibited by mAbs specific for the vWf binding domain of GPIb alpha but not by mAbs against the GPIX subunit, the GPIb alpha subunit, or alternate GPIb alpha epitopes other than the vWf-binding domain. Finally, when whole blood substrate was incubated with a mAb specific for the GPIb binding epitope of vWf, MCF-7-induced platelet aggregation was virtually abolished in comparison with control specimens (N = 8; p < 0.0009). These findings (1) confirm the synthesis and expression of an MCF-7 protein with homology to platelet GPIb alpha, (2) confirm that the functional activity of this MCF-7-immunorelated GPIb alpha differs from that of platelet GPIb alpha, and (3) suggest that MCF-7-immunorelated GPIb alpha in its adhesive interactions with plasma vWf may constitute an initial event in MCF-7-induced platelet aggregation.

Anti-Bacterial Agents↗
PubMed2003

Structural analysis of the epitope of the anti-HIV antibody 2F5 sheds light into its mechanism of neutralization and HIV fusion.

Inhibition of human immunodeficiency virus (HIV) fusion with the host cell has emerged as a viable therapeutic strategy, and rational design of inhibitors and vaccines, interfering with this process, is a prime target for antiviral research. To advance our knowledge of the structural biology of HIV fusion, we have studied the membrane-proximal region of the fusogenic envelope subunit gp41, which includes the epitope ELDKWA of the broadly neutralizing human antibody 2F5. The structural evidence available for this region is contradictory, with some studies suggesting an overall helical conformation, while the X-ray structure of the ELDKWAS peptide bound to the antibody shows it folded in a type I beta turn. We used a two-step strategy: Firstly, by a competition binding assay, we identified the proper boundaries of the domain recognized by 2F5, which we found considerably larger than the ELDKWAS hexapeptide. Secondly, we studied the structure of the resulting 13 amino acid residue peptide by collecting NMR data and analyzing them by our previously developed statistical method (NAMFIS). Our study revealed that the increase in binding affinity goes in parallel with stabilization of specific local and global conformational propensities, absent from the shorter epitope. When compounded with the available biological evidence, our structural analysis allows us to propose a specific role for the membrane-proximal region during HIV fusion, in terms of a conformational transition between the turn and the helical structure. At the same time, our hypothesis offers a structural explanation for the mechanism of neutralization of mAb 2F5.

Amino Acid Sequence↗
PubMed1985

The search for functional correspondences in molecular structure between two dissimilar molecules.

Where structural similarities between molecular ligands are suspected in competitive binding, these similarities may be represented by a set of correspondences between analogous ligand points in the two molecules. This paper describes an algorithm which searches through all possible sets of this form, to find the one which gives the best geometric fit between matched points. Two parameters are supplied to the algorithm, giving the number of correspondences required and the tolerance permitted in the fit. A tree-search technique is used, and methods are described which prune the tree and thus limit the search. The marine neurotoxins, saxitoxin and tetrodotoxin, are used to test a computer implementation of the procedure.

Binding, Competitive↗
PubMed1997

Inhibition of 17 beta-estradiol and progesterone activity in human breast and endometrial cancer cells by carbamate insecticides.

Using a combination of in vitro assays we have examined the capacities of contemporary-exposure chemicals to modulate human estrogen and human progesterone receptor (hER and hPR) activity in human breast and endometrial cancer cells. The carbamate insecticides aldicarb, Baygon (propoxur), bendiocarb, carbaryl, methomyl, and oxamyl were used in this study. The carbamates alone weakly activated estrogen- or progesterone-responsive reporter genes in breast and endometrial cancer cells. All of the carbamates decreased estradiol- or progesterone-induced reporter gene activity in the breast and endometrial cancer cells. In whole cell competition binding assays, the carbamates demonstrated a limited capacity to displace radiolabeled estrogen or progesterone from ER or PR. Based on the results presented here, the carbamate insecticides may represent a class of chemicals which function through a mechanism separate from ligand-binding and, therefore, may act as general endocrine modulators in mammalian cells.

Binding, Competitive↗
PubMed1998

Possible direct effect of gonadotropin releasing hormone on human endometrium and decidua.

Decidualization of endometrial tissues, which is essential for implantation and the continuation of pregnancy, is induced by pituitary hormones that are regulated by gonadotropin releasing hormone (GnRH). Our objective was to determine the role of a direct action of GnRH on endometrial tissues by comparing the characteristics of receptors for GnRH in human endometrial and decidual tissues. Competitive binding studies were performed with the protease-resistant GnRH analogues, buserelin and [125I] buserelin. The effects of buserelin on phosphoinositol turnover were determined by the measurement of inositol 1,4,5-triphosphate(IP3). The values for the dissociation constant (Kd) and number of binding sites (Bmax) per unit protein versus buserelin for endometrial tissues did not differ from the values for decidual tissues. However, the Bmax per unit DNA was significantly higher in endometrial tissues. Also, buserelin induced a significant increase in IP3 in decidual tissue. These results indicate that GnRH may be a potential modulator of the function in human endometrium and decidua. The signal transduction mechanism for GnRH action appeared to involve the accelerated turnover of phosphoinositol.

Binding, Competitive↗
PubMed2003

Attentional effects of nicotinic agonists in rats.

Nicotine can increase stimulus detection, response rate and speed in the five-choice serial reaction time task, a rodent test of attention. In the present experiments, four other nicotinic agonists with different pharmacological profiles were compared in the same procedure. The response profile of epibatidine resembled that previously obtained with nicotine in that response accuracy was enhanced and omission errors and correct response latency decreased. ABT-418 transiently increased accuracy in the first 10 min of test sessions and reduced response latency. Isoarecolone caused a dose-related increase in accuracy, but had no effect on omissions or response latency. This absence of effects on response rate- or speed-related measures may be related to its previously reported reduced ability to release dopamine as compared with nicotine. The alpha7-agonist AR-R17779 was without effect on any measure, indicating that this receptor subtype may not mediate nicotinic effects on attention. Affinity constants of compounds, determined in competition binding assays targeting the alpha4beta2, alpha7, alpha3beta4 and alpha3beta2* nAChR subtypes, could not explain the differential behavioural effects observed. Differences in their functional efficacy at nAChR subtypes may instead be responsible. The finding that attentional performance and response rate and speed can be selectively modulated by nicotinic agonists is encouraging for the development of drugs with therapeutic properties similar to those of nicotine but with reduced unwanted effects.

Animals↗
PubMedDate not supplied

In vitro characterization of trimegestone: a new potent and selective progestin.

Trimegestone (TMG) is a novel 19-norpregnane progestin under development for hormone replacement therapy and oral contraception. The objective of the current study was to characterize the potency and steroid receptor selectivity of TMG in several in vitro assays and to compare its activity to that of medroxyprogesterone acetate (MPA). TMG and MPA had a similar competitive binding affinity for human and rabbit progesterone receptor (PR). However, TMG had a significantly higher affinity for rat PR (IC(50) = 3.3 nM) than MPA (IC(50) = 53.3 nM). In T47D cells, both compounds increased alkaline phosphatase activity and cell proliferation with comparable potencies (EC(50s) of 0.1 nM and of 0.02 nM, respectively). To further characterize the progestational activity and steroid receptor selectivity, we established an immortalized human endometrial stromal cell line (HESC-T). This cell line lacks endogenous estrogen receptor (ER) and PR but does have functional glucocorticoid receptors (GR). When ER is transiently expressed in the cells, 17beta-estradiol (E(2)) induces PR, allowing the study of PR-regulated genes. In HESC-T cells expressing exogenous ER, and therefore PR, both TMG and MPA increased HRE-tk-luciferase activity tenfold with an EC(50) of 0.2 nM. In HESC-T cells without exogenous ER, and therefore no PR, TMG did not induce HRE-tk-luciferase activity, whereas MPA induced the reporter activity with an EC(50) of about 10 nM. This MPA-induced reporter activity is believed to be mediated through GR. The steroid receptor selectivity of TMG was further evaluated using the HRE-tk-luciferase assay in the human lung carcinoma cell line A549, which contains GR but no PR. In these cells TMG had no effect on luciferase activity, whereas MPA increased the reporter activity in a dose-dependent manner with an EC(50) of approximately 30 nM. Furthermore, HRE-tk-luciferase assay in mouse fibroblast cell line L929, which expresses androgen receptor (AR) but no PR, showed that TMG had weak antiandrogenic activity whereas MPA had androgenic activity. In summary, data from several in vitro assays demonstrate that TMG is a potent progestin with a better receptor selectivity profile than MPA.

Adult↗
PubMed1996

Expression of the somatostatin receptor subtype-2 gene predicts response of human pancreatic cancer to octreotide.

BACKGROUND: Somatostatin inhibits proliferation of many solid tumors. The current study examines whether inhibition of the growth of pancreatic cancer by the somatostatin analog, octreotide, requires tumor expression of somatostatin receptors. METHODS: We studied five human pancreatic cancer cell lines, Capan-1, Capan-2, CAV, MIA PaCa-2, and Panc-1. Solid tumors were established in nude mice (n = 20/cell line) by flank injection of tumor cells. Subcutaneous octreotide (500 micrograms/kg/day) was administered by osmotic pumps to 10 of the animals in each group, and the other 10 received control infusions of saline solution. On day 36, the tumors were excised and weighed. Plasma levels of the putative trophic peptides cholecystokinin, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin were assessed by radioimmunoassay. Each of the five cell lines was assayed for the presence of cell surface somatostatin receptors by using whole cell competitive binding assays with 125I-somatostatin. Expression of the somatostatin receptor subtype-2 (SSR2) gene was determined with reverse transcriptase-polymerase chain reactions. Southern blot hybridization was used to assess the presence of the SSR2 gene. RESULTS: Octreotide inhibited tumor growth in the MIA PaCa-2 group (512 +/- 75 mg control versus 285 +/- 71 mg treated; p < 0.05) but had no significant effect on tumor weight in the other four cell lines. Plasma levels of cholecystokinin, epidermal growth factor, insulin-like growth factor-1, and insulin were not altered by chronic octreotide infusion. Cell surface somatostatin receptors and SSR2 gene expression were detected only in the MIA PaCa-2 tumors. The gene for the SSR2 receptor was found in all five tumor lines. CONCLUSIONS: Octreotide-mediated inhibition of pancreatic cancer growth is dependent on expression of somatostatin receptors. The expression of somatostatin receptors should be considered in the design and interpretation of clinical trials with somatostatin analogs for treatment of pancreatic cancer.

Adenocarcinoma↗
PubMed2000

Antigenic characterization of the nucleocapsid protein of Newcastle disease virus by means of a new panel of monoclonal antibodies.

Nine monoclonal antibodies (MAB) against nucleocapsid protein (NP) of Newcastle disease virus (NDV) have been prepared and characterized. All the MABs were classified into three groups by means of the competitive binding assay. At least three antigenic sites were delineated on the NP. The 1st site includes two closely located epitopes; the 2nd site includes two related and two distinct epitopes; the 3rd site includes two closely related and one distinct epitopes.

Antibodies, Monoclonal↗
PubMed2002

Cell death in the inner nuclear layer of the retina is modulated by BDNF.

Developing amacrine cells in the vertebrate retina undergo naturally-occurring cell death which is accentuated by the early removal of retinal ganglion cells. We show that providing BDNF or decreasing endogenous BDNF via competitive binding with soluble TrkB receptors in a whole-retina culture assay modulates the frequency of dying cells in the amacrine cell layer. Ganglion cells synthesize BDNF, and amacrine cells express TrkB receptors, suggesting a likely signaling mechanism.

Aging↗