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Effect of Quinoline-Type Antimalarial Drugs on the Binding of Oestradiol-17β and Progesterone by Rabbit and Human Uterine Cytosols
Abstract
Rabbit and human uterine cytosol, prepared and tested in phosphate buffer, bound less oestradiol-17β or progesterone than cytosol from the same source prepared and tested in Tris-HCl buffer. Dissociation constants were the same in both buffer systems, and the difference in binding was due to a difference in the number of binding sites. Three quinoline-type antimalarial drugs, chloroquine, quinine and mefloquine, and the quinoline derivative, 4-(4'-hydroxy-l'-methylbutylamino)-7-chloroquinoline, increased the steroid binding capacity of phosphate-buffered cytosol to that of Tris-buffered cytosol, the optimal concentration of quinoline derivative being 1.4–1.6 mM. Tris (50 mM) increased the binding capacity of phosphate-buffered cytosol to that of Tris-buffered cytosol. The effects of Tris and quinoline derivatives were not additive. By gel chromatography and sucrose density gradient centrifugation it was shown that the molecular size and sedimentation behaviour of the oestradiol and progesterone receptors were not affected by the quinoline derivatives. Two types of binding site are proposed, one requiring the presence of low molecular weight, basic compounds. The uterine levels of chloroquine attained by normal pharmacological doses of the drug are potentially capable of influencing the binding of oestradiol-17β and progesterone in the uterine cytosol.
Objective
To provide morphological descriptions of microphthalmia or anophthalmia in eight pythons using microcomputerized tomography (μCT), magnetic resonance imaging (MRI), and histopathology.Animals studiedSeven Burmese pythons (Python bivittatus) and one ball python (P. regius) with clinically normal right eyes and an abnormal or missing left eye.ProcedureAt the time of euthanasia, four of the eight snakes underwent necropsy. Hereafter, the heads of two Burmese pythons and one ball python were examined using μCT, and another Burmese python was subjected to MRI. Following these procedures, the heads of these four pythons along with the heads of an additional three Burmese pythons were prepared for histology.ResultsAll eight snakes had left ocular openings seen as dermal invaginations between 0.2 and 2.0 mm in diameter. They also had varying degrees of malformations of the orbital bones and a limited presence of nervous, glandular, and muscle tissue in the posterior orbit. Two individuals had small but identifiable eyes. Furthermore, remnants of the pigmented embryonic framework of the hyaloid vessels were found in the anophthalmic snakes. Necropsies revealed no other macroscopic anomalies.Conclusions
Eight pythons with unilateral left-sided microphthalmia or anophthalmia had one normal eye and a left orbit with malformed or incompletely developed ocular structures along with remnants of fetal structures. These cases lend further information to a condition that is often seen in snakes, but infrequently described.
Dilution at 0 degrees of rat liver cytosol incubated with [3H]triamcinolone acetonide provoked an enhanced binding of steroid-receptor complexes to nuclei. The explanation of this phenomenon was found to be an "activation" of the complexes. Dilution acted by decreasing the concentration of a cytosol inhibitor. This reaction was irreversible at 0 degrees: once activated the complexes could not be reversed to the nonactivated state by the addition of inhibitor. The presence of hormone was necessary, since hormone-free receptor molecules could not be activated by dilution. Removal of the inhibitor did not lead to activation of all complexes: after 24 h a "plateau" was attained where 55 to 70% of the complexes were activated. The inhibitor was shown to be a low molecular weight molecule by dialysis, Sephadex G-25 chromatography, ammonium sulfate precipitation, and ultrafiltration. Thus [3H]triamcinolone acetonide-receptor complexes present in a cytosol from which the inhibitor had been removed by Sephadex G-25 chromatography became spontaneously activated at low ionic strength and at 0 degrees. The inhibitor is not a steroid (at least of usual polarity) since it cannot be extracted by methylene chloride or adsorbed by activated charcoal. It is thermostable (resists to 30 min at 100 degrees). Its removal by incubation with a cation exchange resin suggests that it may be positively charged, however it is not complexed by EDTA. This inhibitor must be distinguished from a previously described inhibitor of steroid-receptor complexes binding to nuclei. The latter compound has been shown in various systems to be responsible for an artifactual saturation of nuclear acceptor by steroid-receptor complexes. It inhibits the binding to nuclear acceptors of already activated complexes and is probably a macromolecule. It is thus different from the low molecular weight activation inhibitor described in the present paper.
The degradation of cellular proteins in fibroblasts, both those of rapid and those of slow turnover rates, was inhibited by low concentrations of chloroquine or neutral red in the medium. Cells inhibited by chloroquine can be inhibited further by fluoride. Chloroquine was taken up by the fibroblasts and the concentration in the cells reached several hundred times that in the medium. Isopycnic fractionation studies showed that within the cells the chloroquine was concentrated in the lysosomes, and that these chloroquine-containing lysosomes had a lower equilibrium density than the lysosomes of untreated cells. Chloroquine, at concentrations attained inside the lysosomes, inhibited cathepsin B(1) but not cathepsin D. It is concluded that chloroquine impairs the breakdown of cellular proteins after these have entered the lysosome system, probably through inhibition of cathepsin B(1).
An assay for proteins in solution is described that depends on the conversion of Coomassie brilliant blue G250 in dilute acid from a brownish-orange to an intense blue color. As soon as the dye solution is mixed with a protein sample, the absorbance of the mixture can be measured. The absorbance of the solution is stable for 60–90 min at room temperature. The test can be used with a variety of proteins and polypeptides with molecular weights greater than 3000. The assay has high reproducibility and can detect less than 1.0 μg of albumin. The single reagent required is very stable, and free amino acids and several chemicals that interfere with the Lowry protein assay cause no interference. The test is adaptable to a micromethod, which is also described.
A patient with a benign hepatic neoplasm developing after treatment with estrogenic hormones is described. After excision, the neoplastic tissue was analyzed for the presence of cycosol estrogen and progeterone binding proteins. The noeplasm was classified as focal nodular hyperplasia of the liver and was demonstrated to contain high-affinity cytosol estrogen and progesterone hormone receptors. The estrogen0binding affinity of the neoplasm was three times greater than that of normal liver.
Further investigation of cytosol hormone receptors in estrogen associated hepatic neoplasms will be required to define the role of these binding proteins in the possible etiology of certain liver tumors.
The kinetics of mefloquine hydrochloride were studied in 20 healthy adult male subjects after single oral doses of 250, 500, 1,000, and 1,500 mg. Whole blood concentrations of the drug were measured periodically for 83 days after drug administration. The resulting concentration/time data were analyzed to determine the rates of absorption and elimination of the drug. There was considerable variation in all of the estimated kinetic parameters, but differences did not appear to be related to the administered dose. Absorption was slow after administration of a tablet of the drug (mean ka, 4.37/day). When the drug was given in an aqueous suspension it was more rapidly and more completely absorbed, as indicated by a mean Ka of 15.34/day and an area under the blood concentration/time curve 35% greater than with the same dose in tablet form. The volume of distribution was quite large (mean value for V/f 13.30 1/kg). The therapeutic efficacy and prolonged duration of prophylactic effect of a single dose of mefloquine was consistent with the finding of a mean whole blood half-life (t 1 2 of 13.89 days. There was considerable variation in the t 1 2 within each dose group, with a range of 6.48 to 22.65 days. Because of the wide individual variation in the kinetics of mefloquine, it is anticipated that occasional treatment failures may occur when the drug is used for single-dose therapy on a large scale.
The plasma derived proteins albumin and sex hormone binding globulin were studied for their effects on the measurement of estradiol receptor binding parameters. The influence of sex hormone binding globulin was found to be the major factor likely to lead to erroneous values. Testosterone prevents this interference.
Binding sites for estrogen receptor in isolated nuclei from several target and nontarget tissues are shown to be unsaturable, even when the nuclei have bound several times more receptor than found at maximal in vivo stimulation of uterus. Previous indications of saturability resulted from varying concentrations of total cytosol protein, which at higher levels depresses receptor binding. When cytosol protein is held constant, nuclear binding becomes strictly proportional to the total receptor concentration. Partially purified receptor also yields the same result. In addition, an excess of receptor charged with unlabeled estradiol does not compete with labeled estradiol-charged receptor for binding sites. In agreement with results from intact cells, we therefore conclude that the nuclear acceptor sites for estrogen receptor are present in large numbers and bind receptor with relatively low affinity.
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The effect of chloroquine on the binding of oestradiol-17β in the uterine cytosol of rabbit
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