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T C Spelsberg

Publications and source records attributed to T C Spelsberg.

At least 163 records · Page 9Linked to original sources
PubMed1981

Absence of high-affinity binding of progesterone (R 5020) in human placenta and fetal membranes.

Increased levels of maternal serum progesterone occur during the last stages of human gestation. The function of the high level of this steroid is unknown. The presence of a progesterone receptor in the placenta was investigated to determine whether progesterone action on the placenta might serve as one function of the high level of this steroid. Cytosol and nuclear fractions, derived from human placentae and fetal membranes, were examined for the presence of progesterone receptors by conducting exchange assays, using tritiated R 5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) as the radiolabelled ligand. High-affinity, low-capacity binding, characteristic of steroid receptors, was estimated as the difference between binding of radiolabelled ligand in the presence of no unlabelled ligand and that in the presence of a 100-fold excess of unlabelled ligand. These exchange assays were conducted during a 24-hour period at 0 degrees C, to allow maximal stability of the receptor, and during a 3-hour period at 20 degrees C, to allow the rapid exchange of radiolabelled ligand for any bound endogenous progesterone. The assays of all fractions showed no specific binding of the R 5020, thus indicating the absence of progesterone receptors in the cytosol and nuclei of the human placenta and the fetal membranes.

Extraembryonic Membranes↗
PubMed1980

Factors affecting the binding of chick oviduct progesterone receptor to deoxyribonucleic acid: evidence that deoxyribonucleic acid alone is not the nuclear acceptor site.

Studies on the interaction of the chick oviduct progesterone-receptor complex (P-R) with various nuclear components revealed a variable, nonsaturable binding of P-R to pure deoxyribonucleic acid (DNA). In contrast, a receptor-dependent, saturable, high level of binding of P-R was observed with a nonhistone protein-DNA complex called nucleoacidic protein (NAP). Three categories of factors were identified which affected the binding of P-R to the DNA. These were (1) the conditions of the binding assay, (2) the properties of the receptor, and (3) the state of the DNA. The conditions in the binding assay which affect DNA binding are the choice of the blanks, the salt concentration, and the pH of the assay. The receptor preparations display their own characteristic levels of binding to native DNA. The basis of this DNA binding capacity by each preparation is unknown. Lastly, the purity and the integrity of the DNA itself determine the level of binding of the P-R. Protein impurities, moderate degradation of the DNA by enzymatic or physical fragmentation, and ultraviolet (UV) light treatment greatly enhance the receptor binding to the DNA. The extent of binding to DNA depends on the degree of damage. Interestingly, totally denatured (single-stranded) DNA displays little or no binding of the P-R. Seasonal differences which are observed for the binding of P-R to chromatin in vivo and in vitro and to NAP in vitro do not occur with DNA whether it is undamaged or damaged. It is concluded from these studies that under controlled conditions and by using DNA preparations as native as possible, minimal binding of P-R to pure DNA occurs. The numerous reports in the literature describing marked binding of the steroid-receptor complex to DNA may well be due to conditions described in this paper. Further, it is concluded that native or partially degraded DNA alone does not appear to represent the native nuclear acceptor sites for the chick oviduct P-R. In contrast, the DNA-nonhistone protein (acceptor protein) complexes do show characteristics of the native-like acceptor sites.

Animals↗
PubMed1980

Identification of biologically active and inactive steroid receptors.

Steroids enter target cells and bind to specific receptor proteins. These complexes translocate to the nuclei, bind to the chromatin, and alter gene expression. Recently, inactive progesterone receptors of the chick oviduct have been identified in this laboratory during the late winter which are not capable of translocating and binding to nuclear acceptor sites either in vivo or in a cell free assay. During this period the oviduct remains unresponsive to the steroid. The nuclear binding activity does return at the end of the season with a corresponding return of oviduct responsiveness to the steroid. Analysis of the active and inactive receptors of progesterone reveals no difference in sedimentation rates in high salt or in the affinity of the steroid for the receptor. The tissue levels of the inactive receptor, however, are about-one-half those of the active receptor. Quantitative analysis of the molecular species of the progesterone receptor separated by isoelectric focusing reveals two species for the active receptor preparations (an A species focusing at a pH of 7 and a B species focusing at a pH of 6). The A species was absent in the active receptor preparations which explains the lower amounts of total receptor in this group. Further studies have shown inactive progesterone receptors in the undeveloped oviducts and in the oviducts of estrogen withdrawn chicks. In these instances, the B species of the receptor is missing. The results suggest: (1)a novel regulation of steroid action may exist which acts by modulating the levels of one of the two receptor species (or possible subunits of a dimer); (2)the presence of a steroid receptor does not necessarily reflect that the receptor is functional; and (3)the biological activity of a steroid may be assessed via cell free nuclear binding assays or via analysis of the molecular species.

Animals↗
PubMed1980

Isolation and characterization of the estrogen receptor in hen oviduct: evidence for two molecular species.

Isolation of the estrogen receptor was undertaken for comparison with the progesterone receptor with respect to seasonal variations in receptor level and function and the specificity of nuclear binding sites in the hen oviduct. The estrogen receptor in the chick and hen oviduct has been only partially characterized, with conflicting or incomplete information on its properties. We have isolated a binding protein and have partially purified it by ammonium sulfate precipitation. The binding protein displays a single, high affinity class of sites (Kd similar to 10(-10) M) with a low capacity (35 fmol/mg protein) for 17beta-estradiol, shows a marked specificity for estrogenic compound, and is tissue specific. The protein sediments at approximately 8S in low salt and at approximately 4S in high salt conditions. The protein elutes from molecular sieve chromatography (agarose-0.5 m) in high salt conditions (0.3 M KCI) in a molecular weight range of approximately 60,000. DEAE chromatography and isoelectric focusing suggest that there are two molecular species, one focusing at pH 6.8 and the other at pH 7.3. Estrogen complexed to this protein will bind to nuclear acceptor sites in a cell-free assay in which free estrogen, estrogen bound to serum proteins, and [3H]estradiol receptor exchanged with a 100-fold excess of cold estradiol do not bind. Binding to nuclei is saturable and displaceable by an excess of nonradioactive estrogen receptor complexes under conditions of constant protein concentration. The above results represent the most detailed characterization to date of the cytosol estrogen receptor of hen oviduct This is the first demonstration of saturable, receptor-dependent, cell-free binding to target organ nuclei.

Animals↗
PubMed1980

Circannual rhythms in steroid receptor concentration and nuclear binding in the chick oviduct.

For 3 yr, 1-yr synchronized circannual rhythms were found for the chick oviduct weights, amounts of soluble protein, concentrations of the progesterone receptor in this organ, and capacity of this steroid-receptor complex in cytosol in bind in vitro to deoxyribonucleoprotein acceptor sites. By fitting the data with 1-yr cosine curves, statistically significant circannual rhythms were described for all of these rhythms, with high values occurring in the late summer and low ones in the late winter. Interestingly, no statistically significant rhythms were described for the binding of the progesterone-receptor complex to pure DNA. By analyzing both receptor and nuclear protein preparations isolated at different times of the year, changes in the receptor were found to underlie the rhythms in nuclear binding in vitro. Studies in vivo revealed similar rhythms for the nuclear translocation and binding of both progesterone and estrogen and for the progesterone-induced changes in RNA polymerase II activity. In each instance, marked reductions in nuclear binding and steroid-induced changes in the polymerase activity occurred in the late winter. Blood levels of the 3H-labeled steroids did not vary throughout the year, so differences in whole body metabolism and disposition of the steroids were ruled out as factors in the in vivo rhythms. These results support the validity of the cell-free binding assays and the hypothesis that certain protein-DNA complexes, and not pure DNA, represent the natural acceptor sites for the progesterone receptor. Furthermore, these rhythms emphasize the fact that steroid receptors are not constant entities but change in both amount and function. Although the exact cause of these rhythms in the progesterone receptor is not understood, a rhythm in estrogen action on the oviduct is strongly suggested. These rhythms in the steroid receptors may play a role in seasonal reproduction and molting in animals.

Animals↗
PubMed1979

Analysis of the molecular species of the chick oviduct progesterone receptor using isoelectric focusing.

Conditions are described for the preparative isoelectric focusing in flat beds of Sephadex of the progesterone receptor from the chick oviduct. The method allows the fractionation of the receptor into two molecular species, one focusing at pI 6 and the other at pI 7 with good purification and recovery. The pI 6 and pI 7 receptor species were purified 2- and 26-fold, respectively. The assaying of the focused fractions with the charcoal binding method provides an accurate identification and quantitation of the [3H]progesterone receptor. The method is reproducible in recovery, quantitation, and resolution of the two receptor species. The receptor with an apparent pI of 6 sediments at approximately 4 S on linear sucrose gradients, while the receptor with an apparent pI of 7 sediments at approximately 3.5 S. On the basis of the sedimentation values and elution patterns from diethylaminoethyl (DEAE) chromatography, the pI 6 component is equivalent to the "B" receptor species and the pI 7 component is equivalent to the "A" receptor species described previously [schrader, W, T., 7 O'Malley, B. W. )1972) J. Biol. Chem. 241, 51--59].

Animals↗
PubMed1979

Circannual rhythms in progesterone receptor levels and functions.

Studies in this laboratory on the nuclear binding sites (acceptors) for progesterone receptor in the developed chick oviduct have resulted in the detection of seasonal variations in the levels and functions of the receptor. Cytosol preparations obtained from the chick oviducts during the winter/spring period between January and May display reduced receptor levels as well as a loss of the capacity of the receptor to bind to nuclear "acceptor" sites in vitro. The binding of [3H]P-R to whole chromatin or purified acceptor proteins reannealed to DNA display the same rhythm. No such rhythm is detected for the binding of P-R to pure DNA. Computer analysis of the data, using least squares method to fit the data to cosine curves, shows a significant fit indicating a circannual rhythm in P-R binding to the acceptor protein-DNA complex but not to pure DNA. The nuclear binding in vivo, achieved by injecting [3H]progesterone into the wing vein and analyzing the radioactivity localized in the oviduct nuclei, also displays a similar rhythm. These results support that native nuclear acceptor sites for progesterone in the chick oviduct represent protein-DNA complexes and not pure DNA. The failure of P-R to bind the nuclear acceptor sites in vivo and in vitro during this period can be explained by the two subunit hypothesis of Schrader and O'Malley, whereby one of the two subunits is absent or inactive during this period.

Animals↗
PubMed1978

Visualization of nonfunctioning adrenal adenomas with iodocholesterol: possible relationship to subcellular distribution of tracer.

[131I] 19-iodocholesterol (I-131 C) correctly located adrenocortical adenomas in four patients who had no clinical or biochemical evidence of excessive steroid production. Three of the four "nonfunctioning" adenomas showed significant quantities of lipid histologically. To clarify the discordance between uptake and adrenal-steroid excretion, the subcellular location of I-131 C was studied. Normal rats and rats treated with ACTH or aminogluthimide (AG) were injected intravenously with I-131 C or [3H] cholesterol (H-3 C) and killed after three days. The homogenized adrenals were subjected to subcellular fractionation. Treatment with AG increased both the amount and the percentage of both I-131 C and H-3 C contained in the lipid fraction. ACTH treatment decreased H-3 C content but did not change I-131 C content in the lipid layer, suggesting an impairment of I-131 C mobilization from lipid droplets. The data demonstrate that excess steroid production is not necessary for I-131 C uptake and provide an explanation why certain biochemically nonfunctioning adrenocortical adenomas are visualized with I-131 C.

Adenoma↗
PubMed1977

The binding of benzo(alpha)pyrene and N-methyl-N'-nitro-N-nitrosoguanidine to subnuclear fractions of AKR mouse embryo cells in culture.

The marked localization of a carcinogenic polycyclic aromatic hydrocarbon, benzo(alpha)pyrene, and its metabolites and a carcinogenic alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine, to a specific subnuclear fraction (fraction I) from AKR-2B mouse embryo cells in culture is described. Fraction I is isolated by sucrose gradient centrifugation of sheared nuclei from cells exposed to the carcinogens. The association of tritiated benzo(alpha)-pyrene to fraction I consisted of loosely associated radioactivity which is extractable by organic solvents, and of tightly bound (termed "covalently" bound) radioactivity which is not extractable by organic solvents. Increases in the extent of metabolism of benzo(alpha)pyrene and in the amount of "covalently" bound radioactivity occur with increasing periods of incubation of the cells with the labelled carcinogen. This observation, together with the fact that these increases are dramatically reduced by inhibiting polycyclic aromatic hydrocarbon metabolism (using the inhibitor 7,8-benzo-flavone), suggests that a time-dependent metabolism of benzo(alpha)pyrene is required for "covalent" binding to muclear material. Data are presented suggesting that a two-step reaction may be involved in the binding of benzo(alpha)pyrene to subnuclear macromolecules. The fraction I localization of such structurally diverse chemical carcinogens as benzo(alpha)pyrene and N-methyl-N'-nitro-N-nitrosoguanidine suggests that this fraction may localize all species of chemical carcinogens and that this localization may be involved in the chemically induced malignant transformation of cells.

Animals↗
PubMed1977

Feedback regulation of vitamin D metabolism by 1,25-dihydroxycholecalciferol.

Many factors influence the production of 1,25(OH)2D3 (1,25-dihydroxycholecalciferol) by the kidney. One important factor seems to be feedback regulation by 1,25(OH)2D3 itself. Administration of 1,25(OH)2D3 to vitamin D-deficient chicks abolishes renal 25(OH)D3(25-hydroxycholecalciferol)1-hydroxylase activity and induces the appearance of 25(OH)D3 24-hydroxylase activity. It is likely that these effects are mediated via a nuclear effect, as they are prevented by pretreatment with actinomycin D and alpha-amanitin. Further, 1,25(OH)2D3 has a marked effect on gene transcription in the kidney cell, as assessed by measurement of RNA polymerase activities. RNA polymerase I and II activities are 80-90% inhibited by 12.5nmol of 1,25(OH)2D3 within 30min of subcutaneous administration, indicating an immediate and massive decrease in total gene transcription. By 4h RNA polymerase II activity has returned to control values, but RNA polymerase I activity is markedly enhanced. These results are consistent with the view that regulation of cholecalciferol metabolism in the kidney is associated with an effect of the active metabolite on the kidney nucleus.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗