Ultrastructural localization of Thermoplasma acidophilum surface carbohydrate by using concanavalin A

Surface carbohydrate, presumably the lipopolysaccharide, of Thermoplasma acidophilum was visualized by means of the concanavalin A, horseradish peroxidase, and diaminobenzidine cytochemical staining procedure.     

Nucleotide pyrophosphatase of rat liver. A comparative study on the enzymes solubilized and purified from plasma membrane and endoplasmic reticulum.

Studies on the subcellular distribution of rat liver nucleotide pyrophosphatase activity revealed its presence in the plasma membrane and the endoplasmic reticulum only. The enzymes from either source were solubilized specifically with trypsin without an apparent change of their catalytic properties. A 200-fold and 1600-fold purification, respectively, was achieved by a procedure including DEAE-cellulose and affinity-chromatography with AMP as ligand, gel filtration on Sephadex G-200 and gel electrophoresis. Both nucleotide pyrophosphatases were isolated as electrophoretically homogeneous soluble proteins. They were shown to contain carbohydrate moieties. The electrophoretic mobility of both enzymes in polyacrylamide gels was identical at three pH values. Dodecylsulfate gel electrophoresis indicated a molecular weight of 137 000 for both glycoproteins. The enzymes hydrolyze a variety of purine and pyrimidine nucleotides yielding a 5'-nucleoside monophosphate. Adenosine 3':5'-monophosphate, nucleic acids and phosphate monoesters are not cleaved, but p-nitrophenyl-thymidine5'-monophosphate is readily hydrolyzed. In view of their substrate and inhibitor specificities the enzymes are considered nucleotide pyrophosphatases rather than phosphodiesterases.     

Hormone-dependent processing of the avian progesterone receptor

Avian progesterone receptor exists as two forms, A and B, with molecular weights of 79,000 and 110,000 daltons, respectively. The origin and significance of these two forms is an area of active investigation and debate. Monoclonal antibodies produced against these two forms were used to examine receptor stability in cytosol and changes in the receptor forms induced by hormone binding. The lability of hormone binding at elevated temperatures is well documented. Analysis by Western blotting showed the receptor was stable in freshly prepared oviduct cytosol for 2 hr at 37°C, while hormone binding was lost within 30 min. However, loss of receptor through degradation was seen when cytosol was prepared from frozen tissue or when homogenization was excessive. Progesterone was injected into diethylstilbestrol-stimulated chicks to examine, in vivo, effects of hormone treatment on receptor forms in the cytosol and nuclear fractions. Progesterone treatment caused a time- and dose-dependent conversion of the A receptor to a form (A′) with a slower electrophoretic mobility. The cytosolic progesterone receptor was divided equally between the B and A forms, while the nuclear receptor was predominantly A′. The amount of nuclear receptor was consistently less than cytosolic receptor. Receptor phosphorylation was analyzed by incubating tissue minces with [³²P]orthophosphate with or without progesterone followed by immune isolation of receptor forms. Progesterone treatment caused a time-dependent increase in cytosol receptor phosphorylation which was evident after 5 min of treatment. This phosphorylation was observed with both the A and B receptor forms. The results indicate that receptor phosphorylation is a very early event during progesterone action.     

Facile geometric isomerization of phenolic non-steroidal estrogens and antiestrogens: limitations to the interpretation of experiments characterizing the activity of individual isomers

In many estrogen responsive systems the isomers of tamoxifen are known to have different biological character-the trans isomer is generally an antagonist and the cis isomer an agonist. Attempts to similarly characterize the isomers of hydroxytamoxifen (which differ greatly in their affinity for the estrogen receptor) are shown to be complicated by their facile isomerization. This isomerization was studied in cultures of estrogen receptor positive MCF-7 human breast cancer cells and monitored by HPLC under reversed phase conditions. Hydroxytamoxifen isomers that are initially 99% pure, undergo a time and temperature dependent isomerization, so that after 2 days in tissue culture medium at 37 degrees C they have isomerized to the extent of 20%. This isomerization occurs in the cell-free medium alone and cannot be attributed to a metabolic conversion by the cells. The isomerization occurs much more slowly at 4 than at 37 degrees C and can be reduced considerably by various antioxidants (butylated hydroxytoluene, ascorbate, alpha-tocopherol, retinoic acid and retinal); however, at concentrations that block isomerization, these antioxidants are toxic to the cells. Although the medium contains both the cis and trans isomers of hydroxytamoxifen, the MCF-7 cells preferentially accumulate the trans isomer and the material associated with the nuclear estrogen receptor is, in all cases, mainly the higher affinity trans isomer. A similar preference of the estrogen receptor for the trans isomer is seen with diethylstilbestrol, resulting again in almost exclusive accumulation of the trans isomer in the receptor binding site. These experiments indicate the importance of verifying the isomer compositions of easily isomerizable non-steroidal estrogens and antiestrogens, such as diethylstilbestrol and hydroxytamoxifen, both in stock solutions and in experimental samples (especially those derived from receptor-associated material), so as to ascertain that the activity of the individual isomers is being correctly assigned.     

Analysis of genetic changes in radiated and non-radiated bulk oat (Avena sativa L.) populations

Summary In both radiated and non-radiated oat populations inbreeding coefficients increased more slowly than was expected on the assumption of full selfing and equal selective values for homozygotes and heterozygotes. Assuming 1% outcrossing for oats and a selective value of 1.0 for the mean, the heterozygotes for two loci governing crown rust reaction have an advantage of 50% over the homozygotes. This study supports previous observations that the heterozygote often has a decided advantage in predominantly self-pollinated crops.