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At least 811 records · Page 45Linked to original sources
PubMed1995

Metopon and two unique derivatives: affinity and selectivity for the multiple opioid receptors.

5 beta-Methyl-7,8-dihydromorphinone (metopon), an isomer [6aS-(6a alpha,9a alpha, 10 beta)13aS]-1,10-methano-4-hydroxy-11-methyl- 6,6a,8,9,10,11,12,13-octahydro-[1]-benzopyrano[4,3,e]isoquinoline- 7-(9aH)-one (compound 1) derived from a photochemical rearrangement of 5 beta-methylmorphinone, and [6aS-(6a alpha,9a alpha,10 beta)13aS]-1,10-methano-4-hydroxy-11-methyl- 6,6a,8,9, 10,11,12,13-octahydro-[1]-benzopyrano[4,3,e]-14 beta- (p-nitrocinnamoylamino) isoquinoline-7-(9aH)-one (compound 2) were characterized for opioid receptor affinity, selectivity and analgesic properties. In competition binding assays using bovine striatal membranes, the three compounds inhibited the binding of 0.25 nM [3H][D-Ala2,(Me)-Phe4,Gly(ol)5]enkephalin (DAMGO), a mu-selective peptide, with IC50 values less than 5 nM. All three compounds exhibited lower affinity for delta- and kappa-opioid receptors. In the mouse 55 degrees C warm-water tail-flick assay, both metopon and compound 1 displayed antinociception that lasted for 60 min after i.c.v. injection. Morphine sulfate, metopon and compound 1 produced 50% antinociception with i.c.v. doses of 0.83, 2.0 and 4.0 nmol, respectively. The mu-selective, irreversible opioid receptor antagonist beta-funaltrexamine blocked antinociception induced by metopon and compound 1, while delta- and kappa-opioid receptor selective antagonists did not effect antinociception. These findings demonstrate metopon and its isomer bound with high affinity to the mu-opioid receptor and produced antinociception through this receptor.

Analgesics, Opioid↗
PubMed1990

Characterization of vasoactive intestinal peptide receptors in retina.

Vasoactive intestinal peptide (VIP) is a neuropeptide having a wide range of effects on a large number of tissues. To gain insight into the role VIP plays in retinal function, VIP receptors in bovine retinal membranes were analyzed in competition binding assays and by affinity labeling studies and compared to VIP receptors in rat liver membranes. In both membrane preparations, high affinity VIP binding sites (KD approximately 1 nM) were detected. Secretin and glucagon, each having close structural homology to VIP, were found to have negligible effects on [125I]VIP binding in retina. In contrast, secretin (KD = 70 nM) was modestly effective in inhibiting [125I]VIP binding to rat liver membranes. Affinity labeling analysis revealed a VIP binding site of 59 kDa in both bovine retinal and rat liver membranes. Digestion of affinity-labeled receptor proteins with endoglycosidase F generated final cleavage products of approx. 45 kDa for both receptors. These results indicate that the retina expresses a high affinity, highly selective VIP receptor thereby supporting a specific function for VIP in this tissue.

Affinity Labels↗
PubMed1988

Plasmodium falciparum: analysis of B epitopes of a polypeptide antigen expressed in Escherichia coli, using monoclonal antibodies.

Monoclonal antibodies were raised against a recombinant molecule corresponding to the polypeptide 72 kDa, previously described as possibly related to protection in Plasmodium falciparum infection. Selection of hybridoma cell lines was done by immunofluorescence to guarantee the reactivity of the monoclonal antibodies both against the recombinant and the native molecule of the parasite. Monoclonal antibodies were characterized by serological and immunochemical techniques. Competitive binding assays between monoclonal antibodies defined four different B epitopes. One epitope is specific for P. falciparum, a second is also present in P. vivax, while the two others seem to be ubiquitous and are also present in the rodent parasite P. chabaudi. The ubiquitous epitope 72.C is apparently the only one recognized by squirrel monkey sera presenting protective antibodies against the asexual blood infection by P. falciparum.

Animals↗
PubMed1989

Schistosoma mansoni: insulin independence.

Several authors have reported that schistosomes are affected by host hormones, including insulin. We found that insulin in a 10,000-fold range of concentrations failed to affect glucose consumption of males, females, or pairs of Schistosoma mansoni incubated for periods up to 24 hr. Insulin, vasopressin, or tetradecanoyl phorbol acetate (an insulin mimic) did not affect the uptake and incorporation of [14C]glucose and [14C]deoxyglucose over the active transport range. Competitive binding studies using 125I-insulin and 125I-insulin-like growth factor II, with varying concentrations of unlabeled hormones, demonstrated only nonspecific binding to intact worms or subcellular fractions. This generalized nonspecific uptake of label was also shown by autoradiography, suggesting that specific insulin receptors are absent from S. mansoni. We conclude that plasma glucose levels are sufficient to supply the metabolic requirements of schistosomes by diffusion, and that no insulin-dependent mechanisms have evolved in these parasites.

Animals↗
PubMed1983

Epidermal growth factor: intracellular Ca2+ inhibits its association with pancreatic acini and A431 cells.

The uptake of 125I-labeled epidermal growth factor (125I-EGF) by mouse pancreatic acini was inhibited (40-50%) by the secretagogue cholecystokinin octapeptide (CCK8). Analysis of competitive binding data showed that the apparent Kd of EGF binding increased 135% while the binding capacity was only slightly altered (30% increase). That the effect of CCK8 on acini was mediated by intracellular Ca2+ was indicated by the following: (i) Inhibition of 125I-EGF binding to acini was dose-dependent and paralleled the known abilities of CCK8, its analogs, and the cholinergic secretagogue carbachol to induce Ca2+ efflux from acini; and (ii) addition of the Ca2+ ionophore A23187 also inhibited 125I-EGF binding. In addition, EGF association with A431 cells was also inhibited by A23187 in the presence but not the absence of Ca2+.

Animals↗
PubMed1984

Identification and isolation of vinculin from platelets.

A vinculin-like protein was identified in chicken as well as in bovine platelets by ELISA competitive binding assay using antibodies against vinculin from chicken gizzard. By a modified procedure (J. Biol. Chem. (1980) 255, 1194-1199) we succeeded in isolating bovine platelet vinculin to apparent homogeneity. The structural identity of platelet and chicken gizzard vinculin was demonstrated by circular dichroism analysis. It was also shown that platelet vinculin induces a significant decrease in the low shear viscosity of F-actin. Vinculin, in all probability, plays an important role in the organization of actin filaments in platelets, especially in the linkages of microfilaments to the membrane.

Actins↗
PubMed1984

Specific labelling by [125I]helodermin of high-affinity VIP receptors in rat liver membranes.

Helodermin, a newly isolated peptide from Gila Monster venom, is structurally related to VIP and secretin. When used as radioligand, [125I]helodermin bound rapidly and reversibly to crude rat liver membranes, the dissociation being accelerated by GTP. Competition binding curves of [125I]helodermin and [125I]VIP with unlabelled peptides showed the following order of decreasing affinity: VIP greater than helodermin greater than secretin greater than hpGRF(1-29)-NH2. The shape of binding curves and of concurrent adenylate cyclase activation is compatible with the specific labelling, by [125I]helodermin, of a class of high-affinity VIP receptors that is capable to stimulate adenylate cyclase.

Adenylyl Cyclases↗
PubMed1989

Eicosapentaenoic acid and docosahexaenoic acid are antagonists at the thromboxane A2/prostaglandin H2 receptor in human platelets.

The present study investigated the mechanism by which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibit platelet activation induced by thromboxane A2. DHA was found to be more potent than EPA in blocking platelet aggregation induced by the stable thromboxane A2 mimetic, U46619. Furthermore, this inhibition by DHA or EPA was competitive. Binding studies using 3H-U46619 demonstrated that both EPA and DHA interact with the platelet thromboxane receptor. The potency of the inhibition of binding corresponded with that seen for the inhibition of aggregation. These results suggest that thromboxane receptor antagonism may be an important mechanism by which EPA and DHA modulate platelet reactivity in vivo.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1989

Identification of a specific receptor for interleukin-1 on rat bone marrow cells.

The interleukin-1 (IL-1) receptor on rat bone marrow cells was investigated using 125I-labeled IL-1 alpha and -1 beta. These radiolabeled ligands bound to the rat bone marrow cells in a specific and saturable manner with Kd values of 0.36 +/- 0.072 nM and 1.9 +/- 0.27 nM, respectively. In a competitive binding experiment, IL-1 alpha and -1 beta inhibited the binding of 125I-IL-1 alpha with Ki values of 0.35 +/- 0.041 nM and 2.9 +/- 1.0 nM. The binding of 125I-IL-1 beta was inhibited by IL-1 alpha and -1 beta with Ki values of 0.27 +/- 0.020 nM and 0.74 +/- 0.12 nM. In cross-linking experiments, 125I-IL-1 alpha was covalently incorporated into two proteins of 163 kDa and 63 kDa. These results suggested the presence of two binding proteins for IL-1 on the rat bone marrow cells.

Animals↗
PubMed1993

The effect of hydroxyl radical on the antigenicity of native DNA.

Hydroxyl free-radical mediated in vitro modification of native calf thymus DNA showed single-strand breaks, decrease in melting temperature and structural alteration of purine and pyrimidine bases. Experimentally induced antibodies against modified DNA exhibited polyspecificity. Native DNA, RNA and synthetic polynucleotides in B-conformation were found to be an effective inhibitor of induced antibody-immunogen interaction. Naturally occurring human anti-DNA autoantibodies showed enhanced recognition of modified DNA as compared to native polymer. IgG isolated from anti-DNA antibody-positive SLE sera by Protein A-Sepharose 4B chromatography were affinity purified on nDNA-(polylysyl-Sepharose 4B) matrix. In competitive binding experiments, the affinity-isolated IgG showed almost equal recognition of both native DNA and hydroxyl radical modified DNA. The possible role of hydroxyl radicals in the etiopathogenesis of SLE has been discussed.

Animals↗
PubMed1992

Effect of cell culture on rabbit colonic smooth muscle bradykinin receptors.

The aim of this study was to assess the effect of cell culture on the bradykinin receptor of rabbit colon myocytes. In longitudinal muscle strips prepared from distal colon, bradykinin stimulated dose-dependent contraction that was 62% of the maximal response to bethanecol. At 4 degrees C, [3H]bradykinin binding to fresh muscle homogenates from the distal colon was time dependent, saturable, and linearly related to tissue concentration. Specific binding of 0.6 nmol/L [3H]bradykinin was 80% +/- 2% of total binding. In competitive binding studies, Hill coefficients approached unity, suggesting the presence of a single class of receptors. The order of potency was bradykinin greater than [D-Phe7]bradykinin much greater than des-Arg9, [Leu8]bradykinin, which is consistent with results of a B2 receptor subclass. Colon myocytes from the longitudinal muscle layer achieved confluence and were harvested for studies after 12-14 days in culture. Bradykinin receptors were of high affinity [disassociation constant (Kd) = 672 pmol/L] and numbered 10,217 +/- 2567/cell. To show that the receptors on cultured myocytes were functional, the effect of bradykinin was measured (a) on intracellular calcium concentration using Fura 2 and (b) on prostaglandin E2 concentration in the culture media using radioimmunoassay. In cells grown to confluence on cover slips and preloaded with Fura 2, bradykinin stimulated the threshold response at 1 nmol/L and maximal response (increased intracellular calcium concentration from 229 to 633 nmol/L) at 1 mumol/L. Bradykinin, 100 nmol/L, increased Prostaglandin E2 in the culture media threefold. In summary, colon myocytes express functioning bradykinin receptors, which, unlike muscarinic receptors, persist in culture. Bradykinin appears to be a suitable agonist for studies of receptor-mediated intracellular events in cultured colon myocytes.

Animals↗
PubMed1993

Intrinsic photoaffinity labeling of native and recombinant rat pancreatic secretin receptors.

BACKGROUND: Structural characterization of pancreatic secretin receptors has been limited by difficulties in generating suitable radioligands and obtaining sufficient substrate. The aims of this study were to design, synthesize, and characterize high affinity radiolabeled analogues of secretin suitable for "intrinsic" photoaffinity labeling and to clone, express, and characterize the recombinant rat pancreatic secretin receptor. METHODS: The ability of synthetic analogues to stimulate amylase secretion by pancreatic acini was studied. Receptor complementary DNA (cDNA) was cloned by screening a rat pancreatic library with a probe based on the sequence of a neural cell secretin-binding protein. Competition binding and affinity labeling were performed with membranes prepared from rat pancreas and transfected cells. RESULTS: Two probes were fully efficacious secretagogues, which bound in a specific, high-affinity, rapid, and temperature-dependent manner. Only ([125I]Tyr10, pNO2-Phe22) rat secretin 27 covalently labeled a 50,000-62,000-molecular weight pancreatic membrane protein, with labeling inhibited in a concentration-dependent manner by secretin but not vasoactive intestinal polypeptide. Hybridization screening yielded a full-length cDNA identical to the neural clone. Photoaffinity labeling of this recombinant receptor identified a 57,000-62,000-molecular weight protein with specificity similar to that of native pancreas. Both native and recombinant receptors migrated at a molecular weight of 42,000 after endoglycosidase F deglycosylation. CONCLUSIONS: This study provides evidence for the molecular identity of the pancreatic secretin receptor and presents a novel probe important in structural characterization of its agonist-binding domain.

Affinity Labels↗
PubMed1985

Biosynthesis of ecdysteroids from cholesterol by crab Y-organs, and eyestalk suppression of cholesterol uptake and secretory activity, in vitro.

Precursor incorporation studies were conducted in vitro with activated Y-organs from 48-hr de-eyestalked Cancer antennarius donors. When the glands were prelabeled in vivo by systemic injection of [3H]cholesterol 12 hr prior to removal, and subsequently incubated 24 hr in label-free medium, the glands secreted 3H-labeled ecdysone. The glands also secreted an unidentified ecdysteroid with comigrating 3H-label with characteristic retention time on normal-phase HPLC of 4 min (4-min unknown). The compound is less polar, and is secreted in a quantity and apparent specific activity approximately fivefold greater, than ecdysone. Compared with chromatographic retention times and competitive binding curves of authentic standards, the 4-min unknown was determined not to be ponasterone A, inokosterone, makisterone, or several other possible products or intermediates. In contrast with Y-organs from intact donors, those from de-eyestalked crabs exhibit greatly increased cholesterol uptake and secretion of both ecdysone and 4-min unknown in vitro. All three responses were suppressed in dose-dependent manner by eyestalk extract in the dose range, 1-4 eyestalk equivalents/Y-organ. Secretion of 4-min unknown was the response most sensitive to eyestalk extract (requiring the least dose for 50% inhibition).

Animals↗
PubMed1983

Antagonistic effects of aldosterone on corticosterone-mediated changes in exploratory behavior of adrenalectomized rats.

The effect of aldosterone administration on exploratory activity of chronic adrenalectomized (10 days) male rats was investigated. Aldosterone (30 micrograms/100 g body wt sc) administered 1 hr or 30 min prior to the behavioral test failed to normalize disturbed exploratory activity of adrenalectomized rats, in contrast to the restoration observed after corticosterone, the naturally occurring glucocorticoid of the rat. Administration of the mineralocorticoid 30 min prior to corticosterone prevented the normalization of the behavioral response by the latter steroid. Administration of the same dose of aldosterone 30 min prior to a tracer amount of [3H]corticosterone effectively blocked cell nuclear uptake of radioactive-labeled hormone in the hippocampus. The specific action of corticosterone on exploratory behavior corresponds with the stringent specificity of the neuronal hippocampal corticosterone receptor system. Mineralocorticoid receptors do not seem to be involved in effects on this behavior. The antagonistic action of aldosterone is probably exerted by competitive binding to the corticosterone receptor.

Adrenalectomy↗
PubMed1975

Radioimmunoassay for nanogram quantities of DNA.

A direct competitive binding radioimmunoassay for DNA has been developed, using 125I-iododeoxyuridine-labelled DNA as the antigen and the serum from a patient with systemic lupus erythematosus. The assay is sensitive in the range of 25 to 1000 ng/ml of DNA. The sensitivity is determined by the affinity of the antibody: this SLE serum contains a component with an association constant of 9.6 X 10(-5) l/mol active at high dilution (1/10,000). Any biological material, such as serum, synovial fluid or tissue extracts can be tested directly. No interference has been found by DNAse in normal serum, or inhibition by mononucleotides or RNA. Native or denatured DNA from different sources (Escherichia coli, salmon sperm, calf thymus and human placenta) either purified or not, competes equally well for the antibody in this system.

Antibody Specificity↗
PubMed1980

A new solid-phase radioimmunoassay to measure IgG secreted by cultured human lymphocytes.

We describe a simple solid-phase radioimmunoassay (RIA) to detect IgG based on competitive binding between radiolabeled and unlabeled IgG for anti-IgG antibody physically adsorbed to the wells of polyvinyl microtiter plates. The assay is sensitive (1 ng), rapid, and is particularly suited for studies of in vitro IgG secretion by human peripheral blood lymphocytes, since such studies require large numbers of cultures. Conditions which permit measurement, by means of this assay, of helper and suppressor T cell effects on IgG production by human B cells in culture are described.

Animals↗
PubMed1981

Homogeneous substrate-labeled fluorescent immunoassay for IgG in human serum.

A homogeneous substrate-labeled fluorescent immunoassay for IgG has been developed. Purified IgG was covalently labeled with 6-(7-beta-galactosylcoumarin-3-carboxamido)-hexylamine to form a stable conjugate, GU-IgG. The galactosyl residue was hydrolyzed from GU-IgG by beta-galactosidase and the progress of the hydrolysis was monitored by the increase in fluorescence emission at 450 nm with excitation at 400 nm. Antibody to IgG diminished the activity of GU-IgG as a substrate for beta-galactosidase. Competitive binding immunoassays were conducted by allowing added IgG and GU-IgG to compete for a limited number of antibody binding sites. Hence, the fluorescence produced by enzymic hydrolysis increased with the level of added IgG. This method provides a simple and reliable immunoassay for IgG and is applicable to other proteins.

Antibodies↗
PubMed1984

Advantage of monoclonal vs. polyclonal antisera in a beta 2-microglobulin radioimmunoassay.

Unassociated human beta 2-microglobulin (beta 2m) and histocompatibility peptide associated beta 2-microglobulin produce significantly different inhibition curves in a competitive binding assay with rabbit anti-beta 2-microglobulin. This difference disallows the use of the rabbit antisera for measuring MHC-beta 2-microglobulin complex concentration using free beta 2m as a standard. Presumably the close amino acid sequence homology between human and rabbit beta 2-microglobulin results in rabbit antisera against a small number of epitopes, some of which are covered or conformationally altered in the MHC-beta 2-microglobulin complex. A monoclonal anti-beta 2-microglobulin antibody, LG1 reacted equally well with both forms of beta 2-microglobulin and is used to measure the concentration of beta 2-microglobulin in the histocompatibility peptide complex.

Animals↗