Determination of high-affinity oestrogen receptor sites in uterine supernatant preparations.
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ABSTRACT:
SUBMITTER: Mester J
PROVIDER: S-EPMC1179677 | biostudies-other | 1970 Dec
REPOSITORIES: biostudies-other
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Project description:A quantitative method was used to determine the concentration of high-affinity oestradiol-receptor sites in rat uterine supernatant preparations under various physiological conditions. Cyclic changes in concentration were observed during the oestrous cycle, with a maximum occurring in late dioestrus. The changes followed a similar pattern in endometrium and myometrium, although concentrations were higher in the former. In pseudopregnancy the concentration was initially low, rising to a maximum on the tenth day. In early pregnancy a high concentration of receptor was found to be associated with the developing placenta, but this declined in later stages of pregnancy. After ovariectomy or combined ovariectomy and adrenalectomy the receptor concentration remained at a constant low value that could be increased by treatment with oestradiol. The receptor concentration was considerably higher in immature than in adult uteri.
| S-EPMC1179678 | biostudies-other
Project description:Tamoxifen is a potent inhibitor of specific oestrogen-induced yolk protein synthesis by chicken liver. The oestradiol receptor in salt extracts of liver nuclei from oestrogen-treated chicks has a K(d) for oestradiol of 0.7+/-0.2nm. Tamoxifen and its metabolite, monohydroxytamoxifen, compete for binding to the salt-soluble nuclear receptor with K(i) values of 2.6 and 0.1nm respectively. The anti-oestrogens show much less inhibition of [(3)H]oestradiol binding when assays are carried out using intact nuclei. The competition by unlabelled oestradiol for [(3)H]oestradiol binding to receptor is identical in both salt extracts and intact nuclei. This suggests that intact nuclei contain components which bind anti-oestrogens, but not oestradiol. While tamoxifen and desmethyltamoxifen will readily dissociate from the salt-soluble nuclear oestrogen receptor, monohydroxytamoxifen does not dissociate under the conditions generally used for exchange assays. A modified assay was developed in which 60-70% of monohydroxytamoxifen-bound sites were shown to be exchangeable for [(3)H]oestradiol. Soluble receptor preparations were first incubated in a 1.7% charcoal suspension at 37 degrees C for 15min before assay of specific oestradiol binding. This technique was used in examining the effects of tamoxifen and monohydroxytamoxifen given in vivo on the nuclear oestrogen receptor concentration. Despite their 30-fold difference in binding affinity for the receptor, both anti-oestrogens increase nuclear receptor levels to about the same degree. When given with oestradiol, both compounds have the same apparent partial inhibitory effect on the oestrogen-induced increase in nuclear receptor. These data are consistent with the metabolic hydroxylation of tamoxifen before binding to the hepatic oestrogen receptor.
| S-EPMC1158596 | biostudies-other
Project description:The high-affinity triarylethylene anti-oestrogen H1285 [4-(NN-diethylaminoethoxy)-beta-ethyl-alpha-(p-hydroxyphenyl) -4'-methoxystilbene] was tritiated to high specific radioactivity (35 Ci/mmol). Competition experiments between [3H]H1285 and H1285 or oestradiol demonstrated that both compounds would compete with [3H]H1285 for oestrogen-specific binding sites in rat uterine cytosol. [3H]H1285 had at least 10 times the affinity for the receptor compared with oestradiol at the 50% competition level. [3H]H1285 appeared to have at least twice the association rate for the oestrogen receptor compared with [3H]oestradiol. In addition, the dissociation half-life (t1/2) of specific binding of [3H]H1285 to oestrogen receptors at 0 degrees C was about 220 h compared with a value of 60 h for [3H]oestradiol. Because of the extremely slow dissociation of [3H]H1285 from the oestrogen receptor, we were able to compare the sedimentation profiles of [3H]H1285-receptor complexes with those of [3H]oestradiol-receptor complexes in the presence of 0.4 M-KCl on 5-20% sucrose density gradients. [3H]Oestradiol-receptor complexes had a major peak at 4.4 S with a smaller peak at 5.6 S, whereas with [3H]H1285-receptor complexes the 5.6 S peak was always higher than the 4.4 S peak. There was significant variation between the dissociation behaviour at 20 degrees C of [3H]H1285-receptor complexes and [3H]oestradiol-receptor complexes pre-activated at 25 degrees C for 30 min in the presence and in the absence of 10 mM-sodium molybdate. The dissociation t1/2 of [3H]oestradiol-receptor complexes at 20 degrees C decreased from 1.5 h to 0.5 h when molybdate was present during heat treatment whereas the dissociation t1/2 for [3H]H1285-receptor complexes was 5 h for both conditions. These observations indicate that there are fundamental differences in the initial interaction of H1285 and oestradiol with the oestrogen receptor.
| S-EPMC1153286 | biostudies-other
Project description:BackgroundUterine fibroids are the most common non-malignant growths in women of childbearing age. They are associated with heavy menstrual bleeding and subfertility. Herbal preparations are commonly used as alternatives to surgical procedures.ObjectivesTo assess the benefits and risks of herbal preparations for uterine fibroids.Search strategyAuthors searched following electronic databases: the Trials Registers of the Cochrane Menstrual Disorders and Subfertility Group and the Cochrane Complementary Medicine Field, the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2008, Issue 3), MEDLINE, EMBASE, the Chinese Biomedical Database, the Traditional Chinese Medical Literature Analysis and Retrieval System (TCMLARS), AMED, and LILACS. The searches ended on 31st December 2008.Selection criteriaRandomised controlled trials comparing herbal preparations with no intervention, placebo, medical treatment or surgical procedures in women with uterine fibroids. We also included trials of herbal preparations with or without conventional therapy.Data collection and analysisTwo review authors collected data independently. We assessed trial risk of bias according to our methodological criteria . We presented dichotomous data as risk ratios (RR) and continuous outcomes as mean difference (MD), both with 95% confidence intervals (CI).Main resultsWe included two randomised trials (involved 150 women) with clear description of randomisation methods. The methodological risk of bias of the trials varied. There were variations in the tested herbal preparations, and the treatment duration was six months. The outcomes available were not the primary outcomes selected for this review, such as symptom relief or the need for surgical treatment; trials mainly reported outcomes in terms of shrinkage of the fibroids.Compared with mifepristone, Huoxue Sanjie decoction showed no significant difference in the disappearance of uterine fibroids, number of patients with shrinking of uterine fibroids or average volume of uterine fibroids, but less effective than mifepristone on reducing average size of uterus (mean difference 23.23 cm(3),95% confidence interval 17.85 to 28.61). There was no significant difference between Nona Roguy herbal product and GnRH agonist in average volume of uterine fibroids or size of uterus. No serious adverse effects from herbal preparations was reported.Authors' conclusionsCurrent evidence does not support or refute the use of herbal preparations for treatment of uterine fibroids due to insufficient studies of large sample and high quality. Further high quality trials evaluating clinically relevant outcomes are warranted.
| S-EPMC3155698 | biostudies-literature
Project description:IntroductionUterine fibroids (UFs) are benign, monoclonal tumours of the female genital tract that originate from the myometrium. They may be diagnosed in as many as 80% of women depending on the selected population. UFs depend mostly on steroid hormones. Elevated levels of oestrogens and progesterone are believed to be among the most important factors inducing their formation and growth. These facts suggest that oestrogen (ESR) and progesterone receptors are crucial in UF pathophysiology as well. Previous studies have shown that, in some populations, polymorphisms in ESR genes (e.g. PvuII) constitute an important risk factor for UFs.Material and methodsThe aim of our study was to investigate whether ESRα PvuII polymorphism is associated with an increased risk of UFs in Caucasian women of Polish origin. A total of 197 patients (114 UF-positive and 83 controls) were included in this retrospective cohort study. ESRα gene polymorphism PvuII (rs2234693) was assayed with PCR and restriction fragment length polymorphism (RFLP).ResultsOur study found no significant difference in the occurrence of ESR PvuII polymorphism between women with UFs and UF-free controls in the selected population.ConclusionsOur results did not indicate a significant association between ESRα gene PvuII polymorphism and the risk of UFs in Caucasian women of Polish origin. More studies and comparisons between races are necessary to clarify the role of ESRα in the development and progression of UFs.
| S-EPMC6372853 | biostudies-literature
Project description:Growth hormone regulates its biological properties via a sequential hormone-induced receptor homodimerization mechanism. Using a mutagenesis-scanning analysis of 81 single and 32 pairwise double mutations, we show that the hormone's two spatially distal receptor binding sites (Site1 and Site2) are allosterically coupled. These allosteric effects are focused among a relatively few residues centered around the interaction between Asp-116 of the hormone and Trp-169 of the receptor in Site2. A rearrangement of this interaction triggered by mutations in Site1 produces both a major conformation and energetic reorganization of Site2, surprisingly without a reduction in overall binding affinity. Additionally, the data suggest a change in the conformational dynamics of several groups in Site2 that appear to be important in defining the Site2 interaction. Changes in binding energy of the affected Site2 residues usually range in magnitude from 3- to 60-fold, but in one case are as large as 10(4).
| S-EPMC535364 | biostudies-literature
Project description:A method was developed for the determination of the number of specific oestradiol-binding sites in the nuclear fraction of oestrogen-sensitive tissues. The method is based on the exchange of [(3)H]oestradiol with non-labelled oestradiol that is bound to nuclear binding sites. The number of specific nuclear binding sites after the injection of 2.5mug of oestradiol, an amount sufficient to saturate all binding sites, is 1.6-1.7pmol per immature uterus. The number of sites occupied after an injection of physiological amounts of oestradiol (0.1mug) was 0.46pmol. The injection of oestradiol results in an increased number of nuclear binding sites in uterus and vagina, but has no effect on kidney or muscle. Injections of testosterone or progesterone failed to increase the number of uterine nuclear binding sites. This method permits an evaluation of the number of oestradiol-binding sites in the nuclear fraction of various tissues as a function of either endogenous oestradiol or non-labelled oestradiol administered by injection.
| S-EPMC1178412 | biostudies-other
Project description:IntroductionAn emerging hypothesis suggests that cytokines could play an important role in cancer as potential modulators of angiogenesis and leucocyte infiltration.MethodsA novel multiplexed flow cytometry technology was used to measure the expression of 17 cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 [p70], IL-13, IL-17, granulocyte colony-stimulating factor [CSF], granulocyte-macrophage CSF, IFN-gamma, monocyte chemoattractant protein [MCP]-1, macrophage inflammatory protein [MIP]-1beta, tumour necrosis factor [TNF]-alpha) at the protein level in 105 breast carcinoma. B lymphocyte, T lymphocyte and macrophage levels were determined by immunohistochemistry.ResultsFourteen of the 17 cytokines were expressed in breast carcinoma, whereas only nine cytokines could be detected in normal breast. Most cytokines were more abundant in breast carcinoma than in normal breast, with IL-6, IL-8, granulocyte CSF, IFN-gamma, MCP-1 and MIP-1beta being very abundant. IL-2, IL-6, IL-8, IL-10, IFN-gamma, MCP-1, MIP-1beta and TNF-alpha, and to a lesser extent IL-1beta and IL-13 exhibited levels of expression that were inversely correlated to oestrogen receptor and progesterone receptor status. Most cytokines were not correlated with age at cancer diagnosis, tumour size, histological type, or lymph node status. However, IL-1beta, IL-6, IL-8, IL-10, IL-12, MCP-1 and MIP-1beta were more abundant in high-grade tumours than in low-grade tumours. In addition, IL-8 and MIP-1beta were expressed to a greater degree in HER2-positive than in HER2-negative patients. The expression of most of the studied cytokines was correlated to levels of activator protein-1, which is known to regulate numerous cytokines. Overexpression of MCP-1 and MIP-1beta were linked to B lymphocyte, T lymphocyte and macrophage infiltration, whereas high levels of IL-8 were correlated with high macrophage content in tumour. Moreover, IL-8 positive tumours exhibited increased vascularization.ConclusionWe found that multiple cytokines were overexpressed in oestrogen receptor negative breast carcinoma, and that the three major cytokines--MCP-1, MIP-1beta and IL-8--were correlated with inflammatory cell component, which could account for the aggressiveness of these tumours.
| S-EPMC1851386 | biostudies-literature
Project description:The interaction of insulin with its receptor is complex. Kinetic and equilibrium binding studies suggest coexistence of high- and low-affinity binding sites or negative cooperativity. These phenomena and high-affinity interactions are dependent on the dimeric structure of the receptor. Structure-function studies of insulin analogs suggest insulin has two receptor binding sites, implying a bivalent interaction with the receptor. Alanine scanning studies of the secreted recombinant receptor implicate the L1 domain and a C-terminal peptide of the receptor alpha subunit as components of one ligand binding site. Functional studies suggest that the first and second type III fibronectin repeats of the receptor contain a second ligand binding site. We have used structure-directed alanine scanning mutagenesis to identify determinants in these domains involved in ligand interactions. cDNAs encoding alanine mutants of the holo-receptor were transiently expressed in 293 cells, and the binding properties of the expressed receptor were determined. Alanine mutations of Lys(484), Leu(552), Asp(591), Ile(602), Lys(616), Asp(620), and Pro(621) compromised affinities for insulin 2-5-fold. With the exception of Asp(620), none of these mutations compromised the affinity of the recombinant secreted receptor for insulin, indicating that the perturbation of the interaction is at the site of mutation and not an indirect effect on the interaction with the binding site of the secreted receptor. These residues thus form part of a novel ligand binding site of the insulin receptor. Complementation experiments demonstrate that insulin interacts in trans with both receptor binding sites to generate high-affinity interactions.
| S-EPMC2819479 | biostudies-other
Project description:In contrast with several earlier reports, cytosol from cockerel liver contains a significant concentration of a protein that binds oestradiol with high affinity. To demonstrate the activity, certain alterations in the conventional method of preparation of cytosol must be made. Homogenization in sucrose-containing buffer at pH 8.4 in the presence of proteinase inhibitors and rapid fractionation of the cytosol with (NH4)2SO4 enables demonstration of a single class of oestradiol-binding sites with a Kd of about 1 nM and specificity only for oestrogens. The concentration is about 300 sites per cell in liver from 2-week-old cockerels. Oestradiol treatment in vivo decreases the number of exchangeable cytosol oestradiol-binding sites by about 80% for 1--4h, after which time it is gradually restored. Gel filtration of the cytosol preparation in the presence of high salt concentrations reveals that most of the oestradiol-binding activity is in high-molecular-weight aggregates, but a mild trypsin treatment generates a specific binding protein with an approximate mol.wt. of 40 000. This protein may be an oestrogen receptor.
| S-EPMC1161059 | biostudies-other