Characterization of antibodies against oestrone-azo-protein conjugates using 3H and 125I radioligands

This report describes the synthesis of six oestrone-azo-protein immunogens differing in the position of the azo group in the steroid skeleton and in the nature of carrier protein. By nitration of oestrone, 2-nitro- and 4-nitro-isomers were prepared and converted to amino-derivatives by reduction of the nitro group. The diazotized amino-oestrones were coupled to albumin, thyreoglobulin and haemocyanin. The immunogens thus prepared were administered to rabbits to assess the effect of position of the bridge, carrier protein and adjuvant on antibody production and specificity. Very weak immune responses were obtained with both types of albumin immunogen. The presence of the azo group at C-2 position led to a lower antibody production as compared with C-4 derivatives. The relatively high antibody production was achieved by immunization with oestrone-4-azo-thyreoglobulin or oestrone-4-azo-haemocyanin, with the oil component of adjuvant omitted. The antibodies combined with tritiated and iodinated radioligands yielded typical curves showing the sensitivity of 10-20 pg for 3H labelled oestrone and that of 3-10 pg for 125I labelled oestrone. The specificity of the 6 antibodies was tested using 25 related structures. In all these cases there was low cross-reaction with oestradiol, oestriol and their derivatives. On the other hand, the discriminating ability of the antibodies with regard to the opposite end of the molecule, i.e. with regard to the A-ring and partly the B-ring was very low. This makes it possible to use these antibodies for the radioimmunoassay of oestrone alone or some of its metabolites modified in the A-ring. The results of cross reactivity are generalized to the effect that in the oestrone-azo-hapten structure the antigenic determinant area can be ascribed to the size not exceeding 2-3 steroid rings.     

Regulation of podosomes by intracellular pH in avian osteoclasts

The effects of changes in intracellular pH (pH1) on the organization of the clear zone of isolated avian osteoclasts in culture were studied. The distribution of podosomes, the close contact areas that mediate the adhesion of osteoclasts to the substrate, was investigated by decoration of microfilaments with fluorescent phalloidin. Intracellular acidification by butyric acid induces significant increase of podosome formation at the level of the clear zone compared to controls. Conversely, alkalinization by HCO3- reduces the percentage of osteoclasts with podosomes. A role of pH1 on the adhesion of the osteoclasts to the substrate is hypothesized.     

Specificity of the immune response to the oestrone-azo-hapten structure

Comparison of the cross-reactions between 14 related steroids with eight antibodies showed that each antibody had its individual molecular specificity. During the 183-day period covering eight immunizations, cross-reaction between oestrone and 1-methyloestrone showed only very little change with five antibodies, while the great differences in cross-reactivity values for the individual antibodies were retained. With the same five antibodies, the course of cross-reaction between oestrone and 3-sulphooestrone as well as 6-oxooestrone was also fairly constant, the differences in cross-reactivity in relation to the individual antibodies being considerably large. The blood plasma antibody concentrations measured at the same intervals showed considerable fluctuation, whereas the affinity constants of the respective antibodies, except one of them, showed a moderate upward trend. The suggested molecular parameters of binding sites of the antibodies to the oestrone-4-azo-hapten structure were in surprisingly good agreement with those reported in the literature for mouse myeloma immunoglobulin A proteins possessing dinitrophenyl-binding activity. The individual antibodies exhibited highly sensitive radioimmunoassay curves for both oestrone and 3-sulphooestrone.     

Elevation of Striatal Dopamine Receptors by Estrogen: Dose and Time Studies

Administration to male rats of a single dose of 17 beta-estradiol valerate (8-500 micrograms/rat) or implantation of a pellet containing 17 beta-estradiol (0.5-50 mg/rat) increased serum 17 beta-estradiol levels in a dose-dependent relationship when measured on the sixth day after administration. At the same time, after these doses, the serum rat prolactin (rPRL) levels were doubled and the striatal 3,4-dihydroxyphenylethylamine (DA, dopamine) receptor densities were increased 20%. A single dose of 17 beta-estradiol valerate of 4 micrograms/rat or less did not alter serum 17 beta-estradiol or rPRL levels or the striatal DA receptor density. After the single injection of 17 beta-estradiol valerate (125 micrograms/rat) the serum 17 beta-estradiol levels peaked at 1 day, the serum rPRL levels peaked at 2 days, and the striatal DA receptor density elevation peaked from 4 to 8 days. Implantation of a pellet containing 17 beta-estradiol (25 mg/rat) produced a constant elevation of serum 17 beta-estradiol levels from 1 to 10 days. Whereas the serum rPRL levels were continuously elevated about two-fold, the densities of the striatal DA receptors were increased significantly by 20-25% only from 4 to 8 days after pellet implantation. These results indicate that striatal DA receptor density rises and returns to control levels during the constant elevation of serum 17 beta-estradiol and rPRL levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA polymerase associated with human rotaviruses in diarrhea stools.

RNA polymerase activity was detected in six stools which were partially purified by high-speed centrifugation from infants with rotavirus gastroenteritis, but was not detected in five stools which were negative for rotavirus by counterimmunoelectrophoresis and radioimmunoassay. The polymerase activity was associated with the 1.38-g/ml rotavirus band after purification in a CsCl gradient.     

Characteristics of antibodies against 17 beta-oestradiol in homologous and heterologous systems of radioimmunoassay

Directly iodinated oestradiol-2(4)-iodo-[125I] and oestradiol-6-O-(CMO)-[125I]iodohistamine were prepared and used in conjunction with anti-oestradiol-2(4)-azo and anti-oestradiol-6-O-(CMO) sera for the development of various radioimmunoassay (RIA) systems which showed marked differences in sensitivity and relatively small differences in specificity. Whereas the heterologous combination of oestradiol-6-O-(CMO)-[125I]iodohistamine and anti-oestradiol-2(4)-azo-serum showed a sensitivity expressed in femtograms, the homologous combination using oestradiol-6-O-(CMO)-[125I]iodohistamine radioligand exhibited a sensitivity two orders lower. The specificity of both the heterologous and homologous system did not differ significantly from the RIA system using tritiated radioligand. The combination of directly iodinated oestradiol and anti-oestradiol-2(4)-azo-serum showed a higher sensitivity and a lower specificity as compared with tritiated radioligand.     

Identification of the virus-specific proteins of respiratory syncytial virus temperature-sensitive mutants by immunoprecipitation

The proteins of Long strain RSV and three temperature-sensitive (ts) mutants of the A2 strain were compared by pulse labeling virus-infected cells with [35S]methionine and [3H]glucosamine followed by analysis of the cell lysates by polyacrylamide gel electrophoresis. At the permissive temperature (30 degrees) proteins ranging in molecular weight from 24,000 to 50,000 (VP24, VP27, VP33, VP44) could be identified. Immunoprecipitation of viral lysates by immune rabbit serum demonstrated antigenic similarity with VP27, VP44, VP50, and VP67 in all ts mutants and Long strain RSV. [3H]Glucosamine labeling demonstrated glycoproteins of 90,000 (GP90) and 50,000 (GP50) in Long strain and GP90 in the ts mutants.