tions. By means of a table of random numbers, the two lesions that would receive an application from tube A were determined (two of lesions 1, 2, 3, and 4). The other two lesions received an application from tube B. 15. After the two lesions that were to receive an application from tube A were determined, one was randomly selected to be a biopsy site; likewise, the two lesions that were to receive an application from tube B were selected randomly. Therefore, one of the two lesions from each treatment was scheduled to be biopsied but the code (tube A and tube B) was not broken until after biochemical analyses were completed. The two remaining lesions, one having received a glucocorticoid and one a control cream treatment, were not biopsied but were left to be visually evaluated 2 days later. 16. J. J. Voorhees, E. A. Duell, M. Stawiski, E. R.Harrell, Adv. Cyclic Nucleotide Res. 4, 11717. The surgically removed treated or control tissue used as starting material in this report is designated by several terms which are meant to be synonymous. These terms are lesional epithelium,-lesional epidermis, lesional tissue, and diseased tissue. By frozen section histology we estimate that keratinocytic epithelium (epidermis) occupies 80 to 95 percent of the specimen volume. The 80 to 95 percent range is the result of surgical technique, vertical lesional configuration, and the presence of non-keratinocytes (inflammation-associated cells, endothelial cells, fibroblasts, and collagen). Therefore, although 5 to 20 percent of the specimen volume is occupied by collagen and nonepithelial cells, the latter of which probably contain the arachidonic acid transformation cascade (Fig. 2), it seems probable that the data in Fig. I are derived mainly from lesional epithelium. 18.
The transport of retinoic acid in plasma was examined in vitamin A-deficient rats maintained on small doses of radioactively labelled retinoic acid. After ultracentrifugation of serum adjusted to density 1.21, most of the radioactivity (83%) was associated with the proteins of density greater than 1.21, and not with the serum lipoproteins. Gel filtration of the labelled serum on Sephadex G-200 showed that the radioactive label was associated with protein in the molecular-weight range of serum albumin. On polyacrylamide-gel electrophoresis almost all of the recovered radioactivity migrated with serum albumin. Similar esults were obtained with serum from a normal control rat given a single oral dose of [(14)C]retinoic acid. These findings indicate that retinoic acid is transported in rat serum bound to serum albumin, and not by retinol-binding protein (the specific transport protein for plasma retinol). Several tissues and the entire remaining carcase of each rat were extracted with ethanol-acetone to determine the tissue distribution of retinoic acid and some of its metabolites. The total recover of radioactive compounds in in the entire body of the rat was about 7-9mug, representing less than 5% or 10% respectively of the total administered label in the two dosage groups studied. The results confirm that retinoic acid is not stored in any tissue. Most of the radioactive material was found in the carcase, rather than in the specific tissues analysed. Two-thirds of the radioactivity in the carcase appeared to represent unchanged retinoic acid. Of the tissues examined, the liver, kidneys and intestine had relatively high concentrations of radioactive compounds, whereas the testes and fat-pads had the lowest concentrations.
Binding studies of thyroid hormone to submitochondrial fractions from rat liver suggest that the component responsible for high-affinity, low-capacity (saturable) binding of hormones arises from the inner mitochondrial membrane. The partially purified component, approximately 150,000 daltons, appears to be half protein and half lipid, largely phospholipids, tentatively identified as lecithin, phosphatidyl ethanolamine, and cardiolipin. A similar hormone-binding macromolecule was found in mitochondria from rabbit kidney, from human liver and kidney, and from rat kidney, myocardium, skeletal muscle, intestinal mucosa, whole small intestine, adipose tissue, and lung. It was absent from mitochondria of adult rat brain, spleen, and testis, organs calorigenically unresponsive to thyroid hormones injected in vivo, but was present in mitochondria from brains of rats 12 days old and younger. The organ distribution of the hormone-binding protein and its presence in neonatal brain mitochondria supports the biological relevance of the mitochondrial component as a thyroid hormone receptor.
The thyroid hormone, triiodothyronine, has been shown to be bound by the intranuclear chromatin protein associated with active DNA, where it is believed to stimulate transcription. Evidence exists that the thyroid hormones have direct action not only on nuclei, but also on mitochondria. Therefore, specific proteins that bind thyroid hormones in the mitochondria should be demonstrable.Mitochondria were isolated from homogenized rat livers by sedimentation through 0.25 M sucrose solution, followed by washing four times to free them of microsomes The many recent reports from the laboratories of Oppenheimer (8-15), , and de Groot (5-7) provide evidence of specific nuclear receptors for the thyroid hormones, thyroxine (T4) and triiodothyronine (TS), and suggest that the cell nucleus is a significant locus of hormone action. Indeed, as early as 1966, Siegel and Tobias demonstrated the nuclear localization of thyroid hormone by radioautography of tritium-labeled hormone added to cell cultures of human renal epithelial cells, grown in monolayer (31, 32), an observation which forecast the later developments. These authors showed a diminution of the number of tritium grains over the nucleus if actinomycin D had been added to the cell cultures, but an increase after addition of puromycin, a finding compatible with thyroid hormone action at the transcriptional level in the nucleus.Prior to and throughout the same period of time, however, Tapley and his associates (33-42) have been accumulating evidence that suggests direct thyroid hormone action upon the mitochondria of responsive cells. Binding of thyroid hormones by rnitochondrial membranes was confirmed by Tata, Ernster, and Suranyi, who reported microsomal and nuclear binding as well (43). Perhaps the most striking observation has been the demonstration of increased mitochondrial protein synthesis in as short an interval as 3 min after the addition of T4 or T3 to isolated rat liver mitochondria in vitro (35).Recently we have been studying the binding of T4 and T3 to cell proteins (23,24). The present communication concerns the finding of a protein fraction of the mitochondrial membrane which binds T4 and T3 in the physiological range, and is presumed related to hormonal effect upon energy metabolism.After isolation of mitochondria from rat liver and kidney, the binding of labeled hormones by intact mitochondria was studied, followed by examination of the mitochondrial matrix and membranes, and subsequently protein fractions extracted from the mitochondrial membranes.
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