Receptor recycling: liberation of glucocorticoid receptors from liver nuclei in vitro

Dynamics of the steroid receptors seems to be the consequence of receptor recycling. In the present study, as a clue to elucidate the mechanism of receptor recycling, factors which affect the rate of liberation of nuclear bound 3H-glucocorticoids were examined in vitro. Among the factors examined, NAD, NADPH, cAMP and p-nitrophenyl phosphate accelerated the liberation of radioactivity from nuclei in a temperature-dependent manner when added to the incubation mixture. The presence of a large amount of unlabeled dexamethasone (Dex) did not modify the rate of liberation. From these results, it was concluded that the metabolism of ligand bound to the receptor is not a necessary step in the liberation of receptor from nuclei. These agents did not influence the binding process of 3H-Dex-receptor complex to DNA-cellulose. Therefore the stimulation of receptor release does not seem to be mediated by reducing the binding affinity between nuclei and receptor complexes. The liberated radioactivity was eluted on a Sephadex G-100 column in the void volume and in macromolecule-unbound fractions. In both fractions, the majority of the radioactivity comigrated with authentic glucocorticoids on thin-layer chromatography.     

Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

Codon Substitution in Evolution and the "Saturation" of Synonymous Changes

A mathematical model for codon substitution is presented, taking into account unequal mutation rates among different nucleotides and purifying selection. This model is constructed by using a 61 X 61 transition probability matrix for the 61 nonterminating codons. Under this model, a computer simulation is conducted to study the numbers of silent (synonymous) and amino acid-altering (nonsynonymous) nucleotide substitutions when the underlying mutation rates among the four kinds of nucleotides are not equal. It is assumed that the substitution rates are constant over evolutionary time, the codon frequencies being in equilibrium, and, thus, the numbers of synonymous and nonsynonymous substitutions both increase linearly with evolutionary time. It is shown that, when the mutation rates are not equal, the estimate of synonymous substitutions obtained by F. Perler, A. Efstratiadis, P. Lomedico, W. Gilbert, R. Kolodner and J. Dodgson's "Percent Corrected Divergence" method increases nonlinearly, although the true number of synonymous substitutions increases linearly. It is, therefore, possible that the "saturation" of synonymous substitutions observed by Perler et al. is due to the inefficiency of their method to detect all synonymous substitutions.     

Estimates of Denitrification and Nitrification in Coastal and Estuarine Sediments

Denitrification and nitrification in sediments of Tama Estuary and Odawa Bay, Japan, were investigated by the combined use of a continuous-flow sediment-water system and a ¹⁵N tracer technique. At Odawa Bay, the nitrification rate was comparable to the nitrate reduction rate, and 70% of the N₂ evolved originated from nitrogenous oxides (nitrate and nitrite) which were produced by the action of nitrifying bacteria in the sediments. At Tama Estuary, the nitrate reduction rate was 11 to 17 times higher than the nitrification rate, and nitrogenous oxides derived from ammonium accounted for only 6 to 9% of the N₂ evolution by denitrification.     

Separation of the Effects of Photoperiod and Hormones on Sex Expression in Cucumber

The effects of photoperiod on the sex expression of cucumberflowers were examined in relation to the endogenous GA and ethyleneproduction with short day (SD)- and long day (LD)-sensitivecultivars under controlled environmental conditions. Growth of plants was greatly reduced under LD in both typesof cultivars. Reflecting this, GA activity was greater and ethyleneevolution less in plants grown under SD than in those underLD. However, the number of pistillate flowers increased underSD and LD in SD- and LD-sensitive cultivar, respectively, indicatingthat a change in sex expression did not coincide with hormonalchanges in the SD-sensitive cultivar. Thus, the effect of photoperiodon the sex expression was found to be separate, at least inpart, from the effect of GA or ethylene in cucumber. (Received September 16, 1982; Accepted December 7, 1982)     

Changes in Microtubules in Onion Leaf Sheath Cells during Bulb Development

Cortical microtubules are oriented transversely to the cellaxis in leaf sheath cells of onion plants (Allium cepa L. cv.Osaka-Okute) that have not started bulb formation. As the bulbdevelops and the leaf sheath cells swell, the microtubules becomedisoriented and scattered and finally disappear. The microtubuleinhibitors colchicine and cremart [O-ethyl O-(3-methyl-6-nitrophenyl)N-sec-butylphosphorothioamidate]cause swelling of leaf sheath cells and make the basal partof the plant bulbous. The cortical microtubules may have animportant role in regulating bulb development in onion plants. (Received August 21, 1982; Accepted December 6, 1982)     

Effect of TPA on the mutagenicity of caffeine in the soybean mutation test

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a well-known tumor promoter in mouse-skin carcinogenesis. Its effects on mutagenesis in a soybean test system were examined, and the effects were judged from the appearance of spots of various colors on the leaves. When soybean seeds were treated with TPA plus 0.03% caffeine, the frequency of spots per leaf decreased significantly and in proportion to the concentration of TPA. TPA alone at concentrations of 1–20 μg/ml did not induce any mutations. Mutations induced by γ-rays were not affected by administration of TPA either before or after exposure to γ-rays. The mechanism of suppression by TPA of mutations induced by caffeine is discussed.     

The ultrastructural basis of the permeability of arterial endothelium to horseradish peroxidase

The thoracic aorta and basilar artery, in which the incidence of atherosclerosis is known to be different, were examined to elucidate the correlation between the structure of the intercellular cleft junction between adjacent endothelial cells and its permeability to HRP. Tannic acid or HRP in the vessel lumen passed through the intercellular clefts of the thoracic aorta into the subendothelial space, whereas in the basilar artery they were unable to penetrate beyond the tight junction of the intercellular clefts. Freeze-fracture replicas revealed that the tight junctions of the thoracic aorta consisted of one to two junctional strands in most areas of the cleaved planes, with discontinuities in some places, whereas those of the basilar artery consisted of a continuous belt-like meshwork of six anastomosing junctional strands on average. These observations confirm that the structure of endothelial junctions in arteries has a close correlation with the permeability of the intercellular clefts to HRP.