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Biomedical subjects

J Hoebeke

Publications and source records attributed to J Hoebeke.

At least 127 records · Page 7Linked to original sources
PubMed1988

Polyclonal and monoclonal antibodies directed against SK & F 94461, a specific H1 histamine receptor ligand.

SK & F 94461, an aminopentyl analogue of mepyramine, is a recently described H1 receptor antagonist. At variance with the other available H1 receptor ligands, SK & F 94461 offers the possibility of coupling to a protein carrier to render the molecule immunogenic. SK & F 94461 coupled to succinylated bovine serum albumin was used as an immunogen to raise polyclonal antibodies in rabbits and BALB/c mice. In parallel, spleen cells from immunized mice were used to produce hybridomas by somatic cell fusion. Thus, six different murine monoclonal antibodies sharing anti-SK & F 94461 specificity were selected for further detailed characterization of their binding properties. Pharmacologic studies of competitive inhibition using a set of 11 histaminergic agents allowed analysis of the fine specificity of anti-SK & F 94461 antibodies. Both polyclonal and monoclonal anti-SK & F 94461 antibodies showed very high affinity for the immunizing molecule (i.e., Ka values for monoclonal antibodies 8 and 12 were, respectively, 3 X 10(10) and 1.4 X 10(10) M-1). Both types of antibodies bound with high affinity (IC50 ranging from 10(-10) to 10(-12) M) to mepyramine, which has a chemical structure closely resembling that of SK & F 94461. Moreover, these antibodies displayed clear-cut stereoselectivity inasmuch as they bound the d-configuration of chlorpheniramine with significantly higher affinity than the l-form. Thus, all six monoclonal antibodies showed IC50 values 1 to 6 log units lower for d- than for l-chlorpheniramine. For some monoclonal antibodies, spectroscopic and fluorescence spectra studies showed that their different binding capacities correlated with their optical properties. Similarly, polyclonal anti-SK & F 94461 antibodies showed a 500-fold lower affinity for l- than for d-chlorpheniramine. All these results indicate that the polyclonal and the majority of monoclonal anti-SK & F 94461 antibodies recognized with high affinity structural configurations known to be important for the pharmacologic activity of H1 ligands, namely the presence of the dimethylaminoethyl side chain and, with stereochemical selectivity, the d-configuration of chlorpheniramine. These data extend for the first time to an H1 histamine receptor ligand results reported in other hormone systems.

Aminopyridines↗
PubMed1987

Immunologic mimicry by a monoclonal antibody of the tricyclic anti-depressants' binding site on muscarinic acetylcholine receptors.

Inhibition constants of tricyclic anti-depressants and related drugs determined for a monoclonal anti-nortriptyline antibody were close to those previously calculated with the same compounds for the brain acetylcholine muscarinic receptor. A highly significant correlation was found between these two series of inhibition constants when no correlation existed between the inhibition constants for the antibody and those for other receptors. This suggests that the binding site for tricyclic anti-depressants on the antibody mimics the binding site for these ligands on muscarinic receptors. Although nortriptyline reveals a noncompetitive inhibition of N-methyl-scopolamine binding to muscarinic receptors, muscarinic ligands display weak or no binding to the antibody. These findings indicate that the binding site for tricyclic anti-depressants on the receptor is distinct from that for the muscarinic ligands.

Animals↗
PubMed1987

Partial characterization of a Legionella pneumophila serogroup 1 immunodominant antigenic determinant recognized by a monoclonal antibody. Legionella specific antigenic determinant.

A monoclonal antibody was used to characterize a serogroup 1 specific Legionella pneumophila Philadelphia strain 1 antigenic determinant. A quantitative fluorometric assay was developed to quantitate the antibody sites (2.7 +/- 0.4 X 10(5)) on Legionella bacteria and to determine the physico-chemical parameters of the antibody-antigen interaction (at 4 degrees C: delta G = -10.9 Kcal X mol-1, delta H = 1.7 Kcal X mol-1, delta S = 45 cal X K-1 X mol-1). The same method was used to study the modification or the removal of the antigen by chemical and enzymatic means (trypsin, papain, lysozyme, acetone, chloroform-methanol and Tris-EDTA); only Tris-EDTA extraction resulted in a significant decrease in antibody binding sites. Inhibition studies of the fluorescein-labelled antibody binding were performed with different sugars of which only L-fucosylamine was inhibitory, and with other monoclonal antibodies to Legionella pneumophila serogroup 1 in order to compare their fine specificity and affinity. The results indicate that the epitope recognized was an immunodominant carbohydrate including an aminodideoxyhexose and carried by the lipopolysaccharide.

Antibodies, Bacterial↗
PubMed1987

The immune response towards beta-adrenergic ligands and their receptors--VII. Equilibrium and kinetic binding studies of l-alprenolol to a monoclonal anti-alprenolol antibody.

Binding of the catecholamine beta-adrenergic antagonist, l-alprenolol, by the IgGl anti-alprenolol monoclonal antibody 37A4 was examined using the radioligand 3H-dihydroalprenolol as an extrinsic signal and the increase in antibody fluorescence upon l-alprenolol binding as intrinsic signal. Equilibrium binding studies based on both signals indicated that the binding process was exothermic with a positive entropy change. The difference in the affinity constants obtained by radioligand binding studies and by fluorescence analysis could be ascribed to the higher affinity of the hydrogenated tritiated l-dihydroalprenolol compared to the unsaturated l-alprenolol. The association rate constants determined by both signals were 10(4)-10(5)/M/sec and showed a high activation enthalpy (8-10 kcal/mol), thus excluding a diffusion controlled reaction. At low temp (7 degrees C), the fluorescence stopped-flow studies showed non-linear pseudo first order kinetics, indicating the existence of a fast pre-equilibrium of low affinity, followed by a conformational change leading to the tight binding of the ligand. The dissociation rate constants determined using both signals were very similar. Thus, the differences in affinity between the hydrogenated and non-saturated l-alprenolol could be ascribed to the association rate constants. Affinity constants and thermodynamic parameters calculated from the kinetic data were in close agreement with those determined by equilibrium binding. The mechanisms of ligand binding are discussed in terms of the interactions of idiotypes and anti-idiotypes in the anti-catecholamine immune response.

Alprenolol↗
PubMed1987

Redistribution of muscarinic acetylcholine receptors on human fibroblasts induced by regulatory ligands.

Distribution of the muscarinic acetylcholine receptor on human fibroblasts was determined by immunofluorescence and immunoperoxidase staining using the anti-muscarinic receptor antibody M-35b. The receptor appeared to be randomly distributed on the cell surface in 1- or 2-day nonconfluent cultures. Brief exposure to 0.1 mM carbamylcholine (15 min to 1 hr) induced receptor accumulation in several restricted domains of the cell surface. This process was associated with sequestration into uncoated vesicles. Random receptor distribution was restored by incubation in ligand-free medium for 4 hr after carbamylcholine treatment, and vesicular profiles were no longer detectable. When incubation with the agonist was prolonged (3 hr at 37 degrees C), endocytotic 'smooth vesicles' fused and formed multivesicular structures presumably implicated in receptor down-regulation. Conversely, when nonconfluent cells were exposed to the muscarinic antagonist atropine, receptor redistribution was revealed, leading to the formation of clusters where receptor accumulated. Muscarinic receptor redistribution induced by atropine therefore does not involve the sequestration process seen in carbamylcholine-treated cells.

Antibodies, Monoclonal↗
PubMed1987

Immunochemical studies of the muscarinic acetylcholine receptor.

Muscarinic receptors have been purified from calf forebrain plasma cell membranes by affinity chromatography on a dexetimide-agarose gel. SDS-PAGE analysis showed a single 70 kDa band. Monoclonal antibodies have been prepared against these affinity purified 70 kDa protein(s). One antibody, M-35, immunoprecipitated up to 80% of digitonin-solubilized muscarinic receptors. M-35 had agonist-like effects on guinea-pig myometrium: it increased the intracellular cyclic GMP content, decreased prostaglandin-induced cyclic AMP accumulation and caused muscle contractions. The two first effects were inhibited by atropine. M-35 was used to visualize muscarinic receptors at the surface of human fibroblastic cells. In the particular cell line used, the receptors have a low affinity for pirenzepine, were negatively coupled to adenylate cyclase and mediated increase in the phosphatidyl-inositol breakdown.

Acetylcholine↗
PubMed1986

Comparative binding of wheat germ agglutinin and its succinylated form on lymphocytes.

We have compared the binding parameters of native wheat germ agglutinin (WGA) and its succinylated form (SWGA) to rat lymphocytes. Scatchard plots were obtained with the fluoresceinated lectins in a concentration range of 10 nM to 0.1 mM. Association and dissociation rate parameters were also measured. The following differences were observed: at low concentration of WGA, binding is positively cooperative with a Hill coefficient of 1.75, whereas binding of SWGA is not. The numbers of high-affinity sites are respectively (2.5 +/- 0.8) X 10(6) and (6.4 +/- 1.3) X 10(5) for WGA and SWGA. Association constants were found to be (4.7 +/- 1.7) X 10(6) l mol-1 for WGA and (1.42 +/- 0.36) X 10(7) l mol-1 for SWGA, which is 35 times higher than for native WGA. Neuraminidase treatment decreases the Hill coefficient as well as the number of sites involved in the cooperative binding of native WGA. Equilibrium data were obtained at three temperatures to determine the thermodynamic parameters (delta H degree and delta S degree). These results are indicative of an oligomerization process dynamically formed at the membrane level before tight binding of the lectin to its receptors could occur.

Animals↗
PubMed1986

Effect of in vivo gamma-irradiation on the binding of wheat germ agglutinin on lymphocyte plasma membranes.

Using quantitative fluorimetry with fluoresceinated wheat germ agglutinin, we have been able to investigate in vivo gamma radiation-induced damage at the outer membrane level of rat splenic lymphocytes, namely damage to the glucosidic moieties of membrane glycoproteins and glycolipids. This paper demonstrates that below an irradiation level of 1 gray (Gy), removal of sialic acid is the major feature leading to new exposed specific binding sites for wheat germ agglutinin, since this lectin is specific for sialic acid and N-acetyl-D-glucosamine. Our studies also suggest that above 1 Gy of irradiation more internal damage occurs, since we observed a striking decrease in wheat germ agglutinin binding sites.

Animals↗
PubMed1986

Enzyme-linked immunosorbent assay for amitriptyline and other antidepressants using a monoclonal antibody.

We describe and evaluate a method to measure amitriptyline and other tricyclic antidepressants by enzyme linked immunosorbent assay, using monoclonal antibody. In this assay, biological samples were first incubated with the antibody; in a second step, free remaining antibody was allowed to bind to lysozyme-nortriptyline coated immunotitration plates. The bound fraction of the monoclonal antibody was revealed with rabbit anti-mouse serum coupled to horseradish peroxidase. The optical density of the reaction product was measured with a colorimeter at 410 nm. Specificity of the antibody was investigated by means of a Farr test showing interferences in therapeutic ranges only for chlorpromazine and phenytoine. Means of intra- and inter-assay variations were 10 and 13%, respectively. The results when compared to those obtained by gas chromatography with a selective nitrogen detector gave a correlation coefficient of 0.897. Finally, the great reliability of the monoclonal antibody, the advantages of a decreased analysis time, low cost and high capacity of the procedure contribute to make this immunoassay most suitable for clinical monitoring and pharmacokinetic studies of tricyclic antidepressants.

Amitriptyline↗
PubMed1986

The immune response towards beta-adrenergic ligands and their receptors. VI. Idiotypy of monoclonal anti-alprenolol antibodies.

Four murine monoclonal antibodies specific for alprenolol, a synthetic beta-adrenergic ligand, with different binding properties towards alprenolol and other beta-adrenergic antagonists and agonists (as described in a previous report) were used to induce anti-idiotypic responses in rabbits and mice. Three of the rabbit anti-idiotypes inhibited, and one increased the binding of tritiated dihydroalprenolol to the Ab1 against which they were induced. The syngeneic mouse anti-idiotypes all had an inhibitory effect on the ligand binding to their corresponding Ab1. Cross-reactivity tests of the xenogeneic and syngeneic anti-idiotypes were positive only for two monoclonal anti-alprenolol antibodies. Cross-reaction could be shown neither on a panel of 15 other monoclonals, nor on polyclonal anti-alprenolol antibodies of the BALB/c and the C57BL/10 mice. These results suggest that the immune response against alprenolol results in antibodies with mostly private idiotypic determinants. Moreover, the properties of the anti-idiotypic response against the same monoclonal antibody seem to be different according to the species used for anti-idiotypic induction.

Alprenolol↗
PubMed1985

Sequence analysis of peptide fragments from the intrinsic membrane protein of calf lens fibers MP26 and its natural maturation product MP22.

Calf lens fiber plasma membranes, containing only the intrinsic membrane protein MP26 and its maturation product MP22 were treated with proteolytic enzymes such as trypsin, protease V8 from S. aureus or with chemical agents as CNBr in formic acid. The cleavage products, purified by electrophoresis, were analysed for their amino acid composition and N-terminal sequences. Proteolysis gave rise to peptides which were mainly shortened at the C-terminal end of the molecules. While the V8 protease produced a fragment with a similar N-terminal sequence as the maturation product MP22, trypsin yielded another cleavage product. Chemical hydrolysis yielded large fragments (11-15 kDa) with hydrophobic N-terminal sequences. Our results suggest that MP26 is characterised by an N-terminal signal sequence and possesses other hydrophobic domains which could function as untranslocated insertion sequences.

Amino Acid Sequence↗
PubMed1985

Altered binding properties of beta-adrenergic receptors and lack of coupling to adenylate cyclase in P815 mastocytoma cells.

P815, a murine mastocytoma cell line, possesses beta-adrenergic binding sites as assessed by using [3H]dihydroalprenolol (antagonist) and [3H]hydroxybenzylisoproterenol (agonist). The number of binding sites per cell was 29 000 for the agonist and 75 000 for the antagonist, as determined by direct binding assays and inhibition experiments on intact cells. On membrane preparations from the same cells, binding of alprenolol was only displaceable by antagonists, while stereospecific binding of hydroxybenzylisoproterenol was only displaceable by agonists. The P815 membranes also possessed an adenylate cyclase stimulated by Gpp(NH)p and NaF but not by 1-isoproterenol. The intracellular cAMP level of intact cells was not modulated by 1-isoproterenol or by 1-epinephrine, but was increased by forskolin. These results suggest that the beta-adrenergic receptor of P815 mastocytoma cells is non-functional. This may explain the failure of agonists to stimulate adenylate cyclase activity in these cells.

Adenylyl Cyclases↗
PubMed1985

beta-Adrenergic agonist activity of a monoclonal anti-idiotypic antibody.

Hybridoma cells bearing monoclonal antibody against the beta-adrenergic ligand alprenolol were used as an immunogen to raise monoclonal anti-idiotypic antibodies. Of six anti-idiotypic antibodies, which inhibit ligand binding, three were able to recognize beta-adrenergic receptors. One of them, mAb2B4, an IgM that could be amplified into ascites, binds to the beta-adrenergic catecholamine receptors of intact epidermoid A431 cells and precipitates receptors solubilized from plasma membranes by digitonin. This antibody identifies the beta 2-adrenergic receptor of A431 cells as a single 55-kDa protein and stimulates adenylate cyclase activity. This stimulation is inhibited by the beta-adrenergic antagonist propranolol.

Adenylyl Cyclases↗
PubMed1985

MP26 in the bovine lens: a post-embedding immunocytochemical study.

Gold immunolabeling of bovine lens tissue embedded in Lowicryl K4M, using a polyclonal antibody specific for a major component of lens fiber plasma membrane of 26 K molecular weight, shows that this constituent is absent from the epithelial cell plasma membrane and associated only with the junctional and non-junctional domains of the lens fiber plasma membrane.

Animals↗
PubMed1985

Structural analysis of the epitope recognized by a monoclonal antibody directed against tricyclic antidepressants.

After somatic cell fusion between splenocytes of immunized BALB/c mice and NS-1 myeloma cells, a clone was obtained that secreted an anti-nortriptyline antibody of the IgG1 kappa isotype. The association constant of this antibody for pharmacologically active tricyclic antidepressant drugs ranged from 0.6 X 10(7) to 3 X 10(7) M-1. From thermodynamic and binding studies as well as tridimensional structures of tested compounds, the epitope recognized by the monoclonal antibody appeared to include both a hydrophobic tricycle in which the two phenyl rings form an angle of 120 to 130 degrees, and a side chain in which the amino group is separated from the two lateral rings of the tricyclic structure by a distance of approximately 5.9 A and 7.5 A, respectively. This conformation seems to be the one interacting with muscarinic acetylcholine brain receptors.

Amino Acid Sequence↗
PubMed1984

Production and detection of monoclonal anti-idiotype antibodies directed against a monoclonal anti-beta-adrenergic ligand antibody.

A new method has been developed to raise monoclonal anti-idiotypic antibodies. Monoclonal anti-idiotypic antibodies were obtained by fusion of NS-1 myeloma cells with splenocytes of mice immunised by intravenous injections of fixed hybridoma cells bearing a monoclonal antibody specific for beta-adrenergic ligands. New screening tests were developed to analyse the resulting hybridoma supernatants for different anti-idiotypic properties. Among 23 hybridoma supernatants recognising the idiotype, 6 were found to inhibit hapten binding and 3 of these recognised beta-adrenergic receptors.

Adrenergic beta-Antagonists↗