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Characterization of the major parathyroid hormone target cell in the endosteal metaphysis of rat long bones.

The majority of in vivo competitive binding of parathyroid hormone (PTH) in the endosteal metaphysis of rat long bones was recently shown to be localized in the intertrabecular tissue to a cell that is distinct from a differentiated osteoblast. In the present report we have further characterized this cell, termed a parathyroid hormone target (PT) cell, by light and electron microscopy using radioautography and histochemical techniques. These studies demonstrate that the PT cell is a mononuclear cell with a large cell body located at times between clusters of differentiated osteoblasts, as well as in other regions of the intertrabecular tissue. Its long cytoplasmic processes extend from the bone matrix through the intertrabecular region toward vascular structures, interdigitating with various cells of the endosteum. A distinctive tubular structure originating in the Golgi system and often associated with long mitochondria and glycogen particles extends throughout the cytoplasmic processes of the PT cell. Based on its capacity to incorporate [3H]thymidine, the PT cell appears to divide rather slowly. The identification of occasional hybrid cells with ultrastructural features of both the PT cell and the differentiated osteoblast and the presence of histochemical evidence for alkaline phosphatase activity suggest that the PT cell is of the osteoblast lineage. These studies therefore morphologically define a major osseous target cell for PTH that, although of the osteoblast lineage, is not a differentiated osteoblast and provide in vivo evidence that characteristics of the "osteoblast phenotype" are not restricted to a sole osseous cell type.

Animals↗

Specificity of an antibody directed against d-methamphetamine. Studies with rigid and nonrigid analogs.

The specificity of an antibody directed against d-(S)-methamphetamine (MA) was determined by competitive binding assay with more than 50 compounds-metabolites, homologs, and analogs of amphetamine. The antibody appears to be specific both for the side chain and the aromatic ring of d-(S)-amphetamine (A). The basic requirements for a compound to be bound to the antibody are (a) an aromatic ring, (b) a basic nitrogen, and (c) a two-carbon chain between the aromatic ring and the nitrogen. A transoid conformation for the phenethylamine skeleton is preferred. The interaction of the antibody with compounds differing from MA or A in side-chain substitutions was directly proportional to the closeness of their structure to MA and/or A. The antibody exhibited greatly reduced affinity for ring-substituted analogs of A; the p-hydroxy metabolite of A did not bind to the antibody. A radioimmunoassay of A is described; it was utilized to study the disposition of A in dogs.

Amphetamines↗

Enzyme coupled immunoassay of insulin using a novel coupling reagent.

A novel coupling reagent, meta-maleimidobenzoyl N-hydroxysuccinimide ester was synthesized. Using this reagent, insulin was conjugated very easily with beta-D-galactosidase [EC 3.2.1.23] in neutral, aqueous solution. No reduction of the enzyme activity was observed during the coupling procedure. The competitive bindings of the conjugate and insulin to anti-insulin serum were tested. The results indicated that the conjugate has enough immune reactivity for use in enzyme coupled immunoassay. Using this assay 20-800 pg of insulin were detectable.

Binding, Competitive↗

LEC induces chemotaxis and adhesion by interacting with CCR1 and CCR8.

Liver-expressed chemokine (LEC) is an unusually large CC chemokine, which is also known as LMC, HCC-4, NCC-4, and CCL16. Previously, LEC was shown to induce leukocyte migration but the responsible signaling receptors were not characterized. We report chemotaxis and competitive binding studies that show LEC binds to and activates CCR1 and CCR8 transfected HEK-293 cells. LEC induced maximal migration of CCR1 and CCR8 transfected cells at 89.3 nmol/L and cell adhesion at 5.6 nmol/L. The molar concentration of LEC required to induce maximum cell migration is 20- to 200-fold greater than that required for RANTES or I309, respectively. All 3 chemokines induced maximal static adhesion at 5 to 7 nmol/L. A neutralizing polyclonal antibody to LEC was developed to demonstrate that the unusually high concentration of LEC required to induce chemotaxis was a property of LEC and not as a result of an irrelevant protein contamination. This study suggests that LEC may be a more effective inducer of cell adhesion than cell migration.

Animals↗

Characterization of bradykinin B2 receptors in adult myocardium and neonatal rat cardiomyocytes.

Specific [125I-Tyr8]bradykinin (BK) binding was observed on myocardial membranes from adult guinea pigs, dogs, rats, and rabbits that was displaced by unlabeled BK with an IC50 between 0.1 and 30 nmol/L. In the adult guinea pig ventricular myocardium, which displays both high- and low-affinity binding, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 mumol/L) eliminated high-affinity binding and reduced total specific [2,3-prolyl-3,4-3H(N)]BK ([3H]BK) binding by > 60%. Agonist competition binding to rat myocardial membranes was characterized as being of one affinity for BK in the nanomolar range, and it was not altered by GTP gamma S. Saturation binding studies with [125I-Tyr8]BK and [3H]BK, performed on cultured neonatal rat cardiac myocytes, revealed a single class of BK binding sites with a Kd and Bmax of 0.24 +/- 0.04 nmol/L and 18.4 +/- 1.1 fmol/mg protein, respectively (approximately 1500 receptors per cell). In competitive binding assays, unlabeled BK, Hoe 140 (a specific BK B2 receptor antagonist), and des-Arg9,[Leu8]BK (a BK B1 receptor antagonist) displaced [125I-Tyr8]BK with an IC50 of 4.3, 0.041, and 307 nmol/L, respectively. In the presence of 100 mumol/L GTP gamma S, [3H]BK binding to myocyte membranes was reduced by 40%, but the IC50 did not change. Cardiac fibroblasts, evaluated in parallel to the myocytes, contain a single class of [3H]BK binding sites (248 +/- 72 fmol/mg) with a 130-fold lower relative affinity (32.4 +/- 11.3 nmol/L) than that determined in rat cardiomyocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Sensitive radioimmunoassay for the broad-spectrum antiviral agent ribavirin.

Ribavirin, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxyamide (Virazole; Viratek, Inc., Covina, Calif.), has a broad spectrum of antiviral activity. However, the study of the absorption, metabolism, and excretion of this compound has been limited by the lack of an appropriate assay for ribavirin and its metabolites. Since ribavirin has definite potential for therapeutic use, we developed a radioimmunoassay to measure ribavirin levels in clinical specimens. To prepare an effective immunogen, ribavirin was monosuccinylated and coupled to ovalbumin. The competitive binding radioimmunoassay, in which tritium-labeled ribavirin and rabbit antiribavirin serum were used, was quantitative for ribavirin at concentrations of 1 pmol/100 microliter in urine or plasma samples. The rabbit antibody cross-reacted with the major metabolite of ribavirin, 1,2,4-triazole-3-carboxamide, at a low level (2 to 5%) which did not interfere with ribavirin binding until concentrations of 1,2,4-triazole-3-carboxamide 10- to 100-fold higher than ribavirin were present in mock samples, a condition not present in biological specimens. We used the ribavirin radioimmunoassay to determine the ribavirin concentration in mouse plasma after intraperitoneal administration, in the sera of adults from Sierra Leone after oral or intravenous administration for treatment of suspected Lassa fever, and in the sera of children in the United States after small-particle aerosol administration. Our experience with the radioimmunoassay indicates that it is sensitive, accurate, and reproducible. The assay will permit studies leading to a better understanding of the pharmacology and pharmacokinetics of this potentially useful antiviral drug.

Adult↗

Displacement of bilirubin from adult and newborn serum albumin by a drug and fatty acid.

Competitive binding of bilirubin and 5-butyl-1-cyclohexylbarbituric acid (bucolome) to human serum albumin was investigated by equilibrium dialysis, with and without added bilirubin (0.5 mol/mol albumin). Further observations were made by peroxidase oxidation kinetics. Finally, cobinding of the two ligands was studied by spectrophotometry. Experiments were performed with defatted adult human serum albumin, with umbilical cord serum, and with adult albumin to which was bound 2 mol palmitate/mol albumin. Bucolome does not induce displacement of bilirubin from binding to defatted adult albumin when one molecule of either ligand is bound. The second molecule of bound bucolome displaces the first bilirubin molecule. With albumin in umbilical cord serum, the first bucolome does decrease binding of the first bilirubin to some extent although the two ligands can bind to the same albumin molecule. The presence of two molecules of bound palmitate together with one of bilirubin causes enhanced bilirubin displacement on binding of bucolome. These observations may be of consequence for practical testing of the bilirubin displacing effect of drugs. Experiments with neonatal albumin and with added fatty acid should be included for a complete study.

Adult↗

Estimation of 1,25 dihydroxyvitamin D by cytoreceptor and competitive protein binding assays without high pressure liquid chromatography.

Using two different cultured rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8) we have investigated the recently described cytoreceptor assay for 1,25-dihydroxyvitamin D (1,25-(OH)2D). The assay method is relatively simple and sensitive to 2.4 fmole per tube. Using either cell line, assay of serum samples, whose only preparation consisted of extraction and purification on a disposable diatomaceous earth column, produced variable values for serum 1,25-(OH)2D. Additional purification, using a disposable silicic acid minicolumn to remove other vitamin D metabolites resulted in consistent values and additional HPLC resulted in no further decrease in the values obtained. Our results show that a single two stage non-HPLC column can purify serum samples for assay in the cytoreceptor assay. The method is also applicable to the competitive protein binding assay employing calf thymus cytosol and the correlation between values obtained by both methods is highly significant. It is a sensitive, simple, and accurate method with technical advantages which allow greater sample throughput than other 1,25-(OH)2D assays.

Animals↗

Examination of the effects of increasing Gs protein on beta2-adrenergic receptor, Gs, and adenylyl cyclase interactions.

We have examined the effect of increased Gs protein levels on the abilities of three different beta2-agonists to induce GTP shifts and stimulate adenylyl cyclase response in an effort to investigate the kinetic association between the beta2-adrenergic receptor Gs and adenylyl cyclase. Agonist competition binding analysis and adenylyl cyclase concentration-response assays revealed that increases in Gs protein resulted in proportional increases in the areas of the GTP shift and adenylyl cyclase activity. Changes in the magnitude of the GTP shift were evaluated with a novel and straightforward approach for analyzing the GTP shift data that allowed us to determine the proportion of high agonist affinity binding receptor population and the apparent dissociation constant between the agonist bound receptor and Gs, regardless of the Gs protein level or the type of beta2-agonist. Using this method, we concluded that increased Gs results in the accumulation of the receptor population displaying high affinity towards agonist (HRGs) by increasing the number of receptor-Gs complexes (to a receptor:Gs protein ratio of about 0.7 at maximal Gs expression) without affecting the affinity between hormone bound receptor and Gs. Using the Gs protein levels determined with our novel analysis, we ran simulations using the theoretical shuttle model equation that relates the EC50 to available Gs. Fitting the simulations to experimental data required a receptor to catalytic unit ratio of 0.45 and revealed at least two distinct stages for beta2-agonist-stimulated adenylyl cyclase activity, namely, the activation of Gs by the beta2-adrenergic receptor (a step whose rate is dependent on the type of agonist used to stimulate activity), and the activation of adenylyl cyclase by active Gs (a step whose rate is independent of the type of agonist).

Adenylyl Cyclases↗

Competition between sigma factors for core RNA polymerase.

The switch of RNA polymerase specificity from early to late promoters of bacteriophage T4 is achieved by substitution of host sigma factor, sigma 70, with the T4 induced factor, sigma gp55. However, overproduction of sigma gp55 from an expression vector is not detrimental to Escherichia coli growth. Direct competition binding assays demonstrate that sigma 70 readily displaces sigma gp55 from RNA polymerase and thereby reverses the promoter specificity of the enzyme. The displacement also occurs with the core enzyme modified by bacteriophage T4 infection. We postulate that an antagonist of sigma 70 should be formed in T4-infected cells to aid sigma gp55 in the early/late switch.

Binding, Competitive↗

Inverse agonism and constitutive activity as functional correlates of serotonin h5-HT(1B) receptor/G-protein stoichiometry.

This study evaluated the influence of receptor/G-protein (R:G) stoichiometry on constitutive activity and the efficacy of agonists, partial agonists, and inverse agonists at human (h) 5-hydroxytryphamine 1B (5-HT(1B)) receptors. Two Chinese hamster ovary cell lines were used; they expressed 8.5 versus 0.4 pmol h5-HT(1B) receptors/mg (determined by [(3)H]GR125,743 saturation analysis) and 3.0 versus 1.5 pmol receptor-activated G-proteins/mg [determined by guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) isotopic dilution], respectively. Thus, they displayed R:G ratios of approximately 3.0 (RGhigh) and approximately 0.3 (RGlow), respectively. In competition-binding experiments, the agonists, 5-HT and sumatriptan, displayed fewer high-affinity (HA)-binding sites and the partial agonists, BMS181, 101 and L775,606, displayed decreased affinity in RGhigh versus RGlow membranes. In contrast, the inverse agonists, SB224,289 and, to a lesser extent, methiothepin, showed increased affinity. In G-protein activation experiments, both basal and 5-HT-activated [(35)S]GTPgammaS binding were higher in RGhigh than in RGlow membranes. Constitutive activity (determined by inhibition of basal [(35)S]GTPgammaS binding with GTPgammaS in the absence of receptor ligands) was more pronounced in RGhigh versus RGlow membranes, as revealed by the >5-fold greater proportion of HA sites. Correspondingly, the negative efficacy of inverse agonists was strikingly augmented, inasmuch as they suppressed approximately two-thirds of HA [(35)S]GTPgammaS binding in RGhigh membranes, but only approximately one-third in RGlow membranes. Furthermore, the efficacy of partial agonists was greater at RGhigh versus RGlow membranes, as estimated by their ability to enhance [(35)S]GTPgammaS binding. In conclusion, an increase in R:G ratios at h5-HT(1B) receptors was associated with an increase in relative efficacy of partial agonists and, most notably, an increase in both constitutive G-protein activation and negative efficacy of inverse agonists.

Animals↗

Cloning, expression, and functional characterization of cynomolgus monkey (Macaca fascicularis) CC chemokine receptor 1.

The CC chemokine receptor 1 (CCR1) has emerged as a relevant factor contributing to inflammatory diseases such as allergic asthma. Commonly used animal models of allergic airway inflammation, especially murine models, have certain limitations. The elaborate, nonhuman, primate models of asthma display the highest comparability with the situation in humans. These models play an important role in the understanding of the pathogenesis of asthma. To improve the understanding in cynomolgus monkey models, we identified and characterized CCR1 in this nonhuman primate. Initially, we cloned the cynomolgus monkey CCR1 (cCCR1) gene, and the sequence analysis revealed high homology at the nucleotide (92%) and amino acid (88.4%) levels with its human counterpart. Human embryonic kidney 293 cells were stably transfected with cCCR1 and used in functional assays. Among those CCR1 ligands tested, CCL14(9-74) was most potent in the induction of intracellular Ca2+ fluxes as observed for human CCR1 (hCCR1). Complete cross-desensitization could be achieved between CCL14(9-74) and CCL15. However, CCL3 could not fully abrogate the response to the potent ligand CCL14(9-74). Competition-binding studies with radiolabeled CCL3 concordantly showed that CCL14(9-74) has a higher affinity to cCCR1 than hCCL3. Moreover, differential tissue-specific expression of cCCR1 was investigated by real-time quantitative polymerase chain reaction, displaying the highest levels in spleen. This study adds basic information needed for the evaluation of the role of CCR1 in the pathophysiology of asthma using the highly relevant cynomolgus monkey model and in addition, aids in the preclinical evaluation of potential novel drugs targeting CCR1.

Amino Acid Sequence↗

Rapid purification and radioimmunoassay of cytosolic malic enzyme.

A very rapid and highly effective procedure has been devised for the isolation of homogeneous malic enzyme from rat liver cytosol. A combination of precipitation with 10 to 20% polyethylene glycol, ion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Procion Red HE-3B Agarose was used to prepare 3 to 4 mg of homogeneous malic enzyme from the livers of two rats in 18 h. In addition to introducing the advantages of simplicity, speed, and high yield (31%) the new method eliminates potentially denaturing steps (heat treatment, ethanol fractionation) and prolonged dialysis procedures used in other purification schemes. Malic enzyme purified by this new method was use to immunize rabbits. The resulting antibodies bound purified rat liver and mouse liver malic enzymes with very similar affinities and also avidly complexed cytosolic malic enzyme from two murine cell lines, 3T3-L1 preadipocytes and 3T3-C2 fibroblasts. When purified malic enzyme was incubated with lactoperoxidase, glucose oxidase and Na 125I 1.8 atoms of 125I were incorporated per molecule of enzyme with full retention of catalytic activity, subunit size, and immunoreactivity. The antiserum, the purified enzyme, and enzymatically iodinated 125I-malic enzyme were used to construct a sensitive, competitive binding radioimmunoassay for the measurement of malic enzyme mass in the range of 1 to 100 ng.

Animals↗

A functional analysis of the antigenicity of streptokinase using monoclonal antibody mapping and recombinant streptokinase fragments.

Streptokinase (SK), a bacterial product of pathogenic Streptococcus species, is now widely used as an effective therapy for the treatment of heart attacks. Because naturally occurring antibody to SK is ubiquitous, serious allergic reactions to SK therapy are common. To begin to identify regions of the molecule that are important for the antigenicity of SK we performed studies using a panel of 51 hybridomas producing anti-SK antibodies, recombinant SK fragments, and assays of SK activity. Antibodies generated from mice hyperimmunized with wild-type SK were shown to fall into six distinct complementation groups by competitive binding studies. Recombinant SK fragments were used to determine the peptide regions recognized by these complementation groups. Correlation of the effects of the mAb on SK function, with knowledge of their SK fragment-binding pattern, suggested regions of the SK molecule that are important for the construction and the catalytic function of the SK-plasminogen activator complex.

Antibodies, Monoclonal↗

125I protein A: applications to the quantitative determination of fluid phase and cell-bound IgG.

An improved method for the preparation of 125I-labelled Protein A (125I PA) of high specific and functional activity is described. 125I PA has been used in combination with purified rabbit IgG bound to a solid support to develop a competitive binding assay capable of detecting Protein A or human, rabbit and guinea pig IgG at the nanogram level. An optimal set of assay conditions was established and levels of IgG measured in normal human, rabbit and strain-2 guinea pig serum. 125I PA has also been used to detect IgG anti-Forssman antibody bound to sheep erythrocytes and to line-1 and line-10 tumor cells and as an indirect assay for tumor associated antigen in the ascitic fluid of tumor-bearing guinea pigs.

Animals↗

Effects of malnutrition on rat myocardial beta-adrenergic and muscarinic receptors.

Malnutrition, as well as malignancy, induces alterations in heart metabolism and performance. Previous studies have implicated adrenergic mechanisms as the cause. The present study was undertaken to investigate if the adenylate cyclase system in the rat heart was affected by malnutrition. Three different animal groups with malnutrition were compared with a control group: rats with acute starvation for 14-96 hours, rats with protein-calorie malnutrition for 2 weeks, and rats with tumors. Stimulation by beta-adrenergic receptors and inhibition by muscarinic receptors of adenylate cyclase activity were not altered by malnutrition. However, conditions used for in vitro adenylate cyclase determinations were, of necessity, not physiological. Neither did the number of beta-adrenergic and muscarinic receptors change. When competition-binding experiments were performed, differences comprising agonist affinity and affinity state distribution were noted among the groups. The myocardial beta-adrenergic receptors formed a reduced number of high-affinity sites in all groups as compared with the control rats. All high-affinity sites displayed a more than 10-fold increase in affinity toward isoproterenol and an impaired sensitivity to guanine nucleotides except in heart membranes derived from rats starved less than 48 hours. While the protein-calorie restricted and the tumor-bearing rats had myocardial beta-adrenergic receptors that were unresponsive to guanine nucleotides, after 48 hours of starvation the rats exhibited an attenuated guanine-nucleotide-induced affinity shift. No changes associated with malnutrition in myocardial membrane levels of the of the stimulatory guanine-nucleotide-binding protein were detected by cholera-toxin-induced ADP-ribosylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

Comparison of three radioligands for the labelling of human beta-adrenoceptor subtypes.

We have compared the ability of three radioligands, [(125)I]-cyanopindolol, [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol, to label the three human beta-adrenoceptor subtypes. Saturation and competition binding experiments were performed using membrane preparations from Chinese hamster ovary cells stably transfected with the three subtypes. While [(3)H]-CGP 12,177 had very similar affinity for beta(1)- and beta(2)-adrenoceptors (about 40 pM), [(125)I]-cyanopindolol and [(3)H]-dihydroalprenolol had 4- to 6-fold higher affinity for beta(2)- as compared to beta(1)-adrenoceptors (10 vs 45 and 187 vs 1,021 pM, respectively). The affinity of [(125)I]-cyanopindolol at beta(3)-adrenoceptors was considerably lower (440 pM) than at the other two subtypes. The beta(3)-adrenoceptor affinity of [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol was so low that it could not be estimated within the tested range of radioligand concentrations (up to 4,000 pM and 30,000 pM for [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol, respectively). We conclude that all three radioligands are ill-suited to label beta(3)-adrenoceptors, particularly in preparations co-expressing multiple subtypes. In the absence of alternatives, [(125)I]-cyanopindolol appears the least unsuitable to label beta(3)-adrenoceptors. There is a need for high-affinity radioligands which are either selective for beta(3)-adrenoceptors or reasonably non-selective among all three beta-adrenoceptor subtypes.

Adrenergic beta-Agonists↗

Chemical force microscopy with active enzymes.

The adhesion forces have been measured between an atomic force microscope tip derivatized with an active enzyme, shikimate kinase, and an ATP mimic immobilized on a gold surface. Experiments with competitive binding of other ligands in solution show that the observed adhesion forces arise predominantly from specific interactions between the immobilized enzyme and surface-bound adenine derivative. These experiments represent a step in the development of a screening methodology based upon chemical force microscopy.

Binding, Competitive↗