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S Bernard

Publications and source records attributed to S Bernard.

At least 163 records · Page 9Linked to original sources
PubMed1985

Virus enzyme-linked cell immunoassay (VELCIA): detection and titration of rotavirus antigen and demonstration of rotavirus neutralizing and total antibodies.

Virus enzyme-linked cell immunoassay (VELCIA) for detection and titration of rotavirus antigen has been developed. Wild-type porcine rotavirus antigen can be detected and titrated directly from the fecal material within 24 h. Porcine OSU strain can be titrated higher than 10(-8). The method has also been introduced for the demonstration of rotavirus neutralizing and total antibodies. In VELCIA the advantages of the cell culture system for virus isolation are combined with enzyme immunodetection and spectrophotometrical reading of the test.

Antibodies, Viral↗
PubMed1985

Nontransformed rabbit liver glucocorticoid receptor: purification, characterization and transformation.

The molybdate-stabilized nontransformed form of the glucocorticoid receptor from rabbit liver has been purified approximately 8,000-fold by a three-step procedure. The first step involved protamine sulfate precipitation which allowed a 5-6-fold purification with 85% yield. The second step, affinity chromatography using a N-(12-dodecyl-amino) 9 alpha-fluoro-16 alpha-methyl-11 beta, 17 alpha-dihydroxy-3-oxo-1,4-androstadiene-17 beta-carboxamide substituted Sepharose gel, purified the receptor 1,500-2,000-fold as calculated by specific radioactivity. The third step involved high performance liquid chromatography resulting in overall purification near 8,000-fold. The final glucocorticoid receptor appeared about 60% pure. The purified nontransformed glucocorticoid receptor had a sedimentation coefficient of 9 S in 0.16 M phosphate containing 5-20% sucrose gradients and the Stokes radius was 6.1-6.3 nm as determined by low pressure gel filtration and HPLC. Binding specificity of the purified receptor was identical to that previously reported in crude rabbit liver cytosol. Isoelectricfocusing and ion-exchange chromatography showed that the purification procedure affected the net charge of the receptor protein. This phenomenon could be related to interactions between the glucocorticoid receptor and cytosolic factors. SDS polyacrylamide gel electrophoresis showed a major Mr = 94,000 protein band which is in good agreement with previously reported values for glucocorticoid receptors. Transformation of the purified receptor was achieved after removal of molybdate by exposure at 25 degrees C to 0.4 M KCl. Characterization of the molecular forms was performed by means of incorporation into isolated nuclei, affinity towards polyanionic exchangers and high pressure size exclusion chromatography. Results show that about 40% of the receptor is in the transformed state.

Adrenalectomy↗
PubMed1984

Evidence for a metalloprotein structure of plasma membrane 5'-nucleotidase.

To point out the metalloprotein structure of bovine liver plasma membrane 5'-nucleotidase, we studied the inhibition mechanism of the purified enzyme by EDTA: this apparently non-competitive inhibition seems to be dependent on EDTA concentration, pH, temperature and incubation time. When the restoration of activity was assayed by addition of divalent cations or by gel filtration, the inhibition became progressively irreversible with time. Incubation of the enzyme with [14C]EDTA allowed us to observe, after gel filtration as well as after sucrose gradient ultracentrifugation, that the chelating agent is bound to 5'-nucleotidase.

5'-Nucleotidase↗
PubMed1984

Resolution of the molecular forms of rat liver glucocorticoid receptor by affinity chromatography on immobilized heparin.

Rat liver cytosolic glucocorticoid receptor labelled with [3H] dexamethasone and stabilized with molybdate was bound to heparin-ultrogel and eluted with NaCl or heparin as a single peak of radioactivity. After heat exposure of cytosol, two steroid receptor complexes could be separated by NaCl or heparin. Characterization of the two forms was performed by means of affinity towards isolated nuclei, sucrose gradient centrifugation and gel exclusion high performance liquid chromatography. The results presented here suggest that the two forms eluted from heparin-agarose correspond to the untransformed and transformed states of the glucocorticoid receptor complex. Taken together, these observations argue in favor of heparin-ultrogel as a suitable procedure to study the mechanism of glucocorticoid-receptor transformation.

Animals↗
PubMed1984

Interactions with lectins indicate differences in the carbohydrate composition of the membrane-bound enzymes acetylcholinesterase and 5'-nucleotidase in different cell types.

We have examined the interactions of the membrane-bound enzymes, 5'-nucleotidase and acetylcholinesterase from bovine tissues with lectins and shown that glycosylation contributes significantly to the polymorphism of these enzymes, in a tissue-specific manner. Lectins which bind 5'-nucleotidase also inhibit its catalytic activity to various degrees. We found different specificities with 5'-nucleotidases from various cell types: for example lymphocyte 5'-nucleotidase did not interact with wheat germ agglutinin, in contrast with 5'-nucleotidases from hepatocyte and caudate nucleus membranes. Treatment with glycohydrolases, alpha-D-mannosidase and neuraminidase, suggested that the latter enzymes possess sialic residues which are absent in the lymphocyte enzyme. Interactions of acetylcholinesterase with lectins were demonstrated by sedimentation analysis and binding to immobilized lectins, but its activity was generally not affected. A notable exception was lymphocyte acetylcholinesterase which was inhibited by the fucose-binding Ulex europeus agglutinin. This inhibition was relieved by alpha-L-fucose but not by alpha-D-fucose and reduced after treatment with alpha-L-fucosidase. In addition this enzyme differs from acetylcholinesterases from other tissues by its higher Km value, although it appears immunologically equivalent. The different forms of acetylcholinesterase from the same tissue may differ in their interactions with lectins. In muscle for example G4 carries carbohydrate chains of the complex type whereas G1 appears to possess only the high mannose type. We discuss the possible relationships between these forms.

5'-Nucleotidase↗
PubMed1984

Influence of molybdate, ionic strength and pH on ligand binding to the glucocorticoid receptor.

The addition of molybdate to rabbit liver cytosol increased significantly the affinity of the glucocorticoid receptor for [3H] dexamethasone without influencing the concentration of binding sites. This effect was concentration dependent. Analysis of the binding data by curve-fitting and Scatchard plot revealed the occurrence of a complex binding process in the presence of molybdate. The pH-dependence curve of the binding was shifted towards alkaline values by the oxyanion. Taken together, these data suggest that molybdate exerts its effects via an interaction with the receptor molecule.

Animals↗
PubMed1984

5'-Nucleotidase from bovine caudate nucleus synaptic plasma membranes: specificity for substrates and cations; study of the carbohydrate moiety by glycosidases.

We studied 5'-nucleotidase in preparations of synaptic plasma membranes from bovine caudate nucleus. The best substrates for this membrane-bound enzyme were purine nucleotides, particularly 5'AMP. Effects of metal cations and chelating agents suggest that 5'-nucleotidase is a metalloprotein. Optimal conditions for solubilization of the 5'-nucleotidase were found by using a low concentration of the zwitterionic detergent sulfobetaine 14. In contrast, another membrane-bound enzyme, acetylcholinesterase, was not solubilized under these conditions, but only in the presence of Triton X-100. The effects of lectins (concanavalin A, Lens culinaris agglutinin, wheat germ agglutinin, and Limulus polyphemus agglutinin) showed that both enzymes are glycoproteins. Sequential hydrolysis with specific glycosidases produced modifications of the effect of lectins on these enzymes. The results suggest the presence of a complex-type glycosylation, with a fucose residue on the internal N-acetyl-D-glucosamine of the pentasaccharide core.

5'-Nucleotidase↗
PubMed1984

[Experimental infection of fattening swine with rotavirus: study of local immunity using coproantibodies].

Fattened pigs were experimentally infected with porcine rotavirus (OSU strain). Viral antigens found in the faeces were analysed over a period of two months (ELISA method). We have found no correlation between virus excretion and the clinical manifestation of disease. The local immunity has been analysed in the faeces by the coproantibodies antirotavirus. The different classes of antibody, G, M, A and immune complexes have been determined using the ELISA method. The level of IgA antibodies was higher (10 fold) than IgG and IgM, and the timing of the appearance of these antibodies antivirus was also different. No correlation was found between local immunity and virus excretion in the faeces.

Animals↗
PubMed1983

Purification and properties of bovine liver plasma membrane 5' nucleotidase.

5'-Nucleotidase from bovine liver plasma membranes has been extracted by the zwitterionic detergent sulfobetaine 14, and purified to apparent homogeneity. Two affinity chromatographies on concanavalin-A-Ultrogel and 5' AMP-Sepharose 4B followed by AcA-54-Ultrogel filtration resulted in a purification of 16000 times relative to the homogenate. Sodium dodecyl sulphate gel electrophoresis indicates that the apparent molecular weight of the subunit is 70000. Cross-linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows a band with an apparent molecular weight of 140000 indicating that the enzyme is a dimer. 5'-Nucleotidase is a glycoprotein and its activity is inhibited to different degrees by various lectins, indicating a direct interaction with the enzyme. The purified enzyme shows a sevenfold greater affinity for AMP than the membrane-bound enzyme. The optimum activity of the purified enzyme occurs at pH 7.5 while the membrane-bound enzyme showed a wide range of pH optimum (7.5-8.3). An Arrhenius plot of the membrane-bound enzyme shows a break at 28 degrees C, which disappears in the purified enzyme. The enzyme was inhibited by EDTA, and this inhibition was reversed by divalent cations. This, as well as other evidence, indicates that the enzyme contains a highly bound metal cation, perhaps Mn2+ or Mg2+.

5'-Nucleotidase↗
PubMed1983

Physicochemical characterization and transformation of the cytosolic glucocorticoid receptor from rabbit liver.

Characterization of the glucocorticoid receptor from rabbit liver cytosol was studied in vitro. Binding of [3H]-dexamethasone showed a high affinity (KD = 2.4 . 10(-9)M) and a concentration of binding sites of 0.3 . 10(-12) mol/mg proteins. Association and dissociation rate constants for [3H]-dexamethasone were respectively 1.6 . 10(5)M-1 . min-1 and 2.5 . 10(-4) min-1. Competition experiments showed that dexamethasone and triamcinolone acetonide were the most effective competitors while progesterone and aldosterone competed very poorly. The stabilities of bound and unbound forms were investigated with and without molybdate and other oxyanions. Calibrated gel filtration gave a stokes radius of 5.6 nm and an apparent mol. wt of 280,000. The untransformed complex sedimented at 9 s in 5-20% sucrose density gradients and the transformed species sedimented near 4 s. The behaviour of the three forms of receptor, untransformed, transformed and molybdate-stabilized was studied on ionic exchangers. Transformation of the [3H]-dexamethasone--receptor complex was shown to occur both with high ionic strength and elevated temperature. The transformation step was almost completely inhibited by tungstate and molybdate while vanadate revealed a very slight inhibitory effect.

Animals↗
PubMed1982

Localization of histone H5 in the subunit organization of chromatin using immunoelectron microscopy.

In avian erythroid cells the erythrocyte-specific histone H5 is involved, like H1, in the packing of nucleosomes in the 25-nm chromatin fibers. In this study the distribution of histone H5 along the polynucleosomal chains was visualized by immunoelectron microscopy. Trinucleosomes from chicken erythrocytes and liver were used in order to test the specificity of the reaction with purified rabbit anti-H5 antibodies at various ionic strengths (5-80 mM). Long-chain chromatin was then reacted with anti-H5 antibodies and with sorted monomeric ferritin conjugate under chosen conditions. The antigenic determinants of histone H5 in the 25-nm fiber of long-chain chromatin (at 80 mM NaCl) are as accessible to the specific antibodies as in trinucleosomes. When the immunocomplexes were examined by electron microscopy in a low-ionic-strength buffer, permitting maximum extension of the chromatin structure on the grid, clusters of compacted nucleosomes were seen, separated by short regions of relaxed nucleosomes. Single nucleosomes enlarged by the antibodies are sometimes visible in the extended domains. We conclude that histone H5 is located primarily on series of adjacent nucleosomes but it can also be found on single nucleosomes located in the H1-enriched extended domains.

Animals↗