Steroids and hematopoiesis. III. The response of granulocytic and erythroid colony-forming cells to steroids of different classes. (1/71)

Selected androgenic and nonandrogenic steroids enhance in vitro granulocytic and erythroid colony formation by mouse marrow cells, but do so by influencing either different target cells or cells in different states of cell cycle. Etiocholanolone, a naturally occurring nonandrogenic testosterone metabolite, permits cells not in active cycle to respond to colony-stimulating factor or erythropoietin. Fluoxymesterone, a synthetic androgen, appears to enhance colony growth by increasing the responsiveness of target cells to tropic stimuli. The majority of cells responding to this androgen are in active DNA synthesis. Direct comparison, however, of etiocholanolone-dependent erythroid or granulocytic colony-forming cells demonstrates nonidentity of the target cells. Thus colony-forming units responding to different classes of steroids are in different states of cell cycle and are physically separable. The enhancement of the in vitro response of colony-forming cells to regulating hormones by steroids such as etiocholanolane suggests a mechanism by which such agents may be therapeutically effective in certain cases of marrow failure in man.  (+info)

Molecular cloning, expression and characterization of a monkey steroid UDP-glucuronosyltransferase, UGT2B19, that conjugates testosterone. (2/71)

Although enzymatic processes involved in the formation of active steroids are well known, less information is available about the enzymes responsible for the metabolism of these hormones. Moreover, the expression of these catabolic enzymes, which include UDP-glucuronosyltransferases, may play a role in the regulation of the level and action of steroid hormones in steroid target tissues. Previous studies have shown that the cynomolgus monkey contains high levels of circulating androgen glucuronides, indicating that it represents the best animal model to study the glucuronidation of steroids in extrahepatic tissues. Two cDNA libraries were constructed from monkey liver and prostate mRNA, and a novel UDP-glucuronosyltransferase UGT2B cDNA, UGT2B19, was isolated from both libraries. The UGT2B19 cDNA is 2108 bp in length and contains an open reading frame of 1584 bp encoding a protein of 528 residues. The UGT2B19 cDNA clone was transfected into HK293 cells and a stable cell line expressing UGT2B19 protein was established. The activity of UGT2B19 on 3alpha-hydroxy and 17beta-hydroxy positions of steroids was demonstrated. The enzyme also conjugates xenobiotics including eugenol, 1-naphthol and p-nitrophenol. Kinetic analysis revealed that UGT2B19 glucuronidates steroids with Km values of 1.6, 2.6 and 4.3 microm for testosterone, etiocholanolone and 5beta-androstane-3alpha,17beta-diol, respectively. UGT2B19 transcript was detected, by specific reverse transcriptase-PCR analysis in the liver, ovary, prostate, colon, spleen, kidney, pancreas, brain, cerebellum, mammary gland and epididymis. The molecular characterization of simian UGT2B19 demonstrates relevance of using monkey as an animal model to study and understand steroid glucuronidation in extrahepatic target tissue.  (+info)

Structure and activity of the murine type 5 17beta-hydroxysteroid dehydrogenase gene(1). (3/71)

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) play a crucial role in the control of active sex steroid intracellular levels. Seven types of 17beta-HSD have been described. In this study, we report the cloning and characterization of the mouse type 5 17beta-HSD belonging to the aldo-keto reductase superfamily, in contrast with types 1, 2, 3, 4, 6, and 7 17beta-HSD which belong to the short-chain alcohol dehydrogenase family. The gene spans 16 kb and contains 9 exons separated by 8 introns. Primer extension analysis identified a major transcription start site beginning 50 nucleotides upstream from the ATG initiation codon. Northern blot analysis showed a high mRNA expression level in the liver and a weaker signal in the kidney. To determine more precisely the substrate specificity of the enzyme, we established a stable cell line expressing mouse type 5 17beta-HSD in transformed human embryonic kidney (293) cells. The transfected cell line preferentially catalyzes the transformation of 4-androstenedione (4-dione) and androstanedione (A-dione) into testosterone (T) and dihydrotestosterone (DHT), respectively. This data is somewhat in contradiction with a previous study that described the enzyme as estradiol 17beta-dehydrogenase. Our results indicate that the rate of transformation of estradiol (E(2)) to estrone (E(1)) represents only 1% of the rate of transformation of 4-dione to T. Mouse type 5 17beta-HSD shares 76% amino acid sequence identity with human type 5 17beta-HSD; 71%, 76%, 76% with rat 3alpha-HSD and human types 1 and 3 3alpha-HSDs, respectively; and 71%, 69% and 77% with mouse, rat and human 20alpha-HSD, respectively.  (+info)

Membrane perturbation: studies employing a calcium-sensitive dye, arsenazo III, in liposomes. (4/71)

A metallochromic dye, arsenazo III [2,7-bis-(2-arsonophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid], has been incorporated into the aquenous interspaces of multilamellar liposomes. multilamellar liposomes. Addition of Ca produced no shift in the absorbance spectrum of dye captured by liposomes, whereas disruption of liposomes by Triton X-100, followed by Ca, produced the spectrum chracteristic of the dye-Ca complex: evidence of latency. Addition of excess ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) reversed the spectal shift. Differences between spectra obtained in this sequence yielded dye efflus. To measure Ca efflux, difference spectra (+/-EGTA) were obtained from cationic liposomes containing Ca after detergent lysis (sensitivity less than 10 mmol/ml). Since liposomes were impermeable either to dye or Ca until perturbed, it was possible to test a variety of membrane-active steroids (diethylstilbesterol, deoxycorticosterone, etiocholanolone) for their capacity to provoke dye efflux from liposomes; preincorporation of cortisol stablized liposomes against dye leak. Immunoglobulin-coated liposomes containing dye were taken up by phagocytes of Mustelus canis, and phagocytic vacuoles stained red-purple after ingestions. Liposomes containing the calcium-sensitive dye constitute a simple, accurate means for determining membrane perturbation and Ca fluxes; their uptake by cells or organelles remains to be exploited further.  (+info)

Hormone profiles in hormone-dependent cancers. (5/71)

Studies on the relationship of urinary excretion of androgen metabolites and estrogens to the natural history of breast cancer are reviewed. The importance of distinguishing between "within-population" studies (i.e., cancer patients versus normal controls) and "between populations" studies (i.e., low-risk versus high-risk populations) is emphasized, and it is pointed out that "qualitative" agreement (i.e. the same direction of differences) between the two types of studies must be present in order to implicate a hormonal parameter as a determinant of the natural history of breast cancer. For reasons detailed in this paper, it is concluded that the reported relationship of low urinary androgen metabolite excretion to increased risk of developing breast cancer and poor response to adrenalectomy or hypophysectomy and the validity of the "estriol hypothesis," namely, that a high urinary ratio of estriol to estrone-plus-estradiol in early life is protective against subsequent development of breast cancer, are both dubious. A new hypothesis concerning the relationship of estrogens to breast cancer risk is presented: "A period of of time, prior to age 30, during which the amount of biological availability of active estrogens' (i.e., estrone and estradiol) is diminished, protects against subsequent development of cancer." This hypothesis is shown to be compatible with the epidemiological and biochemical data. Reports concerning the influence of nutrition on endocrine parameters are reviewed. Inanition and obesity have been shown to alter steroid metabolism but it is not known whether nutritional "microdifferences" (i.e., differences between populations or individuals that are due to cultural, geographic, or socioeconomic factors, but that fall within the range of "normal" or adequate nutrition) can also alter steroid metabolism.  (+info)

Feasibility of on-line supercritical fluid extraction of steroids from aqueous-based matrices with analysis via gas chromatography-mass spectrometry. (6/71)

The solubility of testosterone, boldenone, androstenone, etiocholanolone, and epitestosterone are measured in pure supercritical CO2. Testosterone exhibited the highest solubility in supercritical CO2. The solubility of all steroids except epitestosterone increased by one order of magnitude with increasing pressure from 100 to 400 atm. Epitestosterone had the lowest solubility in supercritical CO2 and its solubility was not affected by pressure. The extraction efficiency of steroids from an aqueous saline environment exceeded 95%. Because of the partial solubility of water in supercritical CO2, the addition of a moisture trap after the aqueous vessel is necessary to prevent the plugging and deterioration of the gas chromatographic (GC) column. It is demonstrated that on-line supercritical fluid extraction-GC-mass spectrometry is feasible for the quantitative extraction and analysis of steroids from both saline and urine solutions. However, it is determined that the adsorbent vessel filled with Hydromatrix is not sufficient to trap all the moisture, and after 3 to 4 extractions, the GC column efficiency lowered.  (+info)

Diethylstilbestrol regulates trophoblast stem cell differentiation as a ligand of orphan nuclear receptor ERR beta. (7/71)

The orphan nuclear receptor ERR beta is expressed in undifferentiated trophoblast stem cell lines and extraembryonic ectoderm, and genetic ablation of ERR beta results in abnormal trophoblast proliferation and precocious differentiation toward the giant cell lineage. Here, we show that the synthetic estrogen diethylstilbestrol (DES) promotes coactivator release from ERR beta and inhibits its transcriptional activity. Strikingly, treatment of trophoblast stem cells with DES led to their differentiation toward the polyploid giant cell lineage. In addition, DES-treated pregnant mice exhibited abnormal early placenta development associated with an overabundance of trophoblast giant cells and an absence of diploid trophoblast. These results define a novel pathway for DES action and provide evidence for steroidlike control of trophoblast development.  (+info)

Steroid induction of delta-aminolevulinic acid synthase and porphyrins in liver. Structure-activity studies and the permissive effects of hormones on the induction process. (8/71)

Quantitative aspects and structure-activity relationships of the inducing effects of natural steroids on delta-aminolevulinic acid (ALA) synthase and porphyrins have been investigated in monolayer cultures of chick embryo liver cells maintained in a serum-free medium as well as in the chick embryo liver in ovo. Many 5 alpha and 5 beta metabolites of neutral C-19 and C-21 hormones and hormone precursors stimulated porphyrin formation and ALA-synthase induction in the cultured liver cells as we have previously described. In these inducing actions a number of 5 beta epimers (A:B cis) were found to be more potent than their corresponding 5 alpha epimers (A:B trans). The structure-activity relationship between 5 beta and 5 alpha steroid epimers with respect to ALA-synthase induction in culture was also found to prevail with respect to induction of this enzyme in chick embryo liver in ovo. Hemin in concentrations of 2 x 10(-7) M inhibited steroid induction of porphyrin formation, and CaMgEDTA enhanced the responsiveness of the cultured liver cells to steroids by approximately 10 times. The addition of insulin, or insulin plus hydrocortisone or insulin plus hydrocortisone plus triiodothyronine, was important for the maintenance of protein synthesis and essential for maximal expression of the ability of steroids to induce porphyrins and ALA-synthase in the "permissive" effect which insulin, hydrocortisone, and triiodothyronine exert on allylisopropylacetamide induction of porphyrins and ALA-synthase also extends to the induction process which is elicited by natural steroids. These findings also strongly suggest that the regulation of hepatic porphyrin-heme biosynthesis by endogenous as well as exogenous chemicals is significantly influenced by the internal hormonal milieu.  (+info)

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