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Sample records for progesterone receptors affinity

  1. Progesterone increases nitric oxide synthesis in human vascular endothelial cells through activation of membrane progesterone receptor-α.

    Science.gov (United States)

    Pang, Yefei; Dong, Jing; Thomas, Peter

    2015-05-15

    Progesterone exerts beneficial effects on the human cardiovascular system by inducing rapid increases in nitric oxide (NO) production in vascular endothelial cells, but the receptors mediating these nongenomic progesterone actions remain unclear. Using human umbilical vein endothelial cells (HUVECs) as a model, we show that progesterone binds to plasma membranes of HUVECs with the characteristics of membrane progesterone receptors (mPRs). The selective mPR agonist Org OD 02-0 had high binding affinity for the progesterone receptor on HUVEC membranes, whereas nuclear PR (nPR) agonists R5020 and medroxyprogesterone acetate displayed low binding affinities. Immunocytochemical and Western blot analyses confirmed that mPRs are expressed in HUVECs and are localized on their plasma membranes. NO levels increased rapidly after treatment with 20 nM progesterone, Org OD 02-0, and a progesterone-BSA conjugate but not with R5020, suggesting that this progesterone action is at the cell surface and initiated through mPRs. Progesterone and Org OD 02-0 (20 nM) also significantly increased endothelial nitric oxide synthase (eNOS) activity and eNOS phosphorylation. Knockdown of mPRα expression by treatment with small-interfering RNA (siRNA) blocked the stimulatory effects of 20 nM progesterone on NO production and eNOS phosphorylation, whereas knockdown of nPR was ineffective. Treatment with PI3K/Akt and MAP kinase inhibitors blocked the stimulatory effects of progesterone, Org OD 02-0, and progesterone-BSA on NO production and eNOS phosphorylation and also prevented progesterone- and Org OD 02-0-induced increases in Akt and ERK phosphorylation. The results suggest that progesterone stimulation of NO production in HUVECs is mediated by mPRα and involves signaling through PI3K/Akt and MAP kinase pathways. Copyright © 2015 the American Physiological Society.

  2. Progesterone receptor modulators in breast cancer

    OpenAIRE

    WIEHLE, Ronald D.

    2015-01-01

    Breast cancer has been treated successfully with selective estrogen receptor antagonists (SERMs) such as tamoxifen, receptor-depleting agents such as fulvestrant, and aromatase inhibitors such as anastrozole. Selective progesterone receptor modulators (SPRMs or PRMs) have not been studied as much and are currently under investigation for inhibition of mammary carcinogenesis in animal models and breast cancer prevention trials in women. They might follow tamoxifen and aromatase inhibitors in t...

  3. Progesterone Receptor Scaffolding Function in Breast Cancer

    Science.gov (United States)

    2012-10-01

    leiomyoma cells in response to RU486 revealed little overlap [101]. PR-A and PR-B are most often co-expressed in the same tissues, and cells that... leiomyoma cells. PLoS One 7 (2012) e29021. [102]P.A. Mote, S. Bartow, N. Tran, C.L. Clarke, Loss of co-ordinate expression of progesterone receptors

  4. Expression of Estrogen and Progesterone Receptors among ...

    African Journals Online (AJOL)

    Study design: This is a descriptive study to detect the level of Estrogen (ER) and Progesterone (PR) receptors in a sample of biopsies from Sudanese women with breast cancer presented at Khartoum teaching Hospital Material and Methods: Forty biopsies from breast cancer patients were examined with immunostaining

  5. Regulation of uterine progesterone receptors by the nonsteroidal anti-androgen hydroxyflutamide

    International Nuclear Information System (INIS)

    Chandrasekhar, Y.; Armstrong, D.T.

    1991-01-01

    The authors have recently reported that the anti-androgen hydroxyflutamide causes delayed implantation and exhibits antideciduogenic activity in the rat. The present experiments were conducted to examine whether hydroxyflutamide binds to the uterine progesterone receptors and/or alters the progesterone binding sites in the uterus. Cytosol and nuclear fractions from decidualized rat uterus were incubated with [3H]-R5020 without or with increasing concentrations of radioinert R5020, RU486, dihydrotestosterone, or hydroxyflutamide. From the log-dose inhibition curves, the relative binding affinity of both hydroxyflutamide and dihydrotestosterone was less than 0.1% and 2%, compared with R5020 (100%) for displacing [3H]-R5020 bound to uterine cytosol and nuclear fractions, respectively. Injection of estradiol-17 beta (1 microgram/rat) to ovariectomized prepubertal rats induced a 1.85-fold increase in uterine weight by 24 h. Hydroxyflutamide at 2.5 or 5.0 mg did not significantly alter the estrogen-induced increase in uterine weight. Compared to vehicle alone, estrogen induced an approximately 5-fold increase in uterine cytosolic progesterone binding sites. Hydroxyflutamide at both 2.5- and 5.0-mg doses significantly attenuated the estrogen-induced elevation in uterine progesterone binding sites. These studies demonstrate that hydroxyflutamide does not bind with high affinity to progesterone receptors, but suppresses the estrogen-induced elevation in progesterone receptor levels in the uterus

  6. Progesterone modulates the LPS-induced nitric oxide production by a progesterone-receptor independent mechanism.

    Science.gov (United States)

    Wolfson, Manuel Luis; Schander, Julieta Aylen; Bariani, María Victoria; Correa, Fernando; Franchi, Ana María

    2015-12-15

    Genital tract infections caused by Gram-negative bacteria induce miscarriage and are one of the most common complications of human pregnancy. LPS administration to 7-day pregnant mice induces embryo resorption after 24h, with nitric oxide playing a fundamental role in this process. We have previously shown that progesterone exerts protective effects on the embryo by modulating the inflammatory reaction triggered by LPS. Here we sought to investigate whether the in vivo administration of progesterone modulated the LPS-induced nitric oxide production from peripheral blood mononuclear cells from pregnant and non-pregnant mice. We found that progesterone downregulated LPS-induced nitric oxide production by a progesterone receptor-independent mechanism. Moreover, our results suggest a possible participation of glucocorticoid receptors in at least some of the anti-inflammatory effects of progesterone. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Contraceptive applications of progesterone receptor modulators.

    Science.gov (United States)

    Chabbert-Buffet, Nathalie; Ouzounian, Sophie; Kairis, Axelle Pintiaux; Bouchard, Philippe

    2008-09-01

    Currently developed progesterone receptor modulators (PRMs) are steroid-derived compounds with mild or potent antiprogestin activity. PRMs may exert a contraceptive activity by different mechanisms such as blockade of ovulation and endometrial desynchronization. Their potential clinical applications are manifold and are very promising in major public health areas, including emergency contraception, long term oestrogen-free contraception (administered alone, or in association with a progestin-only pill to improve bleeding patterns), endometriosis and myoma treatment. The mechanisms of their anti-ovulatory effects and of the endometrial modifications elicited during long term PRM treatment are still not fully elucidated. In future clinical applications, PRMs will be administered orally, via intrauterine systems or vaginal rings.

  8. Selectivity in progesterone and androgen receptor binding of progestagens used in oral contraceptives

    International Nuclear Information System (INIS)

    Kloosterboer, H.J.; Vonk-Noordegraaf, C.A.; Turpijn, E.W.

    1988-01-01

    The relative binding affinities (RBAs) of four progestational compounds (norethisterone, levonorgestrel, 3-keto-desogestrel and gestodene) for the human progesterone and androgen receptors were measured in MCF-7 cytosol and intact MCF-7 cells. For the binding to the progesterone receptor, both Org 2058 and Org 3236 (or 3-keto-desogestrel) were used as labelled ligands. The following ranking (low to high) for the RBA of the nuclear (intact cells) progesterone receptor irrespective of the ligand used is found: norethisterone much less than levonorgestrel less than 3-keto-destogestrel less than gestodene. The difference between the various progestagens is significant with the exception of that between 3-keto-desogestrel and gestodene, when Org 2058 is used as ligand. For the cytosolic progesterone receptor, the same order is found with the exception that similar RBAs are found for gestodene and 3-keto-desogestrel. The four progestagens clearly differ with respect to binding to the androgen receptor using dihydrotestosterone as labelled ligand in intact cells; the ranking (low to high) is: norethisterone less than 3 keto-desogestrel less than levonorgestrel and gestodene. The difference between 3-keto-desogestrel and levonorgestrel or gestodene is significant. The selectivity indices (ratio of the mean RBA for the progesterone receptor to that of androgen receptor) in intact cells are significantly higher for 3-keto-desogestrel and gestodene than for levonorgestrel and norethisterone. From these results we conclude that the introduction of the 18-methyl in norethisterone (levonorgestel) increases both the binding to the progesterone and androgen receptors

  9. Selective suppression of endothelial cytokine production by progesterone receptor

    OpenAIRE

    Goddard, Lauren M.; Ton, Amy N.; Org, Tõnis; Mikkola, Hanna K.A.; Iruela-Arispe, M. Luisa

    2013-01-01

    Steroid hormones are well-recognized suppressors of the inflammatory response, however, their cell- and tissue-specific effects in the regulation of inflammation are far less understood, particularly for the sex-related steroids. To determine the contribution of progesterone in the endothelium, we have characterized and validated an in vitro culture system in which human umbilical vein endothelial cells constitutively express human progesterone receptor (PR). Using next generation RNA-sequenc...

  10. Molecular profiles of progesterone receptor loss in human breast tumors

    NARCIS (Netherlands)

    Creighton, Chad J.; Kent Osborne, C.; van de Vijver, Marc J.; Foekens, John A.; Klijn, Jan G.; Horlings, Hugo M.; Nuyten, Dimitry; Wang, Yixin; Zhang, Yi; Chamness, Gary C.; Hilsenbeck, Susan G.; Lee, Adrian V.; Schiff, Rachel

    2009-01-01

    Background Patient prognosis and response to endocrine therapy in breast cancer correlate with protein expression of both estrogen receptor (ER) and progesterone receptor (PR), with poorer outcome in patients with ER+/PR- compared to ER+/PR+ tumors. Methods To better understand the underlying

  11. Different endothelin receptor affinities in dog tissues

    International Nuclear Information System (INIS)

    Loeffler, B.M.L.; Loehrer, W.

    1991-01-01

    Endothelin (ET) is a long-lasting potent vasoconstrictor-peptide. Here the authors report different binding affinities of endothelin-1 (ET-1) to ET-receptors of various dog tissues. Crude microsomal fractions were prepared after homogenisation of dog tissues in 50 mM Tris/HCl, 20 mM MnCl2, 1 mM EDTA, pH 7.4 by differential centrifugation. Aliquots of microsomal fractions (70 micrograms of protein) were incubated at 25 degrees C for 180 min in the presence of 20 pM 125I-ET-1 and various concentrations of cold ET-1. Four different ET-1 receptor binding affinities were found: adrenals, cerebrum, liver, heart, skeletal muscle and stomach microsomal membranes contained high affinity binding sites (Kd 50 - 80 pM, Bmax 60 - 250 fmol/mg). In cerebellum and spleen medium affinity ET-1 receptors (Kd 350 pM, Bmax 880 and 1200 fmol/mg respectively) were present. In comparison lung and kidney microsomes contained a low affinity ET-1 receptor (Kd 800 and 880 pM, Bmax 1600 and 350 fmol/mg). Receptors of even lower affinity were present in heart, intestine and liver microsomes with Kd values of 3 - 6 nM

  12. Immunohistochemical Expression of Estrogen and Progesterone Receptors in Epulis Fissuratum

    Directory of Open Access Journals (Sweden)

    Maryam Seyedmajidi

    2013-01-01

    Full Text Available Background: Epulis Fissuratum (Epulis Fissuratum (EF or Denture Epulis or inflammatory fibrous hyperplasia is a common hyperplastic tumor-like lesion with reactive nature, related to loose and ill-fitting, full or partial removable dentures and it is more common in women than men. For this reason, hormonal influences may also play role in its creation. The effect of steroid hormones especially sex hormones (Estrogen and progesterone on oral mucosa is identified in some studies. In the present study, the distribution pattern and presence of estrogen and progesterone receptors in epithelial, stromal, endothelial and inflammatory cells in Epulis Fissuratum was investigated. Materials and Methods: This cross-sectional study was carried out on 30 samples of paraffin blocks with Epulis Fissuratum diagnosis and 30 samples of normal mucosal tissues as a control group who have had surgery as a margin beside the above lesions and had been obtained from the oral and maxillofacial pathology departement of Babol Dental School since 2003 up to 2010. Intensity of staining and immunoreactivity were evaluated using subjective index and considering the positive control group (breast carcinoma.Results: Epithelial, stromal, endothelial and inflammatory cells didn’t show reaction with monoclonal antibodies against estrogen and progesterone in none of the samples. Conclusion: It seems that the hypothesis of the existence of estrogen and progesterone receptors in epulis fissuratum and normal oral mucosa is ruled out. The possibility of direct effect of estrogen and progesterone in occurring of epulis fissuratum is rejected.

  13. Prognostic Value of Estrogen Receptor alpha and Progesterone Receptor Conversion in Distant Breast Cancer Metastases

    NARCIS (Netherlands)

    Hoefnagel, Laurien D. C.; Moelans, Cathy B.; Meijer, S. L.; van Slooten, Henk-Jan; Wesseling, Pieter; Wesseling, Jelle; Westenend, Pieter J.; Bart, Joost; Seldenrijk, Cornelis A.; Nagtegaal, Iris D.; Oudejans, Joost; van der Valk, Paul; van Gils, Carla H.; van der Wall, Elsken; van Diest, Paul J.

    2012-01-01

    BACKGROUND: Changes in the receptor profile of primary breast cancers to their metastases (receptor conversion) have been described for the estrogen receptor alpha (ER alpha) and progesterone receptor (PR). The purpose of this study was to evaluate the impact of receptor conversion for ER alpha and

  14. Selective suppression of endothelial cytokine production by progesterone receptor.

    Science.gov (United States)

    Goddard, Lauren M; Ton, Amy N; Org, Tõnis; Mikkola, Hanna K A; Iruela-Arispe, M Luisa

    2013-01-01

    Steroid hormones are well-recognized suppressors of the inflammatory response, however, their cell- and tissue-specific effects in the regulation of inflammation are far less understood, particularly for the sex-related steroids. To determine the contribution of progesterone in the endothelium, we have characterized and validated an in vitro culture system in which human umbilical vein endothelial cells constitutively express human progesterone receptor (PR). Using next generation RNA-sequencing, we identified a selective group of cytokines that are suppressed by progesterone both under physiological conditions and during pathological activation by lipopolysaccharide. In particular, IL-6, IL-8, CXCL2/3, and CXCL1 were found to be direct targets of PR, as determined by ChIP-sequencing. Regulation of these cytokines by progesterone was also confirmed by bead-based multiplex cytokine assays and quantitative PCR. These findings provide a novel role for PR in the direct regulation of cytokine levels secreted by the endothelium. They also suggest that progesterone-PR signaling in the endothelium directly impacts leukocyte trafficking in PR-expressing tissues. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Growth of a progesterone receptor-positive meningioma in a female patient with congenital adrenal hyperplasia.

    Science.gov (United States)

    O'Shea, T; Crowley, R K; Farrell, M; MacNally, S; Govender, P; Feeney, J; Gibney, J; Sherlock, M

    2016-01-01

    Meningioma growth has been previously described in patients receiving oestrogen/progestogen therapy. We describe the clinical, radiological, biochemical and pathologic findings in a 45-year-old woman with congenital adrenal hyperplasia secondary to a defect in the 21-hydroxylase enzyme who had chronic poor adherence to glucocorticoid therapy with consequent virilisation. The patient presented with a frontal headache and marked right-sided proptosis. Laboratory findings demonstrated androgen excess with a testosterone of 18.1 nmol/L (0-1.5 nmol) and 17-Hydroxyprogesterone >180 nmol/L (transsexual patients undergoing therapy with high-dose oestrogen and progestogens. Progesterone receptor positivity has been described previously in meningiomas. 17-Hydroxyprogesterone is elevated in CAH and has affinity and biological activity at the progesterone receptor. Therefore, we hypothesise that patients who have long-standing increased adrenal androgen precursor concentrations may be at risk of meningioma growth. Patients with long-standing CAH (particularly if not optimally controlled) may present with other complications, which may be related to long-standing elevated androgen or decreased glucocorticoid levels.Chronic poor control of CAH is associated with adrenal myelolipoma and adrenal rest tissue tumours.Meningiomas are sensitive to endocrine stimuli including progesterone, oestrogen and androgens as they express the relevant receptors.

  16. The Determinations of Estrogen and Progesterone Receptor in Breast Cancer Cell by Radioimmunoassay Method

    International Nuclear Information System (INIS)

    Kim, Chi Yeul

    1981-01-01

    The estrogen and progesterone receptors which are bound to the cytoplasmic protein of cancer cells were measured in 20 patients with the early breast cancer by means of radioimmunoassay using charcoal. 1) The patients with estrogen receptor positive were 13 (65%) of 20 cases and with progestrone receptor positive were 7 cases (35%) in the early breast cancer. 2) Coexistence of estrogen and progesterone receptor positive was noted in 7 cases (35%). The cases of estrogen receptor positive and progesterone receptor negative were 6 cases (33.3%), while there were no cases of estrogen receptor negative with progesterone receptor positive. 3) Coincidence of estrogen and progesterone negative was noticed in 7 cases (35%). Conclusively it is considered that the measurement of estrogen and progesterone receptors has relevance as predictive value, in the response to hormonal manipulations and chemotherapy for breast cancer patients.

  17. Immunohistochemical assessment of oestrogen and progesterone receptors

    DEFF Research Database (Denmark)

    Grabau, D A; Thorpe, S M; Knoop, A

    2000-01-01

    Two different methods to determine steroid receptors were analysed with respect to their ability to estimate prognosis in primary breast cancer patients. The immunohistochemical assay (IHA) was compared with the dextran-coated charcoal (DCC) method of receptor determination. A random sample of 28...... distinction between benign and malignant tissue is possible using the IHAmethod. Thus, IHAresults appear to be more clinically relevant....

  18. Improving the developability profile of pyrrolidine progesterone receptor partial agonists

    Energy Technology Data Exchange (ETDEWEB)

    Kallander, Lara S.; Washburn, David G.; Hoang, Tram H.; Frazee, James S.; Stoy, Patrick; Johnson, Latisha; Lu, Qing; Hammond, Marlys; Barton, Linda S.; Patterson, Jaclyn R.; Azzarano, Leonard M.; Nagilla, Rakesh; Madauss, Kevin P.; Williams, Shawn P.; Stewart, Eugene L.; Duraiswami, Chaya; Grygielko, Eugene T.; Xu, Xiaoping; Laping, Nicholas J.; Bray, Jeffrey D.; Thompson, Scott K. (GSKPA)

    2010-09-17

    The previously reported pyrrolidine class of progesterone receptor partial agonists demonstrated excellent potency but suffered from serious liabilities including hERG blockade and high volume of distribution in the rat. The basic pyrrolidine amine was intentionally converted to a sulfonamide, carbamate, or amide to address these liabilities. The evaluation of the degree of partial agonism for these non-basic pyrrolidine derivatives and demonstration of their efficacy in an in vivo model of endometriosis is disclosed herein.

  19. A functional polymorphism in the promoter of the progesterone receptor gene associated with endometrial cancer risk

    NARCIS (Netherlands)

    De Vivo, Immaculata; Huggins, Gordon S; Hankinson, Susan E; Lescault, Pamela J; Boezen, Hendrika; Colditz, Graham A; Hunter, David J

    2002-01-01

    Excessive estrogen stimulation unopposed by progesterone strongly predisposes to endometrial cancer. Because the antiproliferative effect of progesterone requires the progesterone receptor (PR), which exists in two isoforms, PR-A and -B, we reasoned that variants in the PR gene may predispose to

  20. Progesterone receptor antagonist CDB-4124 increases depression-like behavior in mice without affecting locomotor ability.

    Science.gov (United States)

    Beckley, Ethan H; Scibelli, Angela C; Finn, Deborah A

    2011-07-01

    Progesterone withdrawal has been proposed as an underlying factor in premenstrual syndrome and postpartum depression. Progesterone withdrawal induces forced swim test (FST) immobility in mice, a depression-like behavior, but the contribution of specific receptors to this effect is unclear. The role of progesterone's GABA(A) receptor-modulating metabolite allopregnanolone in depression- and anxiety-related behaviors has been extensively documented, but little attention has been paid to the role of progesterone receptors. We administered the classic progesterone receptor antagonist mifepristone (RU-38486) and the specific progesterone receptor antagonist CDB-4124 to mice that had been primed with progesterone for five days, and found that both compounds induced FST immobility reliably, robustly, and in a dose-dependent fashion. Although CDB-4124 increased FST immobility, it did not suppress initial activity in a locomotor test. These findings suggest that decreased progesterone receptor activity contributes to depression-like behavior in mice, consistent with the hypothesis that progesterone withdrawal may contribute to the symptoms of premenstrual syndrome or postpartum depression. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. The past, present, and future of selective progesterone receptor modulators in the management of uterine fibroids.

    Science.gov (United States)

    Singh, Sukhbir S; Belland, Liane; Leyland, Nicholas; von Riedemann, Sarah; Murji, Ally

    2017-12-21

    Uterine fibroids are common in women of reproductive age and can have a significant impact on quality of life and fertility. Although a number of international obstetrics/gynecology societies have issued evidence-based clinical practice guidelines for the management of symptomatic uterine fibroids, many of these guidelines do not yet reflect the most recent clinical evidence and approved indication for one of the key medical management options: the selective progesterone receptor modulator class. This article aims to share the clinical experience gained with selective progesterone receptor modulators in Europe and Canada by reviewing the historical development of selective progesterone receptor modulators, current best practices for selective progesterone receptor modulator use based on available data, and potential future uses for selective progesterone receptor modulators in uterine fibroids and other gynecologic conditions. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Convulsant bicuculline modifies CNS muscarinic receptor affinity

    Directory of Open Access Journals (Sweden)

    Rodríguez de Lores Arnaiz Georgina

    2006-04-01

    Full Text Available Abstract Background Previous work from this laboratory has shown that the administration of the convulsant drug 3-mercaptopropionic acid (MP, a GAD inhibitor, modifies not only GABA synthesis but also binding of the antagonist [3H]-quinuclidinyl benzilate ([3H]-QNB to central muscarinic receptors, an effect due to an increase in affinity without modifications in binding site number. The cholinergic system has been implicated in several experimental epilepsy models and the ability of acetylcholine to regulate neuronal excitability in the neocortex is well known. To study the potential relationship between GABAergic and cholinergic systems with seizure activity, we analyzed the muscarinic receptor after inducing seizure by bicuculline (BIC, known to antagonize the GABA-A postsynaptic receptor subtype. Results We analyzed binding of muscarinic antagonist [3H]-QNB to rat CNS membranes after i.p. administration of BIC at subconvulsant (1.0 mg/kg and convulsant (7.5 mg/kg doses. Subconvulsant BIC dose failed to develop seizures but produced binding alteration in the cerebellum and hippocampus with roughly 40% increase and 10% decrease, respectively. After convulsant BIC dose, which invariably led to generalized tonic-clonic seizures, binding increased 36% and 15% to cerebellar and striatal membranes respectively, but decreased 12% to hippocampal membranes. Kd value was accordingly modified: with the subconvulsant dose it decreased 27% in cerebellum whereas it increased 61% in hippocampus; with the convulsant dose, Kd value decreased 33% in cerebellum but increased 85% in hippocampus. No change in receptor number site was found, and Hill number was invariably close to unity. Conclusion Results indicate dissimilar central nervous system area susceptibility of muscarinic receptor to BIC. Ligand binding was modified not only by a convulsant BIC dose but also by a subconvulsant dose, indicating that changes are not attributable to the seizure process

  3. Expression of estrogen and progesterone receptors in astrocytomas: a literature review

    Directory of Open Access Journals (Sweden)

    Cléciton Braga Tavares

    Full Text Available Gliomas are the most common type of primary central nervous system neoplasm. Astrocytomas are the most prevalent type of glioma and these tumors may be influenced by sex steroid hormones. A literature review for the presence of estrogen and progesterone receptors in astrocytomas was conducted in the PubMed database using the following MeSH terms: “estrogen receptor beta” OR “estrogen receptor alpha” OR “estrogen receptor antagonists” OR “progesterone receptors” OR “astrocytoma” OR “glioma” OR “glioblastoma”. Among the 111 articles identified, 13 studies met our inclusion criteria. The majority of reports showed the presence of estrogen and progesterone receptors in astrocytomas. Overall, higher tumor grades were associated with decreased estrogen receptor expression and increased progesterone receptor expression.

  4. Expression of estrogen and progesterone receptors in astrocytomas: a literature review

    Science.gov (United States)

    Tavares, Cléciton Braga; Gomes-Braga, Francisca das Chagas Sheyla Almeida; Costa-Silva, Danylo Rafhael; Escórcio-Dourado, Carla Solange; Borges, Umbelina Soares; Conde, Airton Mendes; da Conceição Barros-Oliveira, Maria; Sousa, Emerson Brandão; da Rocha Barros, Lorena; Martins, Luana Mota; Facina, Gil; da-Silva, Benedito Borges

    2016-01-01

    Gliomas are the most common type of primary central nervous system neoplasm. Astrocytomas are the most prevalent type of glioma and these tumors may be influenced by sex steroid hormones. A literature review for the presence of estrogen and progesterone receptors in astrocytomas was conducted in the PubMed database using the following MeSH terms: “estrogen receptor beta” OR “estrogen receptor alpha” OR “estrogen receptor antagonists” OR “progesterone receptors” OR “astrocytoma” OR “glioma” OR “glioblastoma”. Among the 111 articles identified, 13 studies met our inclusion criteria. The majority of reports showed the presence of estrogen and progesterone receptors in astrocytomas. Overall, higher tumor grades were associated with decreased estrogen receptor expression and increased progesterone receptor expression. PMID:27626480

  5. Progesterone receptor antagonist CDB-4124 increases depression-like behavior in mice without affecting locomotor ability

    Science.gov (United States)

    Beckley, Ethan H.; Scibelli, Angela C.; Finn, Deborah A.

    2010-01-01

    Progesterone withdrawal has been proposed as an underlying factor in premenstrual syndrome and postpartum depression. Progesterone withdrawal induces forced swim test (FST) immobility in mice, a depression-like behavior, but the contribution of specific receptors to this effect is unclear. The role of progesterone’s GABAA receptor-modulating metabolite allopregnanolone in depression- and anxiety-related behaviors has been extensively documented, but little attention has been paid to the role of progesterone receptors. We administered the classic progesterone receptor antagonist mifepristone (RU-38486) and the specific progesterone receptor antagonist CDB-4124 to mice that had been primed with progesterone for five days, and found that both compounds induced FST immobility reliably, robustly, and in a dose-dependent fashion. Although CDB-4124 increased FST immobility, it did not suppress initial activity in a locomotor test. These findings suggest that decreased progesterone receptor activity contributes to depression-like behavior in mice, consistent with the hypothesis that progesterone withdrawal may contribute to the symptoms of premenstrual syndrome or postpartum depression. PMID:21163582

  6. Progesterone receptor isoform A may regulate the effects of neoadjuvant aglepristone in canine mammary carcinoma

    DEFF Research Database (Denmark)

    Guil-Luna, Silvia; Stenvang, Jan; Brünner, Nils

    2014-01-01

    RNA expression of progesterone receptor isoforms A and B in mammary carcinomas in dogs treated with 20 mg/Kg of aglepristone (n¿=¿22) or vehicle (n¿=¿5) twice before surgery.ResultsFormalin-fixed, paraffin-embedded tissue samples taken before and after treatment were used to analyse total progesterone receptor......-receptor positive and isoform-A positive tumours in aglepristone-treated dogs.ConclusionsThese findings suggest that the antiproliferative effects of aglepristone in canine mammary carcinomas are mediated by progesterone receptor isoform A....

  7. Progesterone and progesterone receptor modulators in the management of symptomatic uterine fibroids.

    Science.gov (United States)

    Talaulikar, Vikram Sinai; Manyonda, Isaac

    2012-12-01

    The majority of symptomatic uterine fibroids are currently treated by surgical interventions (myomectomy or hysterectomy) or radiological treatments (uterine artery embolisation or focussed ultrasound surgery). None of these treatments is a panacea, and what is conspicuous is the lack of an effective long-term medical therapy for a disorder so common among women of reproductive age. It has been known for some time that progesterone and its receptors enhance proliferative activity in fibroids and this has raised the possibility that anti-progestins and (PRMs) could be useful in the medical management of fibroids. Some of the compounds which have produced promising results in recent clinical trials or research studies include mifepristone, CDB-4124 (telapristone), CP-8947, J-867 (asoprisnil) and CDB-2914 (ulipristal acetate or UA). UA has recently completed Phase III clinical trials with very encouraging results, and has now acquired a licence for clinical use in Europe. While considerable research has yet to be done on the long-term safety and efficacy of UA there is nevertheless good reason for optimism on the emergence of effective medical therapy in the form of UA and possibly other PRMs. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  8. Microarray-Based Determination of Estrogen Receptor, Progesterone Receptor, and HER2 Receptor Status in Breast Cancer

    NARCIS (Netherlands)

    Roepman, Paul; Horlings, Hugo M.; Krijgsman, Oscar; Kok, Marleen; Bueno-de-Mesquita, Jolien M.; Bender, Richard; Linn, Sabine C.; Glas, Annuska M.; van de Vijver, Marc J.

    2009-01-01

    Purpose: The level of estrogen receptor (ER), progesterone receptor (PR), and HER2 aids in the determination of prognosis and treatment of breast cancer. Immunohistochemistry is currently the predominant method for assessment, but differences in methods and interpretation can substantially affect

  9. The Role of Progesterone and a Novel Progesterone Receptor, Progesterone Receptor Membrane Component 1, in the Inflammatory Response of Fetal Membranes to Ureaplasma parvum Infection.

    Directory of Open Access Journals (Sweden)

    Liping Feng

    Full Text Available Ureaplasma parvum (U. parvum is gaining recognition as an important pathogen for chorioamnionitis and preterm premature rupture of membranes. We aimed to investigate the roles of progesterone (P4 and a novel progesterone receptor, progesterone receptor membrane component 1 (PGRMC1, in the response of fetal membranes to U. parvum. Fetal membrane cells (amnion, chorion and decidua were isolated and confirmed to be free of Mycoplasmataceae. Cells were treated with U. parvum (5x106 CFU, and adherence was quantified by qPCR. Amnion and chorion cells were transfected with scrambled siRNA or validated PGRMC1 siRNA for 72h. Cells were then treated with U. parvum for 4h with or without pretreatment with P4 (10-7 M or ethanol for 1h. Interleukin-8 (IL-8, matrix metalloproteinase 9 (MMP9 and cyclooxygenase (COX-2 mRNA expression were quantified by qRT-PCR. Culture medium was harvested and analyzed for IL-8 and prostaglandin (PGE2 secretion by ELISA and MMP9 activity by zymography. U. parvum had a mean adherence of 15.0±0.6%, 16.9± 3.7% and 4.7±0.3% in cultured amnion, chorion and decidua cells, respectively. Exposure to U. parvum elicited significant inflammatory responses including induction of IL-8, COX-2, PGE2 and MMP9. A possible role of PGRMC1 was identified in the inhibition of U. parvum-stimulated COX-2 and MMP9 mRNA expression in chorion cells and MMP9 activity in amnion cells. On the other hand, it might enhance the U. parvum-stimulated IL-8 protein secretion in amnion cells. P4, mediated through PGRMC1, significantly inhibited U. Parvum-induced MMP9 mRNA and COX-2 mRNA expression in chorion cells. P4 appeared to attenuate U. parvum induced IL-8 mRNA expression in chorion cells, but this P4 effect might not mediated through PGRMC1. In summary, U. parvum preferentially adheres to and induces inflammatory responses in chorion and amnion cells. P4 and PGRMC1 appear to differentially modulate the inflammatory responses induced by U. parvum among

  10. Knockdown of Progesterone Receptor (PGR) in Macaque Granulosa Cells Disrupts Ovulation and Progesterone Production.

    Science.gov (United States)

    Bishop, Cecily V; Hennebold, Jon D; Kahl, Christoph A; Stouffer, Richard L

    2016-05-01

    Adenoviral vectors (vectors) expressing short-hairpin RNAs complementary to macaque nuclear progesterone (P) receptor PGR mRNA (shPGR) or a nontargeting scrambled control (shScram) were used to determine the role PGR plays in ovulation/luteinization in rhesus monkeys. Nonluteinized granulosa cells collected from monkeys (n = 4) undergoing controlled ovarian stimulation protocols were exposed to either shPGR, shScram, or no virus for 24 h; human chorionic gonadotropin (hCG) was then added to half of the wells to induce luteinization (luteinized granulosa cells [LGCs]; n = 4-6 wells/treatment/monkey). Cells/media were collected 48, 72, and 120 h postvector for evaluation of PGR mRNA and P levels. Addition of hCG increased (P < 0.05) PGR mRNA and medium P levels in controls. However, a time-dependent decline (P < 0.05) in PGR mRNA and P occurred in shPGR vector groups. Injection of shPGR, but not shScram, vector into the preovulatory follicle 20 h before hCG administration during controlled ovulation protocols prevented follicle rupture in five of six monkeys as determined by laparoscopic evaluation, with a trapped oocyte confirmed in three of four follicles of excised ovaries. Injection of shPGR also prevented the rise in serum P levels following the hCG bolus compared to shScram (P < 0.05). Nuclear PGR immunostaining was undetectable in granulosa cells from shPGR-injected follicles, compared to intense staining in shScram controls. Thus, the nuclear PGR appears to mediate P action in the dominant follicle promoting ovulation in primates. In vitro and in vivo effects of PGR knockdown in LGCs also support the hypothesis that P enhances its own synthesis in the primate corpus luteum by promoting luteinization. © 2016 by the Society for the Study of Reproduction, Inc.

  11. The PROGINS polymorphism of the human progesterone receptor diminishes the response to progesterone.

    Science.gov (United States)

    Romano, Andrea; Delvoux, Bert; Fischer, Dagmar-Christiane; Groothuis, Patrick

    2007-02-01

    The human progesterone receptor (PR) is a ligand-dependent transcription factor and two isoforms, (PRA and PRB), can be distinguished. PROGINS, a PR polymorphic variant, affects PRA and PRB and acts as a risk-modulating factor in several gynaecological disorders. Little is known about the functional consequences of this variant. Here, we characterise the properties of PROGINS with respect to transcription, mRNA maturation, protein activity and proliferation. PROGINS is characterised by a 320 bp PV/HS-1 Alu insertion in intron G and two point mutations, V660L in exon 4 and H770H (silent substitution) in exon 5. The Alu element contains a half oestrogen-response element/Sp1-binding site (Alu-ERE/Sp1), which acts as an in-cis intronic enhancer leading to increased transcription of the PROGINS allele in response to 17beta-oestradiol. Moreover, Alu insertions in the human genome are frequently methylated. Our data indicate that the PROGINS-Alu does not affect gene transcription due to DNA methylation. However, the Alu element reduced the stability of the PROGINS transcript compared with the CP allele and does not generate splice variants. The amino acid substitution (V600L) in exon 4 leads to differences in PR phosphorylation and degradation in the two PR variants upon ligand binding, most likely as a result of differences in the three-dimensional structures of the two PR variants. As a consequence, the PR-L660 (PROGINS) variant (1) displays decreased transactivation activity in a luciferase reporter system and (2) is less efficient in opposing cell proliferation in hamster ovarian cells expressing human PRA, when compared with the PR-V660 (most common variant). Taken together, our results indicate that the PROGINS variant of PR is less responsive to progestin compared with the most common PR because of (i) reduced amounts of gene transcript and (ii) decreased protein activity.

  12. Cloning and initial characterization of nuclear and membrane progesterone receptors in the Fathead Minnow, Pimephales promelas

    Science.gov (United States)

    Both native progestagens and synthetic progestins have important effects on reproduction that are mediated through progesterone receptors (PRs). They regulate gamete maturation and can serve as precursors for other steroid hormones in vertebrates and act as reproductive pheromone...

  13. Progesterone receptor antagonist CDB-4124 increases depression-like behavior in mice without affecting locomotor ability

    OpenAIRE

    Beckley, Ethan H.; Scibelli, Angela C.; Finn, Deborah A.

    2010-01-01

    Progesterone withdrawal has been proposed as an underlying factor in premenstrual syndrome and postpartum depression. Progesterone withdrawal induces forced swim test (FST) immobility in mice, a depression-like behavior, but the contribution of specific receptors to this effect is unclear. The role of progesterone’s GABAA receptor-modulating metabolite allopregnanolone in depression- and anxiety-related behaviors has been extensively documented, but little attention has been paid to the role ...

  14. Bone growth and turnover in progesterone receptor knockout mice.

    Energy Technology Data Exchange (ETDEWEB)

    Rickard, David J.; Iwaniec, Urszula T.; Evans, Glenda; Hefferan, Theresa E.; Hunter, Jaime C.; Waters, Katrina M.; Lydon, John P.; O' Malley, Bert W.; Khosla, Sundeep; Spelsberg, Thomas C.; Turner, Russell T.

    2008-05-01

    The role of progesterone receptor (PR) signaling in skeletal metabolism is controversial. To address whether signaling through the PR is necessary for normal bone growth and turnover, we performed histomorphometric and mCT analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 weeks of age. These mice possess a null mutation of the PR locus, which blocks the gene expression of A and B isoforms of PR. Body weight gain, uterine weight gain and tibia longitudinal bone growth was normal in PRKO mice. In contrast, total and cortical bone mass were increased in long bones of post-pubertal (12 and 26-week-old) PRKO mice, whereas cancellous bone mass was normal in the tibia but increased in the humerus. The striking 57% decrease in cancellous bone from the proximal tibia metaphysis which occurred between 6 and 26 weeks in WT mice was abolished in PRKO mice. The improved bone balance in aging PRKO mice was associated with elevated bone formation and a tendency toward reduced osteoclast perimeter. Taken together, these findings suggest that PR signaling in mice attenuates the accumulation of cortical bone mass during adolescence and is required for early age-related loss of cancellous bone.

  15. Immunolocalization of progesterone receptors in binucleate trophoblast cells of the buffalo placenta (Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    Carlos Eduardo Ambrósio

    2007-06-01

    Full Text Available The binucleate trophoblast cells (CTBs of the water buffalo placenta (Bubalus bubalis were studied with emphasis on the presence of progesterone receptor. Placentomal tissues from 27 buffalos (2-10 months of pregnancy were processed and embedded in paraplast (Paraplast Embedding Media – Paraplast Plus to locate the progesterone receptors using the immunohistochemistry technique. The immunohistochemical reaction for progesterone receptor through monoclonal antibody PgR Ab2 showed staining of CTBs, caruncular epithelial and estromal cells and blood vessel estromal pericitos present in the placentome throughout the entire gestational period analyzed. These results indicate the production of progesterone with autocrine and paracrine action in the placentome growth, differentiation and functional regulation.

  16. Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue

    International Nuclear Information System (INIS)

    Smith, Robert A; Lea, Rod A; Curran, Joanne E; Weinstein, Stephen R; Griffiths, Lyn R

    2003-01-01

    Previous studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades. RNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance. Analysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor). Statistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue

  17. Photoaffinity labeling of the progesterone receptor from human endometrial carcinoma

    International Nuclear Information System (INIS)

    Clarke, C.L.; Satyaswaroop, P.G.

    1985-01-01

    A nude mouse model for the growth of human endometrial carcinoma and hormonal modulation of the progesterone receptor (PR) was established previously. This study describes the effect of 17 beta-estradiol and tamoxifen (TAM) on growth rate and PR concentration in a hormonally responsive human endometrial tumor (EnCa 101) grown in this experimental system and presents the first characterization of human endometrial carcinoma PR. EnCa 101 was transplanted subcutaneously into ovariectomized, BALB/c, nu/nu athymic mice and grown under 17 beta-estradiol-stimulated, TAM-stimulated, and control conditions. Both 17 beta-estradiol and TAM increased the growth rate of EnCa 101 in nude mice, and a parallel increase in the cytosol PR concentration was observed. PR was partially purified by phosphocellulose and DEAE cellulose chromatography, and the DEAE eluate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photoaffinity labeling with [17 alpha-methyl- 3 H]promegestone ([ 3 H]R5020). Two PR-negative tumors (EnCa K and EnCa V) were also examined in parallel. Photolabeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of EnCa 101 grown in the presence of 17 beta-estradiol or TAM revealed incorporation of [3H]R5020 into proteins of molecular weight approximately 116,000 and 85,000. Labeled proteins of molecular weight 66,000, 45,000, and 35,000 were also observed. No incorporation of [ 3 H]R5020 was observed in EnCa 101 grown in the absence of estrogen, nor was any observed in EnCa K or EnCa V

  18. Effects of ionizing irradiation on the estradiol and progesterone receptors in rat mammary tumors

    International Nuclear Information System (INIS)

    Janssens, J.P.; Wittevrongel, C.; Van Dam, J.; Goddeeris, P.; Lauwerijns, J.M.; De Loecker, W.

    1981-01-01

    The determination of estradiol and progesterone receptor concentrations in mammary tumors is useful in predicting the hormone responsiveness. As this assay is carried out on tumor tissue which may have been subjected to radiotherapy, the possibility of an ionizing irradiation affecting the steroid receptor levels in neoplastic tissue should be taken into account. The steroid receptor concentrations are examined in dimethylbenz(a)anthracene-induced tumors os Sprague-Dawley rats. The estradiol and the progesterone receptor titers become reduced significantly after treatment with 20 Gray while an application with 7 Gray does not affect the titer values. After treatment of the tumor with 20 Gray, the steroid receptor concentrations decrease progressively, reaching a maximal reduction 20 to 30 days after exposure. As radiation treatment affects the receptor concentrations, this should be kept in mind when interpreting the steroid receptor concentrations

  19. Expression of Progesterone and Androgen Receptors in the Breast of Premenopausal Women, Considering Menstrual Phase.

    Science.gov (United States)

    Fahlén, Mia; Zhang, Hua; Löfgren, Lars; Masironi, Britt; VON Schoultz, Eva; VON Schoultz, B O; Sahlin, Lena

    2018-03-01

    Progesterone and androgens are important for normal development and tumorigenesis of the breast. Breast tissue samples from 49 premenopausal women were obtained. The progesterone receptors (PRA, PRB, PGRMC1 and PGRMC2) and the androgen receptor (AR) were determined in malignant and benign breast tumors and control tissues. The PRB and AR mRNA levels were highest in tumors. PGRMC1 and PGRMC2 mRNA levels were higher in malignant tumors compared to their paired normal tissues. PRA protein showed most immunostaining in benign tumors. PRB immunostaining varied according to menstrual phase. AR immunostaining was highest in the glands of malignant tumors. Progesterone and androgen receptors are differently regulated in tumors compared to normal breast tissues. A malignant breast tumor could appear PR-negative if collected in the luteal phase, but positive in the follicular phase. This finding may have clinical implications. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  20. Effect of antiprogestin ZK 98.734 on the ovarian cycle, early pregnancy, and on its binding to progesterone receptors in the myometrium of marmoset Callithrix jacchus

    International Nuclear Information System (INIS)

    Puri, C.P.; Kholkute, S.D.; Pongubala, J.M.; Patil, R.K.; Elger, W.A.; Jayaraman, S.

    1988-01-01

    The antiprogestin ZK 98.734 (11 beta-(4-dimethylaminophenyl-17 beta-hydroxy-17 alpha-(3-hydroxy-prop-1(Z)-enyl-4,9(10)-estradien-3-one) was administered i.m. (5 mg/day) for three consecutive days to two groups of common marmosets. In one group (nonpregnant, n = 6), it was injected during the luteal phase, and to the second group (pregnant, n = 7), it was injected during early pregnancy, on Days 24-26 of the mid-cycle estradiol peak. Administration of ZK 98.734 during the luteal phase caused a sharp drop in plasma progesterone levels. The luteal phase was shortened whether the drug was administered during the early or the late luteal phase. Similarly, administration of ZK 98.734 during early pregnancy caused a significant drop in progesterone levels, and pregnancy was terminated in all of the animals. The post-treatment cycles in both groups of animals were ovulatory and of normal duration. 3 H-ZK 98.734 showed specific binding to myometrial cytosol fraction. ZK 98.734 also displaced the binding of 3 H-progesterone to progesterone receptors. However, progesterone had higher binding affinity than did ZK 98.734. The antifertility action of ZK 98.734 could be a result either of its luteolytic action or of its blocking the progesterone receptors in the target tissue. This study, therefore, indicates that in the common marmoset ZK 98.734 is a progesterone antagonist with a potential to terminate early pregnancy

  1. Dietary acrylamide intake and estrogen and progesterone receptor-defined postmenopausal breast cancer risk

    DEFF Research Database (Denmark)

    Pedersen, Grete S; Hogervorst, Janneke G F; Schouten, Leo J

    2010-01-01

    and risk of postmenopausal breast cancer stratified by estrogen and progesterone receptor status. This study was embedded within the Netherlands Cohort Study on diet and cancer, which was initiated in 1986 enrolling 62,573 women aged 55-69 years at baseline. After 13.3 years of follow-up, 2225 incident...... breast cancer cases were ascertained, with hormone receptor status information for 43%. Cox proportional hazards analysis was applied to determine hazard ratios in quintiles of dietary acrylamide intake stratifying on estrogen receptor (ER) and progesterone receptor (PR) and smoking status....... No association was observed for overall breast cancer or receptor-negative breast cancer risk, irrespective of smoking status. A statistically non-significantly increased risk of ER positive, PR positive and joint receptor-positive breast cancer was found in never-smoking women. The multivariable-adjusted hazard...

  2. Estrogen and progesterone receptor assay using I-125 estradiol and H-3 promegestone as ligands: Results in female mammary carcinoma

    International Nuclear Information System (INIS)

    Glaubitt, D.; Hienz, H.A.; Bettges, G.; Carmanns, B.; Lichtenberg, T.; Akademisches Lehrkrankenhaus, Krefeld

    1984-01-01

    The determination of estrogen and progesterone receptors in the cytosol of carcinoma of the female breast has predictive value as to the success treatment of the patient. An improved estrogen and progesterone receptor assay using 1-125 labelled estradiol and a H-3 tagged synthetic gestagen (H-3 promegestone) as ligands proved to be highly praticable, especially time-saving. (orig.)

  3. Revisiting progesterone receptor (PR) actions in breast cancer: Insights into PR repressive functions.

    Science.gov (United States)

    Proietti, Cecilia J; Cenciarini, Mauro E; Elizalde, Patricia V

    2018-05-01

    Progesterone receptor (PR) is a master regulator in female reproductive tissues that controls developmental processes and proliferation and differentiation during the reproductive cycle and pregnancy. PR also plays a role in progression of endocrine-dependent breast cancer. As a member of the nuclear receptor family of ligand-dependent transcription factors, the main action of PR is to regulate networks of target gene expression in response to binding its cognate steroid hormone, progesterone. Liganded-PR transcriptional activation has been thoroughly studied and associated mechanisms have been described while progesterone-mediated repression has remained less explored. The present work summarizes recent advances in the understanding of how PR-mediated repression is accomplished in breast cancer cells and highlights the significance of fully understanding the determinants of context-dependent PR action. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Progesterone receptor in the prostate: A potential suppressor for benign prostatic hyperplasia and prostate cancer.

    Science.gov (United States)

    Chen, RuiQi; Yu, Yue; Dong, Xuesen

    2017-02-01

    Advanced prostate cancer undergoing androgen receptor pathway inhibition (ARPI) eventually progresses to castrate-resistant prostate cancer (CRPC), suggesting that (i) androgen receptor (AR) blockage is incomplete, and (ii) there are other critical molecular pathways contributing to prostate cancer (PCa) progression. Although most PCa occurs in the epithelium, prostate stroma is increasingly believed to play a crucial role in promoting tumorigenesis and facilitating tumor progression. In the stroma, sex steroid hormone receptors such as AR and estrogen receptor-α are implicated to have important functions, whereas the progesterone receptor (PR) remains largely under-investigated despite the high sequence and structural similarities between PR and AR. Stromal progesterone/PR signaling may play a critical role in PCa development and progression because not only progesterone is a critical precursor for de novo androgen steroidogenesis and an activator of mutant androgen receptors, but also PR functions in a ligand-independent manner in various important pathways. In fact, recent progress in our understanding of stromal PR function suggests that this receptor may exert an inhibitory effect on benign prostatic hyperplasia (BPH), reactive stroma development, and PCa progression. These early findings of stromal PR warrant further investigations as this receptor could be a potential biomarker and therapeutic target in PCa management. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Endometrial changes from short-term therapy with CDB-4124, a selective progesterone receptor modulator.

    Science.gov (United States)

    Ioffe, Olga B; Zaino, Richard J; Mutter, George L

    2009-03-01

    Selective progesterone receptor modulators are a class of drugs with progesterone antagonist activity that may confer therapeutic benefit for reproductive disorders in premenopausal women. Endometrial structure, which is dynamically controlled by circulating sex hormones, is likely to be perturbed by progesterone receptor modulators through their progesterone antagonist properties. We examined endometrial histology in 58 premenopausal women treated with the progesterone receptor modulator CDB-4124 (also known as Proellex) for endometriosis or uterine leiomyomata in two clinical trials. Endometrial biopsies obtained after 3 or 6 months with doses of 12.5, 25, or 50 mg daily oral CDB-4124 were reviewed independently by three pathologists. Consensus diagnoses using the World Health Organization hyperplasia scoring system, comments on specific histologic features, and clinical annotation were collected and analyzed. The majority of the endometrial biopsies (103 of 174 biopsies) contained histologic changes that are not seen during normal menstrual cycles. The histology of CDB-4124-treated patients was generally inactive or atrophic, and less frequently, proliferative or secretory, superimposed upon which were novel changes including formation of cystically dilated glands, and secretory changes coexisting with mitoses and apoptotic bodies. With increasing treatment dose and duration, the cysts became predominant and their lining inactive or atrophic. Cystic glands in the CDB-4124-treated subjects correlated with increased endometrial thickness by ultrasound. None of the CDB-4124-treated patients developed endometrial carcinoma or hyperplasia while on therapy. CDB-4124 therapy for 3-6 months produces histologic changes that are sufficiently novel that they might easily be misinterpreted by pathologists, particularly as disordered proliferative or hyperplastic endometrium. Knowledge of the constellation of endometrial changes associated with this agent and other

  6. Selective progesterone receptor modulators 3: use in oncology, endocrinology and psychiatry.

    Science.gov (United States)

    Benagiano, Giuseppe; Bastianelli, Carlo; Farris, Manuela

    2008-10-01

    A number of synthetic steroids are capable of modulating progesterone receptors with a spectrum of activities ranging from pure antagonism to a mixture of agonism and antagonism. The best known of these are mifepristone (RU 486), asoprisnil (J 867), onapristone (ZK 98299), ulipristal (CDB 2914), Proellex() (CDB 4124), ORG 33628 and ORG 31710. Outside reproduction selective modulators of progesterone receptors have been under investigation for a large variety of indications, for example in oncology as adjuvants in breast, cervical, endometrial, ovarian and prostate cancer, as well as inoperable meningioma and leiomyosarcoma. In addition, they have been used as antiglucocorticoids. It is therefore useful to review the results obtained in these conditions. A careful evaluation of existing major review papers and of recently published articles was carried out for the indications under review, focusing not only on mifepristone but also on those other selective modulators of progesterone receptors for which data are available. In preliminary studies selective modulators of progesterone receptors had some activity on a number of neoplasias. Their antiglucocorticoid activity has been tested with some success in Cushing's syndrome, several psychiatric conditions (e.g., mood disorders and Alzheimer's disease) and acute renal failure. Finally they are being used in a gene regulator system.

  7. Loss of Progesterone Receptor-Mediated Actions Induce Preterm Cellular and Structural Remodeling of the Cervix and Premature Birth

    Science.gov (United States)

    Yellon, Steven M.; Dobyns, Abigail E.; Beck, Hailey L.; Kurtzman, James T.; Garfield, Robert E.; Kirby, Michael A.

    2013-01-01

    A decline in serum progesterone or antagonism of progesterone receptor function results in preterm labor and birth. Whether characteristics of premature remodeling of the cervix after antiprogestins or ovariectomy are similar to that at term was the focus of the present study. Groups of pregnant rats were treated with vehicle, a progesterone receptor antagonist (onapristone or mifepristone), or ovariectomized on day 17 postbreeding. As expected, controls given vehicle delivered at term while rats delivered preterm after progesterone receptor antagonist treatment or ovariectomy. Similar to the cervix before term, the preterm cervix of progesterone receptor antagonist-treated rats was characterized by reduced cell nuclei density, decreased collagen content and structure, as well as a greater presence of macrophages per unit area. Thus, loss of nuclear progesterone receptor-mediated actions promoted structural remodeling of the cervix, increased census of resident macrophages, and preterm birth much like that found in the cervix at term. In contrast to the progesterone receptor antagonist-induced advance in characteristics associated with remodeling, ovariectomy-induced loss of systemic progesterone did not affect hypertrophy, extracellular collagen, or macrophage numbers in the cervix. Thus, the structure and macrophage census in the cervix appear sufficient for premature ripening and birth to occur well before term. With progesterone receptors predominantly localized on cells other than macrophages, the findings suggest that interactions between cells may facilitate the loss of progesterone receptor-mediated actions as part of a final common mechanism that remodels the cervix in certain etiologies of preterm and with parturition at term. PMID:24339918

  8. Methods for quantifying T cell receptor binding affinities and thermodynamics

    Science.gov (United States)

    Piepenbrink, Kurt H.; Gloor, Brian E.; Armstrong, Kathryn M.; Baker, Brian M.

    2013-01-01

    αβ T cell receptors (TCRs) recognize peptide antigens bound and presented by class I or class II major histocompatibility complex (MHC) proteins. Recognition of a peptide/MHC complex is required for initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. Here we discuss methods to quantify the affinities and thermodynamics of interactions between soluble ectodomains of TCRs and their peptide/MHC ligands, focusing on titration calorimetry, surface plasmon resonance, and fluorescence anisotropy. As TCRs typically bind ligand with weak-to-moderate affinities, we focus the discussion on means to enhance the accuracy and precision of low affinity measurements. In addition to further elucidating the biology of the T cell mediated immune response, more reliable low affinity measurements will aid with more probing studies with mutants or altered peptides that can help illuminate the physical underpinnings of how TCRs achieve their remarkable recognition properties. PMID:21609868

  9. Internalisation of membrane progesterone receptor-α after treatment with progesterone: Potential involvement of a clathrin-dependent pathway.

    Science.gov (United States)

    Foster, Helen; Reynolds, Alan; Stenbeck, Gudrun; Dong, Jing; Thomas, Peter; Karteris, Emmanouil

    2010-01-01

    Internalisation and recycling of seven trans-membrane domain receptors is a critical regulatory event for their signalling. The mechanism(s) by which membrane progesterone receptor-α (mPRα) number is regulated on the cell surface is unclear. In this study, we investigated the cellular distribution of mPRα and mechanisms of mPRα trafficking using a cell line derived from a primary culture of human myometrial cells (M11) as an experimental model. RT-PCR and immunofluorescent analysis demonstrated expression of mPRα in M11 cells with mPRα primarily distributed on the cell surface under basal conditions. For the first time, plasma membrane localisation of mPRα was confirmed using immuno-gold transmission electron microscopy. Stimulation of M11 cells with progesterone (P4, 100 nM) resulted in internalisation of mPRα from the plasma membrane to the cytoplasm (10 min) and subsequent partial translocation back to the cell surface (20 min). We investigated potential endocytotic pathways involved in trafficking of mPRα after its internalisation. Partial co-localisation of clathrin with mPRα was obvious after 10 min of P4 treatment. Of note, chlorpromazine (inhibitor of clathrin-mediated pathway) inhibited the endocytosis of mPRα, whereas treatment with nystatin (inhibitor of caveolae-mediated pathway) did not affect internalisation. Collectively, these data suggest that mPRα is expressed on the cell surface of M11 cells and undergoes endocytosis after P4 stimulation primarily via a clathrin-mediated pathway.

  10. Genomic agonism and phenotypic antagonism between estrogen and progesterone receptors in breast cancer

    OpenAIRE

    Singhal, Hari; Greene, Marianne E.; Tarulli, Gerard; Zarnke, Allison L.; Bourgo, Ryan J.; Laine, Muriel; Chang, Ya-Fang; Ma, Shihong; Dembo, Anna G.; Raj, Ganesh V.; Hickey, Theresa E.; Tilley, Wayne D.; Greene, Geoffrey L.

    2016-01-01

    The functional role of progesterone receptor (PR) and its impact on estrogen signaling in breast cancer remain controversial. In primary ER+ (estrogen receptor?positive)/PR+ human tumors, we report that PR reprograms estrogen signaling as a genomic agonist and a phenotypic antagonist. In isolation, estrogen and progestin act as genomic agonists by regulating the expression of common target genes in similar directions, but at different levels. Similarly, in isolation, progestin is also a weak ...

  11. Cytologic assessment of estrogen receptor, progesterone receptor, and HER2 status in metastatic breast carcinoma.

    Science.gov (United States)

    Pareja, Fresia; Murray, Melissa P; Jean, Ryan Des; Konno, Fumiko; Friedlander, Maria; Lin, Oscar; Edelweiss, Marcia

    2017-01-01

    Discordance in the receptor status between primary breast carcinomas (PBC) and corresponding metastasis is well documented. Interrogation of the receptor status of metastatic breast carcinoma (MBC) in cytology material is common practice; however, its utility has not been thoroughly validated. We studied patients with MBC, and evaluated the concordance rates of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) between PBC surgical specimens and corresponding MBC cell blocks (CBs). We correlated the findings with clinicopathologic variables and with the fixation methods used. We searched for patients with MBC diagnosed on cytology from 2007 to 2009 and selected those with ER, PR and HER2 tested in both the PBC surgical specimens and the MBC CBs. We included CBs fixed in formalin and methanol based solution (CytoLyt®). All slides were reevaluated by cytopathologists. Clinical information was retrieved from the medical records. We studied 65 patients with PBC and MBC paired specimens. The concordance rates between PBC and MBC were 78.5%, 58.5% and 96.9%, for ER, PR and HER2, respectively. When discordant, PR status switched from positive (PBC) to negative (MBC) in most cases (23/27). The PR concordance rate was 45.2% for CBs fixed in formalin and 70.6% for those fixed with CytoLyt® (p=0.047). The ER, PR and HER2 concordance rates between the PBC and MBC CBs are similar to those reported in paired surgical specimens. PR status was the most prevalent discordance and was not accompanied by a switch in ER.

  12. Affinity Labeling of Membrane Receptors Using Tissue-Penetrating Radiations

    Directory of Open Access Journals (Sweden)

    Franklin C. Wong

    2013-01-01

    Full Text Available Photoaffinity labeling, a useful in vivo biochemical tool, is limited when applied in vivo because of the poor tissue penetration by ultraviolet (UV photons. This study investigates affinity labeling using tissue-penetrating radiation to overcome the tissue attenuation and irreversibly label membrane receptor proteins. Using X-ray (115 kVp at low doses (<50 cGy or Rad, specific and irreversible binding was found on striatal dopamine transporters with 3 photoaffinity ligands for dopamine transporters, to different extents. Upon X-ray exposure (115 kVp, RTI-38 and RTI-78 ligands showed irreversible and specific binding to the dopamine transporter similar to those seen with UV exposure under other conditions. Similarly, gamma rays at higher energy (662 keV also affect irreversible binding of photoreactive ligands to peripheral benzodiazepine receptors (by PK14105 and to the dopamine (D2 membrane receptors (by azidoclebopride, respectively. This study reports that X-ray and gamma rays induced affinity labeling of membrane receptors in a manner similar to UV with photoreactive ligands of the dopamine transporter, D2 dopamine receptor (D2R, and peripheral benzodiazepine receptor (PBDZR. It may provide specific noninvasive irreversible block or stimulation of a receptor using tissue-penetrating radiation targeting selected anatomic sites.

  13. Marginal activity of progesterone receptor B (PR-B) in dogs but high incidence of mammary cancer

    NARCIS (Netherlands)

    Gracanin, Ana; Voorwald, Fabiana A; van Wolferen, Monique; Timmermans-Sprang, Elpetra; Mol, Jan A

    2014-01-01

    Progesterone plays an important role in the normal development and carcinogenesis of the mammary gland. In vitro studies have shown that the canine progesterone receptor B (cPR-B), which is essential for mammary development in the mouse, does not transactivate reporter constructs containing

  14. Molecular basis of a high affinity murine interleukin-5 receptor.

    OpenAIRE

    Devos, R; Plaetinck, G; Van der Heyden, J; Cornelis, S; Vandekerckhove, J; Fiers, W; Tavernier, J

    1991-01-01

    The mouse interleukin-5 receptor (mIL-5R) consists of two components one of which, the mIL-5R alpha-chain, binds mIL-5 with low affinity. Recently we demonstrated that monoclonal antibodies (Mabs) recognizing the second mIL-5R beta-chain, immunoprecipitate a p130-140 protein doublet which corresponds to the mIL-3R and the mIL-3R-like protein, the latter chain for which so far no ligand has been identified. In this study we show that a high affinity mIL-5R can be reconstituted on COS1 cells by...

  15. Progesterone signaling mediated through progesterone receptor membrane component-1 in ovarian cells with special emphasis on ovarian cancer.

    Science.gov (United States)

    Peluso, John J

    2011-08-01

    Various ovarian cell types including granulosa cells and ovarian surface epithelial cells express the progesterone (P4) binding protein, progesterone receptor membrane component-1 (PGRMC1). PGRMC1 is also expressed in ovarian tumors. PGRMC1 plays an essential role in promoting the survival of both normal and cancerous ovarian cell in vitro. Given the clinical significance of factors that regulate the viability of ovarian cancer, this review will focus on the role of PGRMC1 in ovarian cancer, while drawing insights into the mechanism of PGRMC1's action from cell lines derived from healthy ovaries as well as ovarian tumors. Studies using PGRMC1siRNA demonstrated that P4's ability to inhibit ovarian cells from undergoing apoptosis in vitro is dependent on PGRMC1. To confirm the importance of PGRMC1, the ability of PGRMC1-deplete ovarian cancer cell lines to form tumors in intact nude mice was assessed. Compared to PGRMC1-expressing ovarian cancer cells, PGRMC1-deplete ovarian cancer cells formed tumors in fewer mice (80% compared to 100% for controls). Moreover, the number of tumors derived from PGRMC1-deplete ovarian cancer cells was 50% of that observed in controls. Finally, the tumors that formed from PGRMC1-deplete ovarian cancer cells were about a fourth the size of tumors derived from ovarian cancer cells with normal levels of PGRMC1. One reason for PGRMC1-deplete tumors being smaller is that they had a poorly developed microvasculature system. How PGRMC1 regulates cell viability and in turn tumor growth is not known but part of the mechanism likely involves the regulation of genes that promote cell survival and inhibit apoptosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Preclinical evaluation of a positron emitting progestin ([18F]fluoro-16 alpha-methyl-19-norprogesterone) for imaging progesterone receptor positive tumours with positron emission tomography

    NARCIS (Netherlands)

    Verhagen, Aalt; Luurtsema, Gert; PESSER, JW; DEGROOT, TJ; OOSTERHUIS, JW; Vaalburg, Willem; Wouda, S.

    Three 21-fluoro-progestins were investigated as potential imaging agents for the in vivo assessment of human progesterone receptor positive neoplasms with positron emission tomography. In competitive binding assays these compounds demonstrated high specificity, competing only for progesterone

  17. A Role of Endogenous Progesterone in Stroke Cerebroprotection Revealed by the Neural-Specific Deletion of Its Intracellular Receptors.

    Science.gov (United States)

    Zhu, Xiaoyan; Fréchou, Magalie; Liere, Philippe; Zhang, Shaodong; Pianos, Antoine; Fernandez, Neïké; Denier, Christian; Mattern, Claudia; Schumacher, Michael; Guennoun, Rachida

    2017-11-08

    Treatment with progesterone protects the male and female brain against damage after middle cerebral artery occlusion (MCAO). However, in both sexes, the brain contains significant amounts of endogenous progesterone. It is not known whether endogenously produced progesterone enhances the resistance of the brain to ischemic insult. Here, we used steroid profiling by gas chromatography-tandem mass spectrometry (GC-MS/MS) for exploring adaptive and sex-specific changes in brain levels of progesterone and its metabolites after MCAO. We show that, in the male mouse brain, progesterone is mainly metabolized via 5α-reduction leading to 5α-dihydroprogesterone (5α-DHP), also a progesterone receptor (PR) agonist ligand in neural cells, then to 3α,5α-tetrahydroprogesterone (3α,5α-THP). In the female mouse brain, levels of 5α-DHP and 3α,5α-THP are lower and levels of 20α-DHP are higher than in males. After MCAO, levels of progesterone and 5α-DHP are upregulated rapidly to pregnancy-like levels in the male but not in the female brain. To assess whether endogenous progesterone and 5α-DHP contribute to the resistance of neural cells to ischemic damage, we inactivated PR selectively in the CNS. Deletion of PR in the brain reduced its resistance to MCAO, resulting in increased infarct volumes and neurological deficits in both sexes. Importantly, endogenous PR ligands continue to protect the brain of aging mice. These results uncover the unexpected importance of endogenous progesterone and its metabolites in cerebroprotection. They also reveal that the female reproductive hormone progesterone is an endogenous cerebroprotective neurosteroid in both sexes. SIGNIFICANCE STATEMENT The brain responds to injury with protective signaling and has a remarkable capacity to protect itself. We show here that, in response to ischemic stroke, levels of progesterone and its neuroactive metabolite 5α-dihydroprogesterone are upregulated rapidly in the male mouse brain but not in the

  18. Cloning and initial characterization of nuclear and four membrane progesterone receptors in the fathead minnow(Pimephales promelas)

    Science.gov (United States)

    Both native progestagens and synthetic progestins have important effects on reproduction that are mediated through progesterone receptors (PRs). Progestagens regulate gamete maturation in vertebrates, are critical regulators of placental mammal pregnancy, and act as reproductive ...

  19. Biochemical and immunohistochemical estrogen and progesterone receptors in adenomatous hyperplasia and endometrial carcinoma: correlations with stage and other clinicopathologic features

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, A L; Lyndrup, J

    1992-01-01

    OBJECTIVE: This study investigates clinicopathologic associations of estrogen and progesterone receptor content in endometrial carcinoma. STUDY DESIGN: One hundred fifty-two patients with endometrial cancer and 12 with adenomatous hyperplasia were included. Dextran-coated charcoal receptor assay...... receptor dextran-coated charcoal values and immunohistochemical histologic scores correlated inversely (p dextran-coated charcoal values was independent...

  20. Mineralocorticoid receptor haplotype, estradiol, progesterone and emotional information processing.

    Science.gov (United States)

    Hamstra, Danielle A; de Kloet, E Ronald; Quataert, Ina; Jansen, Myrthe; Van der Does, Willem

    2017-02-01

    Carriers of MR-haplotype 1 and 3 (GA/CG; rs5522 and rs2070951) are more sensitive to the influence of oral contraceptives (OC) and menstrual cycle phase on emotional information processing than MR-haplotype 2 (CA) carriers. We investigated whether this effect is associated with estradiol (E2) and/or progesterone (P4) levels. Healthy MR-genotyped premenopausal women were tested twice in a counterbalanced design. Naturally cycling (NC) women were tested in the early-follicular and mid-luteal phase and OC-users during OC-intake and in the pill-free week. At both sessions E2 and P4 were assessed in saliva. Tests included implicit and explicit positive and negative affect, attentional blink accuracy, emotional memory, emotion recognition, and risky decision-making (gambling). MR-haplotype 2 homozygotes had higher implicit happiness scores than MR-haplotype 2 heterozygotes (p=0.031) and MR-haplotype 1/3 carriers (pemotion recognition test than MR-haplotype 1/3 (p=0.001). Practice effects were observed for most measures. The pattern of correlations between information processing and P4 or E2 differed between sessions, as well as the moderating effects of the MR genotype. In the first session the MR-genotype moderated the influence of P4 on implicit anxiety (sr=-0.30; p=0.005): higher P4 was associated with reduction in implicit anxiety, but only in MR-haplotype 2 homozygotes (sr=-0.61; p=0.012). In the second session the MR-genotype moderated the influence of E2 on the recognition of facial expressions of happiness (sr=-0.21; p=0.035): only in MR-haplotype 1/3 higher E2 was correlated with happiness recognition (sr=0.29; p=0.005). In the second session higher E2 and P4 were negatively correlated with accuracy in lag2 trials of the attentional blink task (pemotional information processing. This moderating effect may depend on the novelty of the situation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Characterization of estrogen receptor-negative/progesterone receptor-positive breast cancer.

    Science.gov (United States)

    Shen, Tiansheng; Brandwein-Gensler, Margaret; Hameed, Omar; Siegal, Gene P; Wei, Shi

    2015-11-01

    Despite the controversies, estrogen receptor-negative/progesterone receptor-positive (ER-/PR+) breast cancers have a reported incidence of 1% to 4%. These tumors are less well defined, and it is unclear whether ER-/PR+ represents a distinct subtype. Thus, we analyzed 5374 consecutive breast cancers to characterize the clinicopathological features of this underrecognized subset of tumors. The ER-/PR+ tumors, constituting 2.3% of the total, were mostly high grade and significantly seen in younger patients and African American women when compared with the ER+/PR+ and ER+/PR- groups, but similar to that of ER-/PR- phenotype (P < .0001). A significantly prolonged relapse-free survival (RFS) was associated with the ER+/PR+ subtype when compared with the ER+/PR- (P = .0002) or ER-/PR+ (P = .0004) tumors, whereas all 3 groups showed a superior outcome to that of the ER-/PR- phenotype. In the subset of patients receiving endocrine therapy, those with ER+/PR+ tumors had a significantly prolonged RFS (P = .001) and disease-specific survival (P = .005) when compared with the group with an ER+/PR- phenotype, but did not significantly differ from those with ER-/PR+ tumors. No significant survival advantage was found between the ER+/PR- and ER-/PR+ tumors in any group of patients analyzed. Furthermore, a higher PR expression was associated with a favorable RFS and disease-specific survival in the patients with ER-/PR+ tumors. Therefore, the ER-/PR+ tumors demonstrate a similar, if not higher than, response rate to endocrine therapy when compared with the ER+/PR- tumors and thus are important to identify. Routine PR testing remains necessary in assisting clinical decision making in the pursuit of precision medicine. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. ANTAGONISM OF PROGESTERONE RECEPTOR SUPPRESSES CAROTID BODY RESPONSES TO HYPOXIA AND NICOTINE IN RAT PUPS

    OpenAIRE

    JOSEPH, V.; NIANE, L. M.; BAIRAM, A.

    2012-01-01

    We tested the hypothesis that antagonism of progesterone receptor (PR) in newborn rats alters carotid body and respiratory responses to hypoxia and nicotinic receptor agonists. Rats were treated with the PR antagonist mifepristone (daily oral gavage 40 μg/g/d) or vehicle between post-natal days 3 and 15. In 11–14-day-old rats, we used in vitro carotid body/carotid sinus nerve preparation and whole body plethysmography to assess the carotid body and ventilatory responses to hypoxia (65 mmHg in...

  3. The progesterone receptor Val660→Leu polymorphism and breast cancer risk

    International Nuclear Information System (INIS)

    De Vivo, Immaculata; Hankinson, Susan E; Colditz, Graham A; Hunter, David J

    2004-01-01

    Recent evidence suggests a role for progesterone in breast cancer development and tumorigenesis. Progesterone exerts its effect on target cells by interacting with its receptor; thus, genetic variations, which might cause alterations in the biological function in the progesterone receptor (PGR), can potentially contribute to an individual's susceptibility to breast cancer. It has been reported that the PROGINS allele, which is in complete linkage disequilibrium with a missense substitution in exon 4 (G/T, valine→leucine, at codon 660), is associated with a decreased risk for breast cancer. Using a nested case-control study design within the Nurses' Health Study cohort, we genotyped 1252 cases and 1660 matched controls with the use of the Taqman assay. We did not observe any association of breast cancer risk with carrying the G/T (Val660→Leu) polymorphism (odds ratio 1.10, 95% confidence interval 0.93–1.30). In addition, we did not observe an interaction between this allele and menopausal status and family history of breast cancer as reported previously. Overall, our study does not support an association between the Val660→Leu PROGINS polymorphism and breast cancer risk

  4. TRPV1 channels and the progesterone receptor Sig-1R interact to regulate pain.

    Science.gov (United States)

    Ortíz-Rentería, Miguel; Juárez-Contreras, Rebeca; González-Ramírez, Ricardo; Islas, León D; Sierra-Ramírez, Félix; Llorente, Itzel; Simon, Sidney A; Hiriart, Marcia; Rosenbaum, Tamara; Morales-Lázaro, Sara L

    2018-02-13

    The Transient Receptor Potential Vanilloid 1 (TRPV1) ion channel is expressed in nociceptors where, when activated by chemical or thermal stimuli, it functions as an important transducer of painful and itch-related stimuli. Although the interaction of TRPV1 with proteins that regulate its function has been previously explored, their modulation by chaperones has not been elucidated, as is the case for other mammalian TRP channels. Here we show that TRPV1 physically interacts with the Sigma 1 Receptor (Sig-1R), a chaperone that binds progesterone, an antagonist of Sig-1R and an important neurosteroid associated to the modulation of pain. Antagonism of Sig-1R by progesterone results in the down-regulation of TRPV1 expression in the plasma membrane of sensory neurons and, consequently, a decrease in capsaicin-induced nociceptive responses. This is observed both in males treated with a synthetic antagonist of Sig-1R and in pregnant females where progesterone levels are elevated. This constitutes a previously undescribed mechanism by which TRPV1-dependent nociception and pain can be regulated.

  5. Estrogen Receptor and Progesterone Receptor Expression in Normal Terminal Duct Lobular Units Surrounding Invasive Breast Cancer

    Science.gov (United States)

    Yang, Xiaohong R.; Figueroa, Jonine D.; Hewitt, Stephen M.; Falk, Roni T.; Pfeiffer, Ruth M.; Lissowska, Jolanta; Peplonska, Beata; Brinton, Louise A.; Garcia-Closas, Montserrat; Sherman, Mark E.

    2014-01-01

    Introduction Molecular and morphological alterations related to carcinogenesis have been found in terminal duct lobular units (TDLUs), the microscopic structures from which most breast cancer precursors and cancers develop, and therefore, analysis of these structures may reveal early changes in breast carcinogenesis and etiologic heterogeneity. Accordingly, we evaluated relationships of breast cancer risk factors and tumor pathology to estrogen receptor (ER) and progesterone receptor (PR) expression in TDLUs surrounding breast cancers. Methods We analyzed 270 breast cancer cases included in a population-based breast cancer case-control study conducted in Poland. TDLUs were mapped in relation to breast cancer: within the same block as the tumor (TDLU-T), proximal to tumor (TDLU-PT), or distant from (TDLU-DT). ER/PR was quantitated using image analysis of immunohistochemically stained TDLUs prepared as tissue microarrays. Results In surgical specimens containing ER-positive breast cancers, ER and PR levels were significantly higher in breast cancer cells than in normal TDLUs, and higher in TDLU-T than in TDLU-DT or TDLU-PT, which showed similar results. Analyses combining DT-/PT TDLUs within subjects demonstrated that ER levels were significantly lower in premenopausal women vs. postmenopausal women (odds ratio [OR]=0.38, 95% confidence interval [CI]=0.19, 0.76, P=0.0064) and among recent or current menopausal hormone therapy users compared with never users (OR=0.14, 95% CI=0.046–0.43, Ptrend=0.0006). Compared with premenopausal women, TDLUs of postmenopausal women showed lower levels of PR (OR=0.90, 95% CI=0.83–0.97, Ptrend=0.007). ER and PR expression in TDLUs was associated with epidermal growth factor receptor (EGFR) expression in invasive tumors (P=0.019 for ER and P=0.03 for PR), but not with other tumor features. Conclusions Our data suggest that TDLUs near breast cancers reflect field effects, whereas those at a distance demonstrate influences of breast

  6. A dynamical systems model of progesterone receptor interactions with inflammation in human parturition.

    Science.gov (United States)

    Brubaker, Douglas; Barbaro, Alethea; R Chance, Mark; Mesiano, Sam

    2016-08-19

    Progesterone promotes uterine relaxation and is essential for the maintenance of pregnancy. Withdrawal of progesterone activity and increased inflammation within the uterine tissues are key triggers for parturition. Progesterone actions in myometrial cells are mediated by two progesterone receptor (PR) isoforms, PR-A and PR-B, that function as ligand-activated transcription factors. PR-B mediates relaxatory actions of progesterone, in part, by decreasing myometrial cell responsiveness to pro-inflammatory stimuli. These same pro-inflammatory stimuli promote the expression of PR-A which inhibits the anti-inflammatory activity of PR-B. Competitive interaction between the progesterone receptors then augments myometrial responsiveness to pro-inflammatory stimuli. The interaction between PR-B transcriptional activity and inflammation in the pregnancy myometrium is examined using a dynamical systems model in which quiescence and labor are represented as phase-space equilibrium points. Our model shows that PR-B transcriptional activity and the inflammatory load determine the stability of the quiescent and laboring phenotypes. The model is tested using published transcriptome datasets describing the mRNA abundances in the myometrium before and after the onset of labor at term. Surrogate transcripts were selected to reflect PR-B transcriptional activity and inflammation status. The model coupling PR-B activity and inflammation predicts contractile status (i.e., laboring or quiescent) with high precision and recall and outperforms uncoupled single and two-gene classifiers. Linear stability analysis shows that phase space bifurcations exist in our model that may reflect the phenotypic states of the pregnancy uterus. The model describes a possible tipping point for the transition of the quiescent to the contractile laboring phenotype. Our model describes the functional interaction between the PR-A:PR-B hypothesis and tissue level inflammation in the pregnancy uterus and is a

  7. Progesterone receptor levels independently predict survival in endometrial adenocarcinoma

    DEFF Research Database (Denmark)

    Nyholm, H C; Christensen, Ib Jarle; Nielsen, Anette Lynge

    1995-01-01

    to correlations to cancer-specific survival in a multivariate analysis including histopathological characteristics. Median patient follow-up time was 67 months with 18 cancer deaths. The PR-DCC and ER-DCC values were dichotomized according to levels previously found by us to correspond to the best agreement...... between receptor status as determined by the DCC and ICA methods (130 fmol/mg cytosol protein for ER, 114 fmol/mg for PR). Using these thresholds, we found by multivariate analysis that "high" PR-DCC levels (> 114 fmol/mg) correlated significantly (P = 0.004) with survival, independent of stage risk group...... could not be statistically evaluated due to the number of cases with eligible ICA values. However, we suggest that owing to a close correlation between DCC and ICA results, PR-ICA status may provide significant prognostic information when DCC measurements are not available....

  8. Progesterone receptors (PR) mediate STAT actions: PR and prolactin receptor signaling crosstalk in breast cancer models.

    Science.gov (United States)

    Leehy, Katherine A; Truong, Thu H; Mauro, Laura J; Lange, Carol A

    2018-02-01

    Estrogen is the major mitogenic stimulus of mammary gland development during puberty wherein ER signaling acts to induce abundant PR expression. PR signaling, in contrast, is the primary driver of mammary epithelial cell proliferation in adulthood. The high circulating levels of progesterone during pregnancy signal through PR, inducing expression of the prolactin receptor (PRLR). Cooperation between PR and prolactin (PRL) signaling, via regulation of downstream components in the PRL signaling pathway including JAKs and STATs, facilitates the alveolar morphogenesis observed during pregnancy. Indeed, these pathways are fully integrated via activation of shared signaling pathways (i.e. JAKs, MAPKs) as well as by the convergence of PRs and STATs at target genes relevant to both mammary gland biology and breast cancer progression (i.e. proliferation, stem cell outgrowth, tissue cell type heterogeneity). Thus, rather than a single mediator such as ER, transcription factor cascades (ER>PR>STATs) are responsible for rapid proliferative and developmental programming in the normal mammary gland. It is not surprising that these same mediators typify uncontrolled proliferation in a majority of breast cancers, where ER and PR are most often co-expressed and may cooperate to drive malignant tumor progression. This review will primarily focus on the integration of PR and PRL signaling in breast cancer models and the importance of this cross-talk in cancer progression in the context of mammographic density. Components of these PR/PRL signaling pathways could offer alternative drug targets and logical complements to anti-ER or anti-estrogen-based endocrine therapies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Cloning, mapping and molecular characterization of porcine progesterone receptor membrane component 2 (PGRMC2 gene

    Directory of Open Access Journals (Sweden)

    Congying Chen

    2010-01-01

    Full Text Available Progesterone plays an important role in sow reproduction by stimulating classic genomic pathways via nuclear receptors and non-genomic pathways via membrane receptors such a progesterone receptor membrane component 2 (PGRMC2. In this work, we used radiation hybrid mapping to assign PGRMC2 to pig chromosome 8 and observed that this receptor has two transcripts in pigs. The full-length cDNA of the large transcript is 1858 bp long and contains a 669-bp open reading frame (ORF encoding a protein of 223 amino acids. The shorter transcript encodes a protein of 170 amino acids. The porcine PGRMC2 gene consists of three exons 446 bp, 156 bp and 1259 bp in length. The promoter sequence is GC-rich and lacks a typical TATA box. Several putative cis-regulatory DNA motifs were identified in the 208-bp upstream genomic region. Five single nucleotide polymorphisms (SNPs were detected in introns* and the 3' UTR. RT-PCR indicated that the PGRMC2 gene is expressed ubiquitously in all pig tissues examined.

  10. Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

    Science.gov (United States)

    He, Mingyue; Hamon, Maureen; Liu, Hong; Corper, Adam L; Taussig, Michael J

    2006-09-01

    The crystal structures of the Fab' fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 10(4)-fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position.

  11. Nuclear progesterone receptors are up-regulated by estrogens in neurons and radial glial progenitors in the brain of zebrafish.

    Directory of Open Access Journals (Sweden)

    Nicolas Diotel

    Full Text Available In rodents, there is increasing evidence that nuclear progesterone receptors are transiently expressed in many regions of the developing brain, notably outside the hypothalamus. This suggests that progesterone and/or its metabolites could be involved in functions not related to reproduction, particularly in neurodevelopment. In this context, the adult fish brain is of particular interest, as it exhibits constant growth and high neurogenic activity that is supported by radial glia progenitors. However, although synthesis of neuroprogestagens has been documented recently in the brain of zebrafish, information on the presence of progesterone receptors is very limited. In zebrafish, a single nuclear progesterone receptor (pgr has been cloned and characterized. Here, we demonstrate that this pgr is widely distributed in all regions of the zebrafish brain. Interestingly, we show that Pgr is strongly expressed in radial glial cells and more weakly in neurons. Finally, we present evidence, based on quantitative PCR and immunohistochemistry, that nuclear progesterone receptor mRNA and proteins are upregulated by estrogens in the brain of adult zebrafish. These data document for the first time the finding that radial glial cells are preferential targets for peripheral progestagens and/or neuroprogestagens. Given the crucial roles of radial glial cells in adult neurogenesis, the potential effects of progestagens on their activity and the fate of daughter cells require thorough investigation.

  12. Asymmetric binding of histone H1 stabilizes MMTV nucleosomes and the interaction of progesterone receptor with the exposed HRE.

    Science.gov (United States)

    Vicent, Guillermo P; Meliá, María J; Beato, Miguel

    2002-11-29

    Packaging of mouse mammary tumor virus (MMTV) promoter sequences in nucleosomes modulates access of DNA binding proteins and influences the interaction among DNA bound transcription factors. Here we analyze the binding of histone H1 to MMTV mononucleosomes assembled with recombinant histones and study its influence on nucleosome structure and stability as well as on progesterone receptor (PR) binding to the hormone responsive elements (HREs). The MMTV nucleosomes can be separated into three main populations, two of which exhibited precise translational positioning. Histone H1 bound preferentially to the 5' distal nucleosomal DNA protecting additional 27-28 nt from digestion by micrococcal nuclease. Binding of histone H1 was unaffected by prior crosslinking of protein and DNA in nucleosomes with formaldehyde. Neither the translational nor the rotational nucleosome positioning was altered by histone H1 binding, but the nucleosomes were stabilized as judged by the kinetics of nuclease cleavage. Unexpectedly, binding of recombinant PR to the exposed distal HRE-I in nucleosomes was enhanced in the presence of histone H1, as demonstrated by band shift and footprinting experiments. This enhanced PR affinity may contribute to the reported positive effect of histone H1 on the hormonal activation of MMTV reporter genes.

  13. Reduced hypoxic ventilatory response in newborn mice knocked-out for the progesterone receptor.

    Science.gov (United States)

    Potvin, Catherine; Rossignol, Orlane; Uppari, NagaPraveena; Dallongeville, Arnaud; Bairam, Aida; Joseph, Vincent

    2014-11-01

    Recent studies showed that progesterone stimulates the hypoxic ventilatory response and may reduce apnoea frequency in newborn rats, but so far we still do not know by what mechanisms and whether endogenous progesterone might contribute to respiratory control in neonates. We therefore determined the role of the nuclear progesterone receptor (PR; member of the steroid receptor superfamily) by using wild-type (WT) and PR knock-out (PRKO) mice at postnatal days (P) 1, 4 and 10. We measured the hypoxic ventilatory response (14 and 12% O2, 20 min each) and apnoea frequency in both male and female mice by using whole-body plethysmography. In response to hypoxia, WT male mice had a marked hypoxic ventilatory response at P1 and P10, but not at P4. At P1 and P10, PRKO male mice had a lower hypoxic ventilatory response than WT males. Wild-type female mice had a marked hypoxic ventilatory response at P10, but not at P1 and P4. At P1 and P10, PRKO female mice had a lower hypoxic ventilatory response than WT females. In basal conditions, apnoea frequency was similar in WT and PRKO mice at P1, P4 and P10. During hypoxia, apnoea frequency was higher in WT male mice compared with PRKO male mice and WT female mice at P1. We conclude that PR is a key contributor to the hypoxic ventilatory response in newborn mice, but PR deletion does not increase the frequency of apnoea during normoxia or hypoxia. © 2014 The Authors. Experimental Physiology © 2014 The Physiological Society.

  14. CDB-4124, a progesterone receptor modulator, inhibits mammary carcinogenesis by suppressing cell proliferation and inducing apoptosis.

    Science.gov (United States)

    Wiehle, Ronald; Lantvit, Daniel; Yamada, Tohru; Christov, Konstantin

    2011-03-01

    CDB-4124 (Proellex or telapristone acetate) is a modulator of progesterone receptor (PR) signaling, which is currently employed in preclinical studies for prevention and treatment of breast cancer and has been used in clinical studies for treatment of uterine fibroids and endometriosis. Here we provide evidence for its action on steroid hormone-signaling, cell cycle-regulated genes and in vivo on mammary carcinogenesis. When CDB-4124 is given to rats at 200 mg/kg for 24 months, it prevents the development of spontaneous mammary hyperplastic and premalignant lesions. Also, CDB-4124 given as subcutaneous pellets at two different doses suppressed, dose dependently, N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis. The high dose (30 mg, over 84 days) increased tumor latency from 66 ± 24 days to 87 ± 20 days (P CDB-4124 inhibited cell proliferation and induced apoptosis in MNU-induced mammary tumors, which correlated with a decreased proportion of PR(+) tumor cells and with decreased serum progesterone. CDB-4124 did not affect serum estradiol. In a mechanistic study employing T47D cells we found that CDB-4124 suppressed G(1)/G(0)-S transition by inhibiting CDK2 and CDK4 expressions, which correlated with inhibition of estrogen receptor (ER) expression. Taken together, these data indicate that CDB-4124 can suppress the development of precancerous lesions and carcinogen-induced ER(+) mammary tumors in rats, and may have implications for prevention and treatment of human breast cancer.

  15. Remodeling of the Cervix and Parturition in Mice Lacking the Progesterone Receptor B Isoform1

    Science.gov (United States)

    Yellon, Steven M.; Oshiro, Bryan T.; Chhaya, Tejas Y.; Lechuga, Thomas J.; Dias, Rejane M.; Burns, Alexandra E.; Force, Lindsey; Apostolakis, Ede M.

    2011-01-01

    Withdrawal of progestational support for pregnancy is part of the final common pathways for parturition, but the role of nuclear progesterone receptor (PGR) isoforms in this process is not known. To determine if the PGR-B isoform participates in cervical remodeling at term, cervices were obtained from mice lacking PGR-B (PGR-BKO) and from wild-type (WT) controls before or after birth. PGR-BKO mice gave birth to viable pups at the same time as WT controls during the early morning of Day 19 postbreeding. Morphological analyses indicated that by the day before birth, cervices from PGR-BKO and WT mice had increased in size, with fewer cell nuclei/area as well as diminished collagen content and structure, as evidenced by optical density of picrosirius red-stained sections, compared to cervices from nonpregnant mice. Moreover, increased numbers of resident macrophages, but not neutrophils, were found in the prepartum cervix of PGR-BKO compared to nonpregnant mice, parallel to findings in WT mice. These results suggest that PGR-B does not contribute to the growth or degradation of the extracellular matrix or proinflammatory processes associated with recruitment of macrophages in the cervix leading up to birth. Rather, other receptors may contribute to the progesterone-dependent mechanism that promotes remodeling of the cervix during pregnancy and in the proinflammatory process associated with ripening before parturition. PMID:21613631

  16. Studies on the mechanism of functional cooperativity between progesterone and estrogen receptors.

    Science.gov (United States)

    Bradshaw, M S; Tsai, S Y; Leng, X H; Dobson, A D; Conneely, O M; O'Malley, B W; Tsai, M J

    1991-09-05

    Steroid response elements (SREs) cooperate with many different cis-acting elements including NF-1 sites, CACCC boxes, and other SREs to induce target gene expression (Schule, R., Muller, M., Otsuka-Murakami, H., and Renkawitz, R. (1988) Nature 332, 87-90; Strahle, U., Schmid, W., and Schutz, G. (1988) EMBO J. 7, 3389-3395). Induction of gene expression can be additive or synergistic with respect to the level of activation by either transactivators. Two mechanisms have been proposed for how synergism occurs: 1) cooperative binding of transcriptional activators to DNA or 2) simultaneous interaction of individually bound activators with a common target protein. We have shown previously that cooperative binding of receptors is important for synergism between two progesterone response elements (PREs). Here we showed that an estrogen response element (ERE) and a PRE can also functionally cooperate and this synergism between an ERE and a PRE is not contributed by cooperative DNA binding. Furthermore, we have demonstrated that the activation domains of the progesterone receptor (PR) (C1Act) are required for synergism between two PREs and sufficient for confirming cooperative binding. However these two activation domains of PR are not sufficient for synergism between an ERE and a PRE. Additional regions within the NH2-terminal and COOH-terminal domains are also required for synergistic interaction between two heterologous SREs.

  17. Distinct functions and regulation of epithelial progesterone receptor in the mouse cervix, vagina, and uterus.

    Science.gov (United States)

    Mehta, Fabiola F; Son, Jieun; Hewitt, Sylvia C; Jang, Eunjung; Lydon, John P; Korach, Kenneth S; Chung, Sang-Hyuk

    2016-04-05

    While the function of progesterone receptor (PR) has been studied in the mouse vagina and uterus, its regulation and function in the cervix has not been described. We selectively deleted epithelial PR in the female reproductive tracts using the Cre/LoxP recombination system. We found that epithelial PR was required for induction of apoptosis and suppression of cell proliferation by progesterone (P4) in the cervical and vaginal epithelium. We also found that epithelial PR was dispensable for P4 to suppress apoptosis and proliferation in the uterine epithelium. PR is encoded by the Pgr gene, which is regulated by estrogen receptor α (ERα) in the female reproductive tracts. Using knock-in mouse models expressing ERα mutants, we determined that the DNA-binding domain (DBD) and AF2 domain of ERα were required for upregulation of Pgr in the cervix and vagina as well as the uterine stroma. The ERα AF1 domain was required for upregulation of Pgr in the vaginal stroma and epithelium and cervical epithelium, but not in the uterine and cervical stroma. ERα DBD, AF1, and AF2 were required for suppression of Pgr in the uterine epithelium, which was mediated by stromal ERα. Epithelial ERα was responsible for upregulation of epithelial Pgr in the cervix and vagina. Our results indicate that regulation and functions of epithelial PR are different in the cervix, vagina, and uterus.

  18. Effect of progesterone receptor status on maspin synthesis via nitric oxide production in neutrophils in human breast cancer.

    Science.gov (United States)

    Ganguly Bhattacharjee, Karabi; Bhattacharyya, Mau; Halder, Umesh Chandra; Jana, Pradipta; Sinha, Asru K

    2014-09-01

    Although progesterone receptor (PR) status, similarly to estrogen receptor status, is of prognostic importance in breast cancer, the involvement of the PR in breast cancer remains obscure. Studies were conducted to determine the function of the PR in neutrophils in the nitric oxide-induced synthesis of maspin, an anti-breast-cancer protein produced in nonmalignant mammary cells and in neutrophils in the circulation. PR status was determined by immunohistochemistry. Maspin synthesis was determined by in-vitro translation of messenger RNA and quantified by enzyme-linked immunosorbent assay. Nitric oxide was determined by the methemoglobin method. It was found that PR status in neutrophils was identical with that in malignant breast tissues. A Scatchard plot for progesterone binding to normal and PR-positive (PR+) neutrophils revealed that whereas normal neutrophils had 11.5 × 10(10) PR sites/cell with K d = 47.619 nM, PR+ neutrophils had 6.6 × 10(10) PR sites/cell with K d = 47.619 nM. The progesterone negative (PR-) neutrophils failed to bind to progesterone. Incubation of normal and PR+ neutrophils with 25 nM progesterone produced 1.317 μM NO and 2.329 nM maspin; the PR+ neutrophils produced 0.72 μM NO and 1.138 nM maspin. The PR- neutrophils failed to produce any NO or maspin in the presence of progesterone. Inhibition of progesterone-induced NO synthesis led to complete inhibition of maspin synthesis in all neutrophils. These results suggest that estrogen and progesterone complement each other in NO-induced maspin synthesis, and do not necessarily antagonize in the synthesis of the anti-breast-cancer protein.

  19. Membrane progesterone receptor alpha as a potential prognostic biomarker for breast cancer survival: a retrospective study.

    Directory of Open Access Journals (Sweden)

    Mingxuan Xie

    Full Text Available Classically, the actions of progesterone (P4 are attributed to the binding of nuclear progesterone receptor (PR and subsequent activation of its downstream target genes. These mechanisms, however, are not applicable to PR- or basal phenotype breast cancer (BPBC due to lack of PR in these cancers. Recently, the function of membrane progesterone receptor alpha (mPRα in human BPBC cell lines was studied in our lab. We proposed that the signaling cascades of P4→mPRα pathway may play an essential role in controlling cell proliferation and epithelial mesenchymal transition (EMT of breast cancer. Using human breast cancer tissue microarrays, we found in this study that the average intensity of mPRα expression, but not percentage of breast cancer with high level of mPRα expression (mPRα-HiEx, was significantly lower in the TNM stage 4 patients compared to those with TNM 1-3 patients; and both average intensities of mPRα expression and mPRα-HiEx rates were significantly higher in cancers negative for ER, as compared with those cancers with ER+. However, after adjusting for age at diagnosis and/or TNM stage, only average intensities of mPRα expression were associated with ER status. In addition, we found that the rates of mPRα-HiEx were significantly higher in cancers with epithelial growth factor receptor-1 (EGFR+ and high level of Ki67 expression, indicating positive correlation between mPRα over expression and EGFR or Ki67. Further analysis indicated that both mPRα-HiEx rate and average intensity of mPRα expression were significantly higher in HER2+ subtype cancers (i.e. HER2+ER-PR- as compared to ER+ subtype cancers. These data support our hypothesis that P4 modulates the activities of the PI3K and cell proliferation pathways through the caveolar membrane bound growth factor receptors such as mPRα and growth factor receptors. Future large longitudinal studies with larger sample size and survival outcomes are necessary to confirm our

  20. A novel progesterone receptor membrane component (PGRMC) in the human and swine parasite Taenia solium: implications to the host-parasite relationship.

    Science.gov (United States)

    Aguilar-Díaz, Hugo; Nava-Castro, Karen E; Escobedo, Galileo; Domínguez-Ramírez, Lenin; García-Varela, Martín; Del Río-Araiza, Víctor H; Palacios-Arreola, Margarita I; Morales-Montor, Jorge

    2018-03-09

    We have previously reported that progesterone (P 4 ) has a direct in vitro effect on the scolex evagination and growth of Taenia solium cysticerci. Here, we explored the hypothesis that the P 4 direct effect on T. solium might be mediated by a novel steroid-binding parasite protein. By way of using immunofluorescent confocal microscopy, flow cytometry analysis, double-dimension electrophoresis analysis, and sequencing the corresponding protein spot, we detected a novel PGRMC in T. solium. Molecular modeling studies accompanied by computer docking using the sequenced protein, together with phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is from parasite origin. Our results show that P 4 in vitro increases parasite evagination and scolex size. Using immunofluorescent confocal microscopy, we detected that parasite cells showed expression of a P 4 -binding like protein exclusively located at the cysticercus subtegumental tissue. Presence of the P 4 -binding protein in cyst cells was also confirmed by flow cytometry. Double-dimension electrophoresis analysis, followed by sequencing the corresponding protein spot, revealed a protein that was previously reported in the T. solium genome belonging to a membrane-associated progesterone receptor component (PGRMC). Molecular modeling studies accompanied by computer docking using the sequenced protein showed that PGRMC is potentially able to bind steroid hormones such as progesterone, estradiol, testosterone and dihydrodrotestosterone with different affinities. Phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is related to a steroid-binding protein of Echinoccocus granulosus, both of them being nested within a cluster including similar proteins present in platyhelminths such as Schistocephalus solidus and Schistosoma haematobium. Progesterone may directly act upon T. solium cysticerci probably by binding to PGRMC. This research has implications in the

  1. Estrogen and progesterone receptor testing in breast carcinoma: concordance of results between local and reference laboratories in Brazil

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    Sheila Cristina Lordelo Wludarski

    Full Text Available CONTEXT AND OBJECTIVE: Breast cancer accounts for approximately one quarter of all cancers in females. Estrogen and progesterone receptor testing has become an essential part of the clinical evaluation of breast carcinoma patients, and accurate results are critical in identifying patients who may benefit from hormone therapy. The present study had the aim of investigating the concordance of the results from hormone receptor tests between a reference laboratory and local (or community laboratories in Brazil. DESIGN AND SETTING: Retrospective study at a reference pathology laboratory. METHODS: The concordance in the results from hormone receptor tests between a reference laboratory and 146 local laboratories in Brazil was compared in relation to 500 invasive breast carcinoma cases, using immunohistochemistry. RESULTS: There was concordance in 89.4% (447/500 cases and 85.0% (425/500 cases of the results from estrogen (κ = 0.744, P < 0.001 and progesterone (κ = 0.688, P < 0.001 receptor tests, respectively, between local and reference laboratories. This was similar to findings in other countries. The false negative rates from estrogen and progesterone receptor tests in local laboratories were 8.7% and 14.4%, respectively. The false positive rates from estrogen and progesterone receptor tests in local laboratories were 15.5% and 16.0%, respectively. CONCLUSION: Technical and result interpretation issues may explain most of the discordances in hormone receptor testing in local laboratories. Validation of estrogen and progesterone receptor tests at local laboratories, with rigorous quality control measures, is strongly recommended in order to avoid erroneous treatment of breast cancer patients.

  2. Sulfhydryl group content of chicken progesterone receptor: effect of oxidation on DNA binding activity

    International Nuclear Information System (INIS)

    Peleg, S.; Schrader, W.T.; O'Malley, B.W.

    1988-01-01

    DNA binding activity of chicken progesterone receptor B form (PRB) and A form (PRA) has been examined. This activity is strongly dependent upon the presence of thiols in the buffer. Stability studies showed that PRB was more sensitive to oxidation that was PRA. Receptor preparations were fractionated by DNA-cellulose chromatography to DNA-positive and DNA-negative subpopulations, and sulfhydryl groups were quantified on immunopurified receptor by labeling with [ 3 H]-N-ethylmaleimide. Labeling of DNA-negative receptors with [ 3 H]-N-ethylmaleimide showed 21-23 sulfhydryl groups on either PRA or PRB form when the proteins were reduced and denatured. A similar number was seen without reduction if denatured DNA-positive receptor species were tested. In contrast, the DNA-negative PRB had only 10-12 sulfhydryl groups detectable without reduction. A similar number (12-13 sulfhydryl groups) was found for PRA species that lost DNA binding activity after exposure to a nonreducing environment in vitro. The authors conclude that the naturally occurring receptor forms unable to bind to DNA, as well as receptor forms that have lost DNA binding activity due to exposure to nonreducing environment in vitro, contain 10-12 oxidized cysteine residues, likely present as disulfide bonds. Since they were unable to reduce the disulfide bonds when the native DNA-negative receptor proteins were treated with dithiothreitol (DTT), they speculate that irreversible loss of DNA binding activity of receptor in vitro is due to oxidation of cysteine residues that are not accessible to DTT in the native state

  3. Oestrogen and progesterone receptor assays in breast tumours. The Prince Henry's Hospital experience, 1983-1990.

    Science.gov (United States)

    Pearce, P T; Myles, K M; Funder, J W

    1993-08-16

    To present and analyse the results of eight years of experience (1983-1990) in breast tumour receptor analysis. All female primary breast tumour samples received (4683) were analysed for seasonal variation, patient age, relative risk index, oestrogen receptor (ER) and progesterone receptor (PR) status, ER and PR status as a function of age, ER and PR levels as a function of age, and ER and PR levels as a function of month of analysis. The assays were done at the Medical Research Centre, Prince Henry's Hospital, Melbourne, as a non-profit service to surgeons, oncologists and pathologists. The numbers of samples referred for assay increased progressively each year, from 473 in 1983 to 1097 in 1990, but the receptor status (ER +/-, PR +/-) appeared not to vary from year to year. ER+PR+ tumours were the most common in all age groups, steadily increasing from between 50% and 60% in premenopausal women to 70% or more in those aged over 80. In postmenopausal women, levels of ER in ER+ tumours were three times those in premenopausal women; PR levels in PR+ tumours, however, were bimodal, with higher levels in the age groups 35-49 and 70-89 years than in women aged 50-69 years. No significant seasonal variation was seen, and the overall patterns of receptor status are similar to those seen in Northern hemisphere studies.

  4. Affinity of Iresine herbstii and Brugmansia arborea extracts on different cerebral receptors.

    Science.gov (United States)

    Nencini, Cristina; Cavallo, Federica; Bruni, Giancarlo; Capasso, Anna; De Feo, Vincenzo; De Martino, Laura; Giorgi, Giorgio; Micheli, Lucia

    2006-05-24

    Iresine herbstii Hook. (Amaranthaceae) and Brugmansia arborea (L.) Lagerheim (Solanaceae) are used in the northern Peruvian Andes for magic-therapeutical purposes. The traditional healers use Iresine herbstii with the ritual aim to expel bad spirits from the body. Furthermore, Iresine herbstii was used in association with other plants, such as Trichocereus pachanoi Britt. et Rose, for divination, to diagnose diseases, and to take possession of another identity. Also, species of Brugmansia have been reported to be used during ritual practices for magical and curative purposes. Given the above evidence, the aim of the present study is to evaluate if the central effects of Iresine herbstii and Brugmansia arborea could be associated with interaction with SNC receptors. Two Iresine herbstii extracts (methanolic and aqueous) and one Brugmansia arborea aqueous extract were tested for in vitro affinity on 5-HT(1A), 5-HT(2A), 5-HT(2C), D1, D2, alpha(1), and alpha(2) receptors by radioligand binding assays. The biological materials for binding assay (cerebral cortex) were taken from male Sprague-Dawley rats. The extracts affinity for receptors is definite as inhibition percentage of radioligand/receptor binding and measured as the radioactivity of remaining complex radioligand/receptor. The data obtained for Iresine extracts have shown a low affinity for the 5-HT(1A) receptor and no affinity for 5-HT(2A) receptor. Otherwise the methanolic extract showed affinity for 5-HT(2C) receptor (IC(50): 34.78 microg/ml) and for D1 receptor (IC(50): 19.63 microg/ml), instead the Iresine aqueous extract displayed a lower affinity for D1 (48.3% at the maximum concentration tested) and a higher value of affinity for D2 receptors (IC(50): 32.08 microg/ml). The Brugmansia aqueous extract displayed affinity for D1 receptors (IC(50): 17.68 microg/ml), D2 receptors (IC(50): 15.95 microg/ml) and weak affinity for the serotoninergic receptors. None of the three extracts showed relevant affinity

  5. Efficient Prediction of Progesterone Receptor Interactome Using a Support Vector Machine Model

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    Ji-Long Liu

    2015-03-01

    Full Text Available Protein-protein interaction (PPI is essential for almost all cellular processes and identification of PPI is a crucial task for biomedical researchers. So far, most computational studies of PPI are intended for pair-wise prediction. Theoretically, predicting protein partners for a single protein is likely a simpler problem. Given enough data for a particular protein, the results can be more accurate than general PPI predictors. In the present study, we assessed the potential of using the support vector machine (SVM model with selected features centered on a particular protein for PPI prediction. As a proof-of-concept study, we applied this method to identify the interactome of progesterone receptor (PR, a protein which is essential for coordinating female reproduction in mammals by mediating the actions of ovarian progesterone. We achieved an accuracy of 91.9%, sensitivity of 92.8% and specificity of 91.2%. Our method is generally applicable to any other proteins and therefore may be of help in guiding biomedical experiments.

  6. Expression of progesterone receptor membrane component-1 in bovine reproductive system during estrous cycle

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    A.M. Luciano

    2011-09-01

    Full Text Available Several reports suggest the participation of progesterone receptor membrane component 1 (PGRMC1 in progesterone signaling in the reproductive system. This study aimed at investigating the presence and localization of PGRMC1 in bovine ovary, oviduct and uterus, during the follicular and luteal phases of the estrous cycle. In the ovary, PGRMC1 has been detected in surface germinal epithelium, granulosa cells, theca cells and in the germinal vesicle of the oocytesat all stages of folliculogenesis. In the corpus luteum the expression of PGRMC1 was influenced by the stage of the estrous cycle. In the oviducts and in the uterus horns, PGRMC1 was immunolocalized in the luminal epithelium, in the muscle layer cells and in the endothelial cells. In the uterus, PGRMC1 was intensely localized also in the glandular endometrium. However, in the oviducts and in the uterus horns, the localization of PGRMC1 was independent on the stage of the estrous cycle and on whether evaluating the ipsilateral or the contralateral organ. In conclusion, the present immunohistochemical study showed that PGRMC1 is located in various compartments of the bovine female reproductive organs. With the exception of the corpora lutea, PGRMC1 localization showed similar pattern during different stage of the estrous cycle.

  7. Interactions of p60, a mediator of progesterone receptor assembly, with heat shock proteins hsp90 and hsp70

    DEFF Research Database (Denmark)

    Chen, S; Prapapanich, V; Rimerman, R A

    1996-01-01

    Previous studies on the assembly of progesterone receptor (PR) complexes in vitro have suggested that PR assembly is a dynamic, ordered process involving at least eight nonreceptor proteins. One of these proteins, p60, appears transiently during assembly and is not a component of functionally...

  8. ANTAGONISM OF PROGESTERONE RECEPTOR SUPPRESSES CAROTID BODY RESPONSES TO HYPOXIA AND NICOTINE IN RAT PUPS

    Science.gov (United States)

    JOSEPH, V.; NIANE, L. M.; BAIRAM, A.

    2013-01-01

    We tested the hypothesis that antagonism of progesterone receptor (PR) in newborn rats alters carotid body and respiratory responses to hypoxia and nicotinic receptor agonists. Rats were treated with the PR antagonist mifepristone (daily oral gavage 40 μg/g/d) or vehicle between post-natal days 3 and 15. In 11–14-day-old rats, we used in vitro carotid body/carotid sinus nerve preparation and whole body plethysmography to assess the carotid body and ventilatory responses to hypoxia (65 mmHg in vitro, 10% O2 in vivo) and to nicotinic receptor agonists (as an excitatory modulator of carotid body activity—nicotine 100 μM for in vitro studies, and epibatidine 5 μg/kg, i.p., which mainly acts on peripheral nicotinic receptors, for in vivo studies). The carotid body responses to hypoxia and nicotine were drastically reduced by mifepristone. Compared with vehicle, mifepristone-treated rats had a reduced body weight. The ventilatory response to epibatidine was attenuated; however, the hypoxic ventilatory response was similar between vehicle and mifepristone-treated pups. Immunohistochemical staining revealed that mifepristone treatment did not change carotid body morphology. We conclude that PR activity is a critical factor ensuring proper carotid body function in newborn rats. PMID:22326965

  9. Relationship of oestrus synchronization method, circulating hormones, luteinizing hormone and prostaglandin F-2 alpha receptors and luteal progesterone concentration to premature luteal regression in superovulated sheep.

    Science.gov (United States)

    Schiewe, M C; Fitz, T A; Brown, J L; Stuart, L D; Wildt, D E

    1991-09-01

    Ewes were treated with exogenous follicle-stimulating hormone (FSH) and oestrus was synchronized using either a dual prostaglandin F-2 alpha (PGF-2 alpha) injection regimen or pessaries impregnated with medroxy progesterone acetate (MAP). Natural cycling ewes served as controls. After oestrus or AI (Day 0), corpora lutea (CL) were enucleated surgically from the left and right ovaries on Days 3 and 6, respectively. The incidence of premature luteolysis was related (P less than 0.05) to PGF-2 alpha treatment and occurred in 7 of 8 ewes compared with 0 of 4 controls and 1 of 8 MAP-exposed females. Sheep with regressing CL had lower circulating and intraluteal progesterone concentrations and fewer total and small dissociated luteal cells on Day 3 than gonadotrophin-treated counterparts with normal CL. Progesterone concentration in the serum and luteal tissue was higher (P less than 0.05) in gonadotrophin-treated ewes with normal CL than in the controls; but luteinizing hormone (LH) receptors/cell were not different on Days 3 and 6. There were no apparent differences in the temporal patterns of circulating oestradiol-17 beta, FSH and LH. High progesterone in gonadotrophin-treated ewes with normal CL coincided with an increase in total luteal mass and numbers of cells, which were primarily reflected in more small luteal cells than in control ewes. Gonadotrophin-treated ewes with regressing CL on Day 3 tended (P less than 0.10) to have fewer small luteal cells and fewer (P less than 0.05) low-affinity PGF-2 alpha binding sites than sheep with normal CL. By Day 6, luteal integrity and cell viability was absent in ewes with prematurely regressed CL. These data demonstrate that (i) the incidence of premature luteal regression is highly correlated with the use of PGF-2 alpha; (ii) this abnormal luteal tissue is functionally competent for 2-3 days after ovulation, but deteriorates rapidly thereafter and (iii) luteal-dysfunctioning ewes experience a reduction in numbers of

  10. The relationship of cerb B 2 expression with estrogen receptor and progesterone receptor and prognostic parameters in endometrial carcinomas

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    Kandemir Nilufer

    2010-02-01

    Full Text Available Abstract Background Endometrial carcinoma (EC is the most common malignancy of the female genital tract. Gene alterations and overexpression of various oncogenes are important in tumor development. The human HER 2 neu (c-erbB-2 gene product is a transmembrane receptor with an intracellular tyrosine kinase that plays an important role in coordinating the endometrial growth factor receptor signaling network. The aim of this study was to investigate the expression of c-erbB-2 in endometrial cancer, to study its correlation to established prognostic parameters and estrogen receptor (ER and progesterone receptor (PR status. Methods Immunohistochemical (IHC analyses of ER, PR and c-erbB-2 were performed in 72 EC cases. Results We detected a positive staining with c erbB 2 in 18.1% of the cases and determined a statistically significant relation between c-erbB-2 and PR. We could not find a statistically significant relation between c-erbB-2 staining and ER. There was not a statistically significant difference between c-erbB-2 and histological grade. The highest level of c-erbB-2 was found in grade 2 cases. There was not any statistically significant relation between c-erbB-2 and menstrual status, myometrial invasion, lymph node status, stage and survival. Conclusions Although our study provides additional evidence of the potential prognostic role of c-erbB-2, further prospective and controlled studies are required to validate their clinical usefulness.

  11. Potential contribution of progesterone receptors to the development of sexual behavior in male and female mice.

    Science.gov (United States)

    Desroziers, Elodie; Brock, Olivier; Bakker, Julie

    2017-04-01

    We previously showed that estradiol can have both defeminizing and feminizing effects on the developing mouse brain. Pre- and early postnatal estradiol defeminized the ability to show lordosis in adulthood, whereas prepubertal estradiol feminized this ability. Furthermore, we found that estradiol upregulates progesterone receptors (PR) during development, inducing both a male-and female-typical pattern of PR expression in the mouse hypothalamus. In the present study, we took advantage of a newly developed PR antagonist (ZK 137316) to determine whether PR contributes to either male- or female-typical sexual differentiation. Thus groups of male and female C57Bl/6j mice were treated with ZK 137316 or OIL as control: males were treated neonatally (P0-P10), during the critical period for male sexual differentiation, and females were treated prepubertally (P15-P25), during the critical period for female sexual differentiation. In adulthood, mice were tested for sexual behavior. In males, some minor effects of neonatal ZK treatment on sexual behavior were observed: latencies to the first mount, intromission and ejaculation were decreased in neonatally ZK treated males; however, this effect disappeared by the second mating test. By contrast, female mice treated with ZK during the prepubertal period showed significantly less lordosis than OIL-treated females. Mate preferences were not affected in either males or females treated with ZK during development. Taken together, these results suggest a role for PR and thus perhaps progesterone in the development of lordosis behavior in female mice. By contrast, no obvious role for PR can be discerned in the development of male sexual behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Induction of parturition in the bitch with the progesterone-receptor blocker aglépristone.

    Science.gov (United States)

    Baan, M; Taverne, M A M; Kooistra, H S; de Gier, J; Dieleman, S J; Okkens, A C

    2005-04-15

    The triggering mechanism for parturition in the bitch remains unclear. Consequently, the development of drugs to successfully induce parturition in the dog has been difficult. The aim of this study was to evaluate the efficacy of the progesterone-receptor blocker aglépristone for the induction of parturition in beagle bitches. The course of parturition was therefore investigated in six parturitions induced by aglépristone and in six spontaneous parturitions. In addition, data were collected on pup survival and growth rates. Aglépristone was administered twice with a 9h interval on day 58 of pregnancy. If parturition did not proceed a standard intervention protocol was applied. Expulsion of the first pup occurred between 32 and 56 h after the first treatment with aglépristone, at which time the plasma progesterone concentration was still elevated. Accordingly, the gestation length of the bitches in the induced group (59.5+/-0.2 days) was significantly shorter than that of the spontaneously whelping bitches (62.2+/-0.5 days). The expulsion phase length, the inter-pup interval, the number of puppies born dead, and the number of clinical interventions needed during parturition did not significantly differ between the spontaneously whelping and the induced group. Pup survival and mean birth weights in the two groups did not differ significantly and aglépristone treatment had no significant influence on the growth rates. The results of this study show that aglépristone is an effective drug which can be used safely for the induction of parturition in the dog.

  13. Progesterone receptor expression during prostate cancer progression suggests a role of this receptor in stromal cell differentiation.

    Science.gov (United States)

    Yu, Yue; Yang, Ou; Fazli, Ladan; Rennie, Paul S; Gleave, Martin E; Dong, Xuesen

    2015-07-01

    The progesterone receptor, like the androgen receptor, belongs to the steroid receptor superfamily. Our previous studies have reported that the PR is expressed specifically in prostate stroma. PR inhibits proliferation of, and regulates cytokine secretion by stromal cells. However, PR protein expression in cancer-associated stroma during prostate cancer progression has not been profiled. Since the phenotypes of prostate stromal cells change dynamically as tumors progress, whether the PR plays a role in regulating stromal cell differentiation needs to be investigated. Immunohistochemistry assays measured PR protein levels on human prostate tissue microarrays containing 367 tissue cores from benign prostate, prostate tumors with different Gleason scores, tumors under various durations of castration therapy, and tumors at the castration-resistant stage. Immunoblotting assays determined whether PR regulated the expression of alpha smooth muscle actin (α-SMA), vimentin, and fibroblast specific protein (FSP) in human prostate stromal cells. PR protein levels decreased in cancer-associated stroma when compared with that in benign prostate stroma. This reduction in PR expression was not correlated with Gleason scores. PR protein levels were elevated by castration therapy, but reduced to pre-castration levels when tumors progressed to the castration-resistant stage. Enhanced PR expression in human prostate stromal cells increased α-SMA, but decreased vimentin and FSP protein levels ligand-independently. These results suggest that PR plays an active role in regulating stromal cell phenotypes during prostate cancer progression. © 2015 Wiley Periodicals, Inc.

  14. The impact of luteal phase support on endometrial estrogen and progesterone receptor expression: a randomized control trial

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    Brezina Paul R

    2012-02-01

    Full Text Available Abstract Background To assess the impact of luteal phase support on the expression of estrogen receptor (ER alpha and progesterone receptors B (PR-B on the endometrium of oocyte donors undergoing controlled ovarian hyperstimulation (COH. Methods A prospective, randomized study was conducted in women undergoing controlled ovarian hyperstimulation for oocyte donation. Participants were randomized to receive no luteal support, vaginal progesterone alone, or vaginal progesterone plus orally administered 17 Beta estradiol. Endometrial biopsies were obtained at 4 time points in the luteal phase and evaluated by tissue microarray for expression of ER alpha and PR-B. Results One-hundred and eight endometrial tissue samples were obtained from 12 patients. No differences were found in expression of ER alpha and PR-B among all the specimens with the exception of one sample value. Conclusions The administration of progesterone during the luteal phase of COH for oocyte donor cycles, either with or without estrogen, does not significantly affect the endometrial expression of ER alpha and PR.

  15. [The molecular mechanisms and morphological manifestations of leiomyoma reduction induced by selective progesterone receptor modulators].

    Science.gov (United States)

    Demura, T A; Revazova, Z V; Kogan, E A; Adamyan, L V

    to investigate the molecular mechanisms and morphological substrate of reduced uterine leiomyoma in patients receiving the selective progesterone receptor modulator (SPRM) ulipristal acetate for 3 months, by estimating the immunohistochemical expression of the markers steroid receptor coactivator 1 (SRC-1), nuclear receptor corepressor 1 (NCoR-1), ER, PgR, Ki-67, p16, TGF-β, and VEGF in tumor tissue. The investigation enrolled 75 women with uterine leiomyoma, menorrhagias, and anemia. Group 1 included 40 patients who were treated with ulipristal for 3 months, followed by laparoscopic myomectomy. Group 2 consisted of 35 patients who underwent surgery without previous preparation. The intra- and postoperative parameters and molecular and morphological changes in the myomatous nodules were comparatively analyzed in both groups. After 3 months of therapy initiation, menorrhagia completely ceased, myomatous nodules decreased in size (pleiomyoma reduction was leiomyocyte apoptosis and dystrophy, tumor stroma sclerosis and hyalinosis with diminished Ki-67 expression and elevated p16 in the smooth muscle cells, trophic nodular tissue disorders exhibited by vascular wall sclerosis and lower VEGF and TGF-β expression, and leiomyocyte hormonal reception dysregulation that made itself evident through the reduced expression of SRC-1 with the unchanged expression of PR and ER and the maintained level of NCoR-1. The molecular mechanisms of tumor reduction involved the reduced Ki-67 expression and elevated p16, lower VEGF and TGF-β, diminished SRC-1 expression with the maintained level of PR, ER, and NCoR-1. Overall, this is suggestive of enhanced apoptosis and reduced leiomyoma proliferation and angiogenesis induced by SPRM and indicative of the expediency of using ulipristal acetate as a preoperative agent for organ-sparing surgery in reproductive-aged patients with uterine myoma, menorrhagias, and anemia.

  16. Provision of a simplified methodology for determining estradiol and progesterone receptors in human breast tumours. Internal and external quality control

    International Nuclear Information System (INIS)

    Farinate, Z.

    1990-10-01

    A simplified assay for the detection of progesterone receptors (PR) in human breast tissue is described. Tissue storage is at -20 deg. C rather than -70 deg. C and a centrifugation speed of 20,000 rpm avoids requirement of an ultracentrifuge. Cytosol preparations obtained from homogenized oestradiol benzoate primed wistar rat uteri performed satisfactorily as positive controls with stability of two months in liquid nitrogen. The use of iodinated tracer (progesterone 11 alpha glucuronide 125 I iodotyramine) proved disappointing in the progesterone receptor assay in contrast to 125 I oestradiol which worked well in a oestrogen receptor assay, previously developed. Hydroxyl-apatite was a better separating agent than dextran coated charcoal in both assays and yielded better sensitivity, particularly when protein concentrations were low. Five breast cancer specimens assayed yielded, by Scatchard analysis, Kd values between 12 to 22x10 -9 m|h, comparable to the positive controls. However, two of these had binding site capacity of less than 5 fmol/mg cytosol as compared to the three others and the positive controls where values ranged from 47-196 fmol/mg cytosol. 28 refs, 6 figs, 14 tabs

  17. Gastrin receptor characterization: affinity cross-linking of the gastrin receptor on canine gastric parietal cells

    International Nuclear Information System (INIS)

    Matsumoto, M.; Park, J.; Yamada, T.

    1987-01-01

    The authors applied affinity cross-linking methods to label the gastrin receptor on isolated canine gastric parietal cells in order to elucidate the nature of its chemical structure. 125 I-labeled Leu 15 -gastrin and 125 I-labeled gastrin/sub 2-17/ bound to intact parietal cells and their membranes with equal affinity, and half-maximal inhibition of binding was obtained at an incubation concentration of 3.2 x 10 -10 M unlabeled gastrin. 125 I-gastrin/sub 2-17/ was cross-linked to plasma membranes or intact parietal cells by incubation in disuccinimidyl suberate. The membrane pellets were solubilized with or without dithiothreitol and applied to electrophoresis on 7.5% sodium dodecyl sulfate polyacrylamide gels. Autoradiograms revealed a band of labeling at M/sub r/ 76,000 and labeling of this band was inhibited in a dose-dependent fashion by addition of unlabeled gastrin to the incubation mixture. Dithiothreitol in concentrations as high as 100 mM did not later the electrophoretic mobility of the labeled band. After taking into account the molecular weight of 125 I-gastrin/sub 2-17/, the results suggest that the gastrin receptor on parietal cells is a single protein of M/sub r/ 74,000 without disulfide-linked subunits

  18. Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

    DEFF Research Database (Denmark)

    Andersen, P S; Geisler, C; Buus, S

    2001-01-01

    Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3...... (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency...... correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength....

  19. Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

    Science.gov (United States)

    Yu, Yue; Lee, Jennifer Suehyun; Xie, Ning; Li, Estelle; Hurtado-Coll, Antonio; Fazli, Ladan; Cox, Michael; Plymate, Stephen; Gleave, Martin; Dong, Xuesen

    2014-01-01

    Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR) was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood. Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels. Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1) and interlukin-6 (IL-6) by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion. Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

  20. Clinical utility of progesterone receptor modulators and their effect on the endometrium.

    Science.gov (United States)

    Spitz, Irving M

    2009-08-01

    In view of the spate of recent publications related to mifepristone and some second generation progesterone receptor modulators (PRMs), this appears to be an opportune time to view the clinical status of these compounds. Randomized double-blind placebo-controlled trials have been conducted with mifepristone, CDB-4124 (Proellex), CDB-2914 (VA 2914, Ulipristal) and asoprisnil (J867). All these PRMs are effective in the treatment of uterine fibroids where they are associated with a reduction in pain, bleeding and improvement in quality of life and decrease in fibroid size. CDB-4124 is also efficacious in endometriosis. Long-term treatment with PRMs may be associated with endometrial thickening on ultrasound and there have been reports of endometrial hyperplasia. Several reassuring recent publications have done much to explain the mechanism underlying these endometrial changes. The most common histological finding is cystic glandular dilatation often associated with both admixed estrogen (mitotic) and progestin (secretory) epithelial effects. This histology has not been previously encountered in clinical practice and should not be confused with endometrial hyperplasia. The endometrial thickness is related to this cystic glandular dilatation. At this stage of development, PRMs cannot be administered for longer than 3 or 4 months. Even over this time, there is improvement of symptoms associated with fibroids and endometriosis. Clinicians and pathologists need to be aware that the endometrial thickening and histological appearance do not represent endometrial hyperplasia.

  1. American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer.

    NARCIS (Netherlands)

    Hammond, M.E.; Hayes, D.F.; Dowsett, M.; Allred, D.C.; Hagerty, K.L.; Badve, S.; Fitzgibbons, P.L.; Francis, G.; Goldstein, N.S.; Hayes, M.; Hicks, D.G.; Lester, S.; Love, R.; Mangu, P.B.; McShane, L.; Miller, K.; Osborne, C.K.; Paik, S.; Perlmutter, J.; Rhodes, A.; Sasano, H.; Schwartz, J.N.; Sweep, F.C.; Taube, S.; Torlakovic, E.E.; Valenstein, P.; Viale, G.; Visscher, D.; Wheeler, T.; Williams, R.B.; Wittliff, J.L.; Wolff, A.C.

    2010-01-01

    PURPOSE: To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers. METHODS: The American Society of Clinical Oncology and the College of

  2. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer.

    NARCIS (Netherlands)

    Hammond, M.E.; Hayes, D.F.; Dowsett, M.; Allred, D.C.; Hagerty, K.L.; Badve, S.; Fitzgibbons, P.L.; Francis, G.; Goldstein, N.S.; Hayes, M.; Hicks, D.G.; Lester, S.; Love, R.; Mangu, P.B.; McShane, L.; Miller, K.; Osborne, C.K.; Paik, S.; Perlmutter, J.; Rhodes, A.; Sasano, H.; Schwartz, J.N.; Sweep, F.C.; Taube, S.; Torlakovic, E.E.; Valenstein, P.; Viale, G.; Visscher, D.; Wheeler, T.; Williams, R.B.; Wittliff, J.L.; Wolff, A.C.

    2010-01-01

    PURPOSE: To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers. METHODS: The American Society of Clinical Oncology and the College of

  3. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version).

    NARCIS (Netherlands)

    Hammond, M.E.; Hayes, D.F.; Dowsett, M.; Allred, D.C.; Hagerty, K.L.; Badve, S.; Fitzgibbons, P.L.; Francis, G.; Goldstein, N.S.; Hayes, M.; Hicks, D.G.; Lester, S.; Love, R.; Mangu, P.B.; McShane, L.; Miller, K.; Osborne, C.K.; Paik, S.; Perlmutter, J.; Rhodes, A.; Sasano, H.; Schwartz, J.N.; Sweep, F.C.; Taube, S.; Torlakovic, E.E.; Valenstein, P.; Viale, G.; Visscher, D.; Wheeler, T.; Williams, R.B.; Wittliff, J.L.; Wolff, A.C.

    2010-01-01

    PURPOSE: To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers. METHODS: The American Society of Clinical Oncology and the College of

  4. Bioinformatic analysis of cis-regulatory interactions between progesterone and estrogen receptors in breast cancer

    Directory of Open Access Journals (Sweden)

    Matloob Khushi

    2014-11-01

    Full Text Available Chromatin factors interact with each other in a cell and sequence-specific manner in order to regulate transcription and a wealth of publically available datasets exists describing the genomic locations of these interactions. Our recently published BiSA (Binding Sites Analyser database contains transcription factor binding locations and epigenetic modifications collected from published studies and provides tools to analyse stored and imported data. Using BiSA we investigated the overlapping cis-regulatory role of estrogen receptor alpha (ERα and progesterone receptor (PR in the T-47D breast cancer cell line. We found that ERα binding sites overlap with a subset of PR binding sites. To investigate further, we re-analysed raw data to remove any biases introduced by the use of distinct tools in the original publications. We identified 22,152 PR and 18,560 ERα binding sites (<5% false discovery rate with 4,358 overlapping regions among the two datasets. BiSA statistical analysis revealed a non-significant overall overlap correlation between the two factors, suggesting that ERα and PR are not partner factors and do not require each other for binding to occur. However, Monte Carlo simulation by Binary Interval Search (BITS, Relevant Distance, Absolute Distance, Jaccard and Projection tests by Genometricorr revealed a statistically significant spatial correlation of binding regions on chromosome between the two factors. Motif analysis revealed that the shared binding regions were enriched with binding motifs for ERα, PR and a number of other transcription and pioneer factors. Some of these factors are known to co-locate with ERα and PR binding. Therefore spatially close proximity of ERα binding sites with PR binding sites suggests that ERα and PR, in general function independently at the molecular level, but that their activities converge on a specific subset of transcriptional targets.

  5. Immunohistochemical features of progesterone receptors expression of placental barrier in women with multiple pregnancies resulting from assisted reproduction

    Directory of Open Access Journals (Sweden)

    T. D. Zadorozhna

    2016-01-01

    Full Text Available Hormonal disorders are one of the main known causes of miscarriage and preterm birth in multiple pregnancies resulting from assisted reproductive technology (ART. Progesterone and the number of its receptors play an important role in the preservation and prolongation of pregnancy and it is the pressing issue of our time. The study of placentas, as the main site of synthesis of progesterone, has high informative potential and it is the most important diagnostic object, and information received by its research is essential for the full conclusion on the causes, mechanisms, close and long-term effects of multiple pregnancy pathology. Aim. The aim of our study was to investigate immunohistochemical features of placentas from women with dichorionic diamniotic twin pregnancies in spontaneous fertilization and after use of assisted reproductive technology (ART. Methods and results. According to this goal we examined 94 women, 44 of whom had multiple pregnancies due to ART, 42 with separate multiple pregnancy and 38 women with a singleton pregnancy. We carried out clinical and statistical analysis of the course of pregnancy and childbirth in the studied groups. During the study it was found that multiple pregnancies due to assisted reproduction belong to the high risk of gestation, at which premature births occur much more frequently than in singleton pregnancies. We were the first to carry out the immunohistochemical study of placentas in which the highest expression of progesterone receptors in the nuclei of cells of decidua (45% related to the parent structure of the placenta from women with multiple pregnancies caused by ART is found. It is also found that with increasing gestational age, there has been a significant decrease in the expression of the activity of progesterone receptors (from 45 to 2.5%, regardless of the method of conception and the number of fetuses. Conclusions. The results of the study point to the definitive link of structures of

  6. The effects of black cohosh on the regulation of estrogen receptor (ERα) and progesterone receptor (PR) in breast cancer cells.

    Science.gov (United States)

    Szmyd, Monica; Lloyd, Victoria; Hallman, Kelly; Aleck, Katie; Mladenovik, Viktoria; McKee, Christina; Morse, Mia; Bedgood, Tyler; Dinda, Sumi

    2018-01-01

    The North American plant Cimicifuga racemosa , also known as black cohosh (BC), is a herb that recently has gained attention for its hormonal effects. As the usage of hormone replacement therapy is declining due to its adverse effects in women with cancer, many are turning to herbal remedies like BC to treat menopausal symptoms. It is crucial to determine whether the effects of BC involve estrogen receptor-alpha (ERα). Previous studies from our laboratory have shown ERα to be a possible molecular target for BC. In this study, we examined the effects of BC (8% triterpene glycosides) alone and in combination with hormones and antihormones on the cellular viability, expression of ERα and progesterone receptor (PR)-A/B, and cytolocalization of ERα in ER (+) and PR-A/B (+) T-47D breast cancer cells. Cells were cultured and proteins were extracted and quantified. Western blot analysis revealed alterations in the expression of ERα and PR after treatment with BC (5-100 µM). BC induced a concentration-dependent decrease in ERα and PR protein levels when compared to the control. Image cytometric analysis with propidium iodide staining was used to enumerate changes in T-47D cell number and viability. A decrease in T-47D cell viability was observed upon treatment with 5-100 µM BC. The ideal concentration of BC (100 µM) was used in combination with hormones and antihormones in an effort to further understand the possible similarities between this compound and other known effectors of ERα and PR. After a 24-hour concomitant treatment with and/or in combination of BC, estradiol, ICI 182, 780, and Tamoxifen, downregulation of ERα and PR protein levels was observed. Delineating the role of BC in the regulation of ERα, PR, as well as its mechanisms of action, may be important in understanding the influence of BC on hormone receptors in breast cancer.

  7. Synthetic Receptors for the High-Affinity Recognition of O-GlcNAc Derivatives

    NARCIS (Netherlands)

    Rios, Pablo; Carter, Tom S; Mooibroek, Tiddo J; Crump, Matthew P; Lisbjerg, Micke; Pittelkow, Michael; Supekar, Nitin T; Boons, Geert-Jan|info:eu-repo/dai/nl/088245489; Davis, Anthony P

    2016-01-01

    The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water-soluble carbohydrate receptors ("synthetic lectins"). Both systems show outstanding affinities for derivatives of N-acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside

  8. Inhibition of the 26S proteasome blocks progesterone receptor-dependent transcription through failed recruitment of RNA polymerase II.

    Science.gov (United States)

    Dennis, Andrew P; Lonard, David M; Nawaz, Zafar; O'Malley, Bert W

    2005-03-01

    In the present study, we investigated the involvement of protein degradation via the 26S proteasome during progesterone receptor (PR)-mediated transcription in T-47D cells containing a stably integrated MMTV-CAT reporter construct (CAT0 cells). Progesterone induced CAT and HSD11beta2 transcription while co-treatment with the proteasome inhibitor, MG132, blocked PR-induced transcription in a time-dependent fashion. MG132 treatment also inhibited transcription of beta-actin and cyclophilin, but not two proteasome subunit genes, PSMA1 and PSMC1, indicating that proteasome inhibition affects a subset of RNA polymerase II (RNAP(II))-regulated genes. Progesterone-mediated recruitment of RNAP(II) was blocked by MG132 treatment at time points later than 1 h that was not dependent on the continued presence of PR, associated cofactors, and components of the general transcription machinery, supporting the concept that proteasome-mediated degradation is needed for continued transcription. Surprisingly, progesterone-mediated acetylation of histone H4 was inhibited by MG132 with the concomitant recruitment of HDAC3, NCoR, and SMRT. We demonstrate that the steady-state protein levels of SMRT and NCoR are higher in the presence of MG132 in CAT0 cells, consistent with other reports that SMRT and NCoR are targets of the 26S proteasome. However, inhibition of histone deacetylation by trichostatin A (TSA) treatment or SMRT/NCoR knockdown by siRNA did not restore MG132-inhibited progesterone-dependent transcription. Therefore, events other than histone deacetylation and stability of SMRT and NCoR must also play a role in inhibition of PR-mediated transcription.

  9. Investigational hormone receptor agonists as ongoing female contraception: a focus on selective progesterone receptor modulators in early clinical development.

    Science.gov (United States)

    Nelson, Anita L

    2015-01-01

    As efforts are made to continue to increase the safety of contraceptive methods, those without estrogen have attracted new attention. Progestin-only options are available in many delivery systems, but most cause disturbed bleeding patterns. For gynecologic patients, selective progesterone receptor modulators (SPRMs) have been approved for medical abortion, for ovulation suppression in emergency contraception, and for the treatment of heavy menstrual bleeding due to leiomyoma. This article discusses the role of SPRMs in controlling fertility on an ongoing basis with particular emphasis on mifepristone and ulipristal acetate (UPA), since none of the other compounds has progressed out of early Phase I - II testing. It also discusses important information about the mechanisms of action and safety of these two SPRMs. Of all the investigational hormone agonist/antagonists, SPRMs have demonstrated the greatest potential as ongoing female contraceptives. They have the ability to suppress ovulation after initiation of the luteinizing hormone (LH) surge without affecting ovarian production of estrogen or inducing any significant metabolic changes. SPRMs may well be able to provide longer term contraception as oral agents, vaginal rings, and perhaps even intrauterine devices. UPA has the greatest promise. Current research needs to be expanded.

  10. Estrogen receptor and progesterone receptor status of breast cancer patients of eastern India: A multi-institutional study.

    Science.gov (United States)

    Chatterjee, Koushik; Bhaumik, Gautam; Chattopadhyay, Bhargab

    2018-01-01

    There is a paucity of any significant data on the estrogen receptor (ER) and progesterone receptor (PR) status of breast cancer patients from the eastern part of India. This study aims to document the ER and PR status of breast cancer patients in the eastern Indian population, as catered by two premier tertiary care hospitals in Kolkata. All breast cancer patients registered between January 1, 2013 and December 31, 2015, in the Departments of Oncology, of IPGMER and SSKM Hospitals and R. G. Kar Medical College and Hospital, Kolkata, who had at least undergone a core biopsy or surgery, were analyzed retrospectively for documentation of their ER and PR status, using the 2010 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) interpretation guidelines. Over a period of 3 years, a total of 927 patients were included for the study. A total of 825 (89%) patients had their ER and PR data available for evaluation. ER and PR positive was seen in 312 (37.82%) patients, ER and PR negative in 399 (48.36%) patients, ER positive and PR negative in 71 (8.6%) patients, and ER negative and PR positive results was found in 43 (5.21%) patients. This is the first multi-institutional documentation of ER and PR status from eastern India, having a modest number of patients and one of the earliest documentations using the latest ASCO/CAP interpretation guidelines. These findings resemble the data from the south and also reiterate the fact that majority of the Indian breast cancer patients are still ER and PR negative in spite of the changes in the interpretation guidelines.

  11. Increased progesterone receptor expression in uterine leiomyoma: correlation with age, number of leiomyomas, and clinical symptoms.

    Science.gov (United States)

    Tsigkou, Anastasia; Reis, Fernando M; Lee, Meng H; Jiang, Bingjie; Tosti, Claudia; Centini, Gabriele; Shen, Fang-Rong; Chen, You-Guo; Petraglia, Felice

    2015-07-01

    To investigate the possible correlation between progesterone receptor (PR) expression in uterine leiomyoma or adjacent myometrium and patient's age, size/number of leiomyomas, or clinical symptoms such as dysmenorrhea, acyclic pelvic pain, or menstrual and intermenstrual uterine bleeding. Cross-sectional study. Referral center. Sixty-two Chinese women undergoing elective hysterectomy for uterine leiomyomata. None. Evaluation of PR-total and PR-B mRNA with real-time polymerase chain reaction; PR-A and PR-B proteins quantified by Western blot in leiomyoma tissue and myometrium; symptoms rated by the patients using visual analog scores. The PR-B mRNA and PR-A and PR-B proteins were more concentrated in leiomyomas than in matched myometrium. A direct correlation between PR-B mRNA levels in leiomyoma and age (r = 0.347) and number of tumors (r = 0.295) was found. Conversely, there was an inverse correlation between PR-B mRNA levels in leiomyoma and dysmenorrhea (r = -0.260) and intermenstrual bleeding (r = -0.266). Multiple regression analysis indicated that age (β = 0.363) and the number of myomas (β = 0.296) were independently associated with PR-B mRNA levels in leiomyoma tissue. The levels of PR-B mRNA in leiomyoma tissue are directly associated with the number of tumors and inversely correlated with the intensity of intermenstrual bleeding and dysmenorrhea, suggesting that PR signaling may favor leiomyoma growth while attenuating clinical symptoms. This duality should be taken into account in the clinical management of patients with symptomatic uterine leiomyoma. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  12. Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

    Directory of Open Access Journals (Sweden)

    Yue Yu

    Full Text Available Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood.Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels.Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1 and interlukin-6 (IL-6 by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion.Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

  13. Estrogen and progesterone receptor status in breast cancer in Kuwait female population

    International Nuclear Information System (INIS)

    Paszko, Z.; Padzik, H.; Nasralla, M.Y.; Bouzubar, N.; Omar, Y.T.; Jazzaf, H.; Temmin, L.

    1993-01-01

    The levels cytosol estrogen (ERc) and progesterone (PRc) receptors were determined in 315 primary breast cancers of female Arab patients aged 23-80 years. Most of breast cancers (78%) occurred in women aged 21-50 years, and only 22% were in women aged 51-80 years. Breast cancers containing ERc and PRc concentrations in the range 5-50 fmol/mg of cytosol protein (mg c.p.) were found with with similar frequency in women aged under or over 50 years (53% of ERc and 43% for PRc, respectively). On the other hand, breast cancer with ERc values of >50 and >100 fmol/mg c.p. were twice as frequent in in women aged over 50 years as in women aged under 50 years. The frequency of breast cancers with PRc level of over 50 fmol/mg c.p. in women aged over 50 years was only half that in those aged under 50 years. In breast cancers of Kuwait Arab women the higher values of ERc (>100 fmol/mg c.p.) and PRc (>50 fmol/mg c.p.) were less frequent than in other populations reported in literature. The low frequency of breast cancer on postmenopausal Kuwait women is associated with low proportions of tumors with higher ERc and PRc contents. In contrast to this, data from literature indicate that in the the North Western European and American populations the postmenopausal incidence rise of breast cancers is associated with increased proportions of tumors with higher ERc and PRc levels. (author)

  14. Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin.

    Directory of Open Access Journals (Sweden)

    Jim F White

    2010-09-01

    Full Text Available Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1.To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [(3H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein.Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.

  15. Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W

    2005-01-01

    To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting...... that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization...... of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level....

  16. Progesterone receptor blockade in human breast cancer cells decreases cell cycle progression through G2/M by repressing G2/M genes

    OpenAIRE

    Clare, Susan E.; Gupta, Akash; Choi, MiRan; Ranjan, Manish; Lee, Oukseub; Wang, Jun; Ivancic, David Z.; Kim, J. Julie; Khan, Seema A.

    2016-01-01

    Background The synthesis of specific, potent progesterone antagonists adds potential agents to the breast cancer prevention and treatment armamentarium. The identification of individuals who will benefit from these agents will be a critical factor for their clinical success. Methods We utilized telapristone acetate (TPA; CDB-4124) to understand the effects of progesterone receptor (PR) blockade on proliferation, apoptosis, promoter binding, cell cycle progression, and gene expression. We then...

  17. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    Energy Technology Data Exchange (ETDEWEB)

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  18. Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

    Directory of Open Access Journals (Sweden)

    Sujatha P Koduvayur

    Full Text Available The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

  19. Differential regulation of the human progesterone receptor gene through an estrogen response element half site and Sp1 sites.

    Science.gov (United States)

    Petz, Larry N; Ziegler, Yvonne S; Schultz, Jennifer R; Kim, Hwajin; Kemper, J Kim; Nardulli, Ann M

    2004-02-01

    The progesterone receptor (PR) gene is regulated by estrogen in normal reproductive tissues and in MCF-7 human breast cancer cells. Although it is generally thought that estrogen responsiveness is mediated by interaction of the ligand-occupied estrogen receptor (ER) with estrogen response elements (EREs) in target genes, the human progesterone receptor (PR) gene lacks a palindromic ERE. Promoter A of the PR gene does, however, contain an ERE half site upstream of two adjacent Sp1 sites from +571 to +595, the +571 ERE/Sp1 site. We have examined the individual contributions of the ERE half site and the two Sp1 sites in regulating estrogen responsiveness. Transient transfection assays demonstrated that both Sp1 sites were critical for estrogen-mediated activation of the PR gene. Interestingly, rather than decreasing transcription, mutations in the ERE half site increased transcription substantially suggesting that this site plays a role in limiting transcription. Chromatin immunoprecipitation assays demonstrated that Sp1 was associated with the +571 ERE/Sp1 site in the endogenous PR gene in the absence and in the presence of estrogen, but that ERalpha was only associated with this region of the PR gene after MCF-7 cells had been treated with estrogen. Our studies provide evidence that effective regulation of transcription through the +571 ERE/Sp1 site requires the binding of ERalpha and Sp1 to their respective cis elements and the appropriate interaction of ERalpha and Sp1 with other coregulatory proteins and transcription factors.

  20. Loss of co-ordinate expression of progesterone receptors A and B is an early event in breast carcinogenesis.

    Science.gov (United States)

    Mote, P A; Bartow, S; Tran, N; Clarke, C L

    2002-03-01

    Progesterone receptor (PR) mediates the effects of progesterone in mammary tissues and plays a crucial role in normal breast development and in breast cancer. PR proteins are expressed as two isoforms, PRA and PRB, that have different capacities to activate target genes, yet it is unknown whether progesterone action in normal and malignant breast is mediated by PRA and/or PRB. This study determines the relative expression of PRA and PRB in normal breast and in benign, premalignant and malignant archival breast lesions by dual immunofluorescent histochemistry. In normal breast and in proliferative disease without atypia (PDWA) PRA and PRB were co-expressed within the same cells in comparable amounts, implicating both isoforms in progesterone action. In atypical lesions, however, there was a significant increase in predominant expression of PRA or PRB, with lesion progression from the normal state to malignancy. PR isoform predominance, especially PRA predominance, was evident in a high proportion of ductal carcinomas in situ (DCIS) and invasive breast lesions. In the normal breast and in PDWA, the relative expression of PRA and PRB in adjacent cells was homogenous. There was a significant increase in cell-to-cell heterogeneity of PR isoform expression in ADH and DCIS lesions and in the majority of breast cancers. Heterogeneous cell-to-cell expression of PR isoforms occurred prior to overall predominant expression of one isoform in premalignant breast lesions, demonstrating that loss of control of relative PRA:PRB expression is an early event in the development of breast cancer. PRA:PRB ratios within a breast lesion are likely to be important as both markers and effectors of tumor growth and development, and progressively aberrant PR isoform expression may play a role in the etiology of breast cancer.

  1. Determination of estradiol, estrone and progesterone in serum and human endometrium in correlation to the content of steroid receptors and 17β-hydroxysteroid dehydrogenase activity during menstrual cycle

    International Nuclear Information System (INIS)

    Schmidt-Gollwitzer, M.; Eiletz, J.; Pachaly, J.

    1977-01-01

    A study has been carried out to compare the influence of estradiol estrone and progesterone on the estradiol and progesterone receptor levels and 17β-hydroxysteroid dehydrogenase (17β-HSD) activity in human endometrium. The steroid hormone concentrations were measured simultaneously in both serum and endometrial tissue. The estradiol receptor levels were highest during the early proliferative phase and were inversely correlated to the endometrial tissue and serum concentrations of estradiol and progesterone. The highest progesterone binding capacity was found in endometrical cytosol during the late proliferative phase (midcycle) of the menstrual cycle. The midcycle peak of the progesterone receptor level correlated well with the first peak of the serum and tissue concentrations of estradiol. During,the luteal phase, in contrast to the proliferative phase, the progesterone receptor level decreased whereas serum progesterone concentrations were high. Estrone concentrations were higher in secretory than proliferative endometrium and were correlated to the increase of progesterone receptor content and 17β-HSD activity during early secretory phase. The 17β-HSD activity was approximately 10-fold higher during the early secretory than during the proliferative phase. The progesterone receptor level was highly correlated to the specific 17β-HSD activity of the microsomal fraction whereas a significant inverse correlation between the enzyme activity and the estradiol receptor level was observed. (orig.) [de

  2. Molecular essence and endocrine responsiveness of estrogen receptor-negative, progesterone receptor-positive, and HER2-negative breast cancer.

    Science.gov (United States)

    Yu, Ke-Da; Jiang, Yi-Zhou; Hao, Shuang; Shao, Zhi-Ming

    2015-10-05

    The clinical significance of progesterone receptor (PgR) expression in estrogen receptor-negative (ER-) breast cancer is controversial. Herein, we systemically investigate the clinicopathologic features, molecular essence, and endocrine responsiveness of ER-/PgR+/HER2- phenotype. Four study cohorts were included. The first and second cohorts were from the Surveillance, Epidemiology, and End Results database (n = 67,932) and Fudan University Shanghai Cancer Center (n = 2,338), respectively, for clinicopathologic and survival analysis. The third and fourth cohorts were from two independent publicly available microarray datasets including 837 operable cases and 483 cases undergoing neoadjuvant chemotherapy, respectively, for clinicopathologic and gene-expression analysis. Characterized genes defining subgroups within the ER-/PgR+/HER2- phenotype were determined and further validated. Clinicopathologic features and survival outcomes of the ER-/PgR+ phenotype fell in between the ER+/PgR+ and ER-/PgR- phenotypes, but were more similar to ER-/PgR-. Among the ER-/PgR+ phenotype, 30% (95% confidence interval [CI] 17-42%, pooled by a fixed-effects method) were luminal-like and 59% (95% CI 45-72%, pooled by a fixed-effects method) were basal-like. We further refined the characterized genes for subtypes within the ER-/PgR+ phenotype and developed an immunohistochemistry-based method that could determine the molecular essence of ER-/PgR+ using three markers, TFF1, CK5, and EGFR. Either PAM50-defined or immunohistochemistry-defined basal-like ER-/PgR+ cases have a lower endocrine therapy sensitivity score compared with luminal-like ER-/PgR+ cases (P defined basal-like ER-/PgR+ cases might not benefit from adjuvant endocrine therapy (log-rank P = 0.61 for sufficient versus insufficient endocrine therapy). The majority of ER-/PgR+/HER2- phenotype breast cancers are basal-like and associated with a lower endocrine therapy sensitivity score. Additional studies are needed

  3. Role of T cell receptor affinity in the efficacy and specificity of adoptive T cell therapies

    Directory of Open Access Journals (Sweden)

    Jennifer D. Stone

    2013-08-01

    Full Text Available Over the last several years, there has been considerable progress in the treatment of cancer using gene modified adoptive T cell therapies. Two approaches have been used, one involving the introduction of a conventional alpha-beta T cell receptor (TCR against a pepMHC cancer antigen, and the second involving introduction of a chimeric antigen receptor (CAR consisting of a single-chain antibody as an Fv fragment (scFv linked to transmembrane and signaling domains. In this review, we focus on one aspect of TCR-mediated adoptive T cell therapies, the impact of the affinity of the alpha-beta TCR for the pepMHC cancer antigen on both efficacy and specificity. We discuss the advantages of higher affinity TCRs in mediating potent activity of CD4 T cells. This is balanced with the potential disadvantage of higher affinity TCRs in mediating greater self-reactivity against a wider range of structurally similar antigenic peptides, especially in synergy with the CD8 co-receptor. Both TCR affinity and target selection will influence potential safety issues. We suggest pre-clinical strategies that might be used to examine each TCR for possible on-target and off-target side effects due to self-reactivities, and to adjust TCR affinities accordingly.

  4. N-Oxide analogs of WAY-100635 : new high affinity 5-HT (1A) receptor antagonists

    NARCIS (Netherlands)

    Oberwinkler - Marchais, Sandrine; Nowicki, B; Pike, VW; Halldin, C; Sandell, J; Chou, YH; Gulyas, B; Brennum, LT; Farde, L; Wikstrom, H V

    2005-01-01

    WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide] 1 and its O-des-methyl derivative DWAY 2 are well-known high affinity 5-HT1A receptor antagonists. which when labeled with carbon-II (beta(+): t(1/2) 20.4min) in the carbonyl group are effective

  5. Embryonic exposure to the fungicide vinclozolin causes virilization of females and alteration of progesterone receptor expression in vivo: an experimental study in mice.

    Science.gov (United States)

    Buckley, Jill; Willingham, Emily; Agras, Koray; Baskin, Laurence S

    2006-02-21

    Vinclozolin is a fungicide that has been reported to have anti-androgenic effects in rats. We have found that in utero exposure to natural or synthetic progesterones can induce hypospadias in mice, and that the synthetic progesterone medroxyprogesterone acetate (MPA) feminizes male and virilizes female genital tubercles. In the current work, we selected a relatively low dose of vinclozolin to examine its in utero effects on the development of the genital tubercle, both at the morphological and molecular levels. We gave pregnant dams vinclozolin by oral gavage from gestational days 13 through 17. We assessed the fetal genital tubercles from exposed fetuses at E19 to determine location of the urethral opening. After determination of gonadal sex, either genital tubercles were harvested for mRNA quantitation, or urethras were injected with a plastic resin for casting. We analyzed quantified mRNA levels between treated and untreated animals for mRNA levels of estrogen receptors alpha and beta, progesterone receptor, and androgen receptor using nonparametric tests or ANOVA. To determine effects on urethral length (males have long urethras compared to females), we measured the lengths of the casts and performed ANOVA analysis on these data. Our morphological results indicated that vinclozolin has morphological effects similar to those of MPA, feminizing males (hypospadias) and masculinizing females (longer urethras). Because these results reflected our MPA results, we investigated the effects of in utero vinclozolin exposure on the mRNA expression levels of androgen, estrogen alpha and beta, and progesterone receptors. At the molecular level, vinclozolin down-regulated estrogen receptor alpha mRNA in females and up-regulated progesterone receptor mRNA. Vinclozolin-exposed males exhibited up-regulated estrogen receptor alpha and progesterone receptor mRNA, effects we have also seen with exposure to the synthetic estrogen, ethinyl estradiol. The results suggest that

  6. Embryonic exposure to the fungicide vinclozolin causes virilization of females and alteration of progesterone receptor expression in vivo: an experimental study in mice

    Directory of Open Access Journals (Sweden)

    Baskin Laurence S

    2006-02-01

    Full Text Available Abstract Background Vinclozolin is a fungicide that has been reported to have anti-androgenic effects in rats. We have found that in utero exposure to natural or synthetic progesterones can induce hypospadias in mice, and that the synthetic progesterone medroxyprogesterone acetate (MPA feminizes male and virilizes female genital tubercles. In the current work, we selected a relatively low dose of vinclozolin to examine its in utero effects on the development of the genital tubercle, both at the morphological and molecular levels. Methods We gave pregnant dams vinclozolin by oral gavage from gestational days 13 through 17. We assessed the fetal genital tubercles from exposed fetuses at E19 to determine location of the urethral opening. After determination of gonadal sex, either genital tubercles were harvested for mRNA quantitation, or urethras were injected with a plastic resin for casting. We analyzed quantified mRNA levels between treated and untreated animals for mRNA levels of estrogen receptors α and β, progesterone receptor, and androgen receptor using nonparametric tests or ANOVA. To determine effects on urethral length (males have long urethras compared to females, we measured the lengths of the casts and performed ANOVA analysis on these data. Results Our morphological results indicated that vinclozolin has morphological effects similar to those of MPA, feminizing males (hypospadias and masculinizing females (longer urethras. Because these results reflected our MPA results, we investigated the effects of in utero vinclozolin exposure on the mRNA expression levels of androgen, estrogen α and β, and progesterone receptors. At the molecular level, vinclozolin down-regulated estrogen receptor α mRNA in females and up-regulated progesterone receptor mRNA. Vinclozolin-exposed males exhibited up-regulated estrogen receptor α and progesterone receptor mRNA, effects we have also seen with exposure to the synthetic estrogen, ethinyl

  7. Progesterone receptor isoform analysis by quantitative real-time polymerase chain reaction in formalin-fixed, paraffin-embedded canine mammary dysplasias and tumors

    DEFF Research Database (Denmark)

    Guil-Luna, S.; Stenvang, Jan; Brünner, Nils

    2014-01-01

    and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign...... in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors...

  8. Determine equilibrium dissociation constant of drug-membrane receptor affinity using the cell membrane chromatography relative standard method.

    Science.gov (United States)

    Ma, Weina; Yang, Liu; Lv, Yanni; Fu, Jia; Zhang, Yanmin; He, Langchong

    2017-06-23

    The equilibrium dissociation constant (K D ) of drug-membrane receptor affinity is the basic parameter that reflects the strength of interaction. The cell membrane chromatography (CMC) method is an effective technique to study the characteristics of drug-membrane receptor affinity. In this study, the K D value of CMC relative standard method for the determination of drug-membrane receptor affinity was established to analyze the relative K D values of drugs binding to the membrane receptors (Epidermal growth factor receptor and angiotensin II receptor). The K D values obtained by the CMC relative standard method had a strong correlation with those obtained by the frontal analysis method. Additionally, the K D values obtained by CMC relative standard method correlated with pharmacological activity of the drug being evaluated. The CMC relative standard method is a convenient and effective method to evaluate drug-membrane receptor affinity. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. The role of estrogen and progesterone receptors in response rate to megestrol acetate: conservative treatment of stage Ia endometrial adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Yarandi F

    2010-12-01

    Full Text Available "nBackground: Surgery is the most effective treatment of well-differentiated endometrial cancer. But using systemic progestins, have been evaluated to treat the young patients with well-differentiated endometrial cancer who wish to preserve their fertility. The aim of this study was the evaluation of megestrol acetate on endometrial adenocarcino-ma with regard to the receptors."n "nMethods: This was a quasi-experimental study. In 16 infertile patients with stage Ia well-differentiated endometrial adenocarcinoma. The treatment initiated with 160mg/d of megestrol acetate and continued with 320mg/d for non-responsive cases. All of the patients followed with FD&C and hysteroscopy. The responsive patients were referred to IVF group and they were followed for three years."n "nResults: Of nine patient in the first step of the study, 4 (25% became pregnant. Eight patients underwent Total Abdominal Hysterectomy (TAH, and one was retreated conservatively. Of seven patient of second step of the study, five are under treatment at the time of closing the paper (three cases candidate for IVF and two are under 320 mg/d megestrol acetate, one patient is a candidate for hysterectomy, and one exited of study because of male infertility. All of the patients were progesterone receptor positive, and only one was estrogen receptor negative."n "nConclusion: Conservative treatment of early stage well-differentiated endometrial adenocarcinoma with progestins may be used in highly selected young patients who have not completed their family. Close long- term follow up in this special group of patients is necessary. The evaluation of estrogen and progesterone receptors assay may be useful in predicting response to the treatment.

  10. Guanine nucleotide regulation of dopamine receptor agonist affinity states in rat estradiol-induced pituitary tumors

    Energy Technology Data Exchange (ETDEWEB)

    Di Paolo, T.; Falardeau, P.

    1987-08-31

    The authors have investigated dopamine (DA) receptor agonist high- and low-affinity states in female rate estradiol-induced prolactin (PRL)-secreting pituitary tumors and intact pituitary tissue. Estradiol treatment increased the anterior pituitary weight 9-fold and plasma prolactin levels 74-fold and these measures are correlated (R = 0.745, n = 73, p < 0.001). Competition for (/sup 3/H)-spiperone binding to the DA receptor by apomorphine was compared in normal and adenomatous pituitary tissue. The inhibition constants (Ki) and the proportions of the two apomorphine sites are unchanged in tumors compared to intact pituitary tissue. Guanosine 5'-(..beta..-..gamma..-imino)triphosphate (Gpp(NH)p) causes complete conversion of the high into low affinity dopaminergic agonist site in normal pituitary and in tumors. These results suggest that rats with primary estradiol-induced pituitary tumors have normal and functional DA receptors. 9 references, 2 tables.

  11. Guanine nucleotide regulation of dopamine receptor agonist affinity states in rat estradiol-induced pituitary tumors

    International Nuclear Information System (INIS)

    Di Paolo, T.; Falardeau, P.

    1987-01-01

    The authors have investigated dopamine (DA) receptor agonist high- and low-affinity states in female rate estradiol-induced prolactin (PRL)-secreting pituitary tumors and intact pituitary tissue. Estradiol treatment increased the anterior pituitary weight 9-fold and plasma prolactin levels 74-fold and these measures are correlated (R = 0.745, n = 73, p 3 H]-spiperone binding to the DA receptor by apomorphine was compared in normal and adenomatous pituitary tissue. The inhibition constants (Ki) and the proportions of the two apomorphine sites are unchanged in tumors compared to intact pituitary tissue. Guanosine 5'-[β-γ-imino]triphosphate (Gpp(NH)p) causes complete conversion of the high into low affinity dopaminergic agonist site in normal pituitary and in tumors. These results suggest that rats with primary estradiol-induced pituitary tumors have normal and functional DA receptors. 9 references, 2 tables

  12. Clinicopathologic Characteristics of Oestrogen Receptor-Positive/Progesterone Receptor-Negative/Her2-Negative Breast Cancer According to a Novel Definition of Negative Progesterone Receptor Status: A Large Population-Based Study from China.

    Directory of Open Access Journals (Sweden)

    An-qi Li

    Full Text Available A lack of progesterone receptor (PgR expression in oestrogen receptor-positive (ER+ tumours is associated with worse survival. PgR status is usually defined as positive or negative using 1% positive nuclei as a cut-off point. In this study, we aimed to assess the clinicopathologic characteristics of ER+/PgR-/HER2- tumours by comparing them with ER+/PgR+/HER2- tumours using a PgR cut-off point of 20% as a divisive criterion.We analysed 1,522 patients with primary breast cancer who had undergone surgery at the Cancer Center of Fudan University between 2012 and 2014. Age, grade, tumour size, lymph node status and lymphovascular invasion were assessed. Multinomial logistic regression, linear regression and chi-square test models were applied to assess associations between ER, PR and clinical features.ER+/PgR-/HER2- tumours showed poorer clinicopathologic characteristics relative to ER+/PgR+/HER2- tumours using a PgR threshold of 20% instead of 1%. The clinicopathologic characteristics did not differ between tumours with purely negative PgR expression and tumours with a PgR percentage ranging from 1% to 19%. The prognostic significance of PR expression appeared more pronounced in patients under a high Ki-67 status than those under a low Ki-67 status.Based on these findings, we propose the use of a novel threshold of 20% to define PgR status. Nevertheless, the impact of this new criterion on patient management and clinical treatment requires additional study.

  13. Understanding the Inguinal Sinus in Sheep (Ovis aries—Morphology, Secretion, and Expression of Progesterone, Estrogens, and Prolactin Receptors

    Directory of Open Access Journals (Sweden)

    Graça Alexandre-Pires

    2017-07-01

    Full Text Available Post-parturient behavior of mammalian females is essential for early parent–offspring contact. After delivery, lambs need to ingest colostrum for obtaining the related immunological protection, and early interactions between the mother and the lamb are crucial. Despite visual and auditory cues, olfactory cues are decisive in lamb orientation to the mammary gland. In sheep, the inguinal sinus is located bilaterally near the mammary gland as a skin pouch (IGS that presents a gland that secretes a strong-smelling wax. Sheep IGS gland functions have many aspects under evaluation. The objective of the present study was to evaluate sheep IGS gland functional aspects and mRNA transcription and the protein expression of several hormone receptors, such as progesterone receptor (PGR, estrogen receptor 1 (ESR1, and 2 (ESR2 and prolactin receptor (PRLR present. In addition, another aim was to achieve information about IGS ultrastructure and chemical compounds produced in this gland. All hormone receptors evaluated show expression in IGS during the estrous cycle (follicular/luteal phases, pregnancy, and the post-partum period. IGS secretion is rich in triterpenoids that totally differ from the surrounding skin. They might be essential substances for the development of an olfactory preference of newborns to their mothers.

  14. Syntheses of 7-Substituted α-Cyperone Derivatives for Selective Sigma-1 Receptor over Cannabinoid-1 Receptor Binding Affinities

    Energy Technology Data Exchange (ETDEWEB)

    Park, Juyoung; Shin, Younggyun; Yoon, Sunghwa [Ajou Univ., Suwon (Korea, Republic of); Kim, Keewon; Kwon, Youngbae [ChonBuk National Univ., Jeonju (Korea, Republic of)

    2013-11-15

    We have successfully synthesized seven α-cyperone derivatives and found that the presence of a hydrogen bond donor/acceptor groups at the C7 position of α-cyperone significantly affects specificity and potency of CB{sub 1} receptor binding affinity over sigma-1 receptor binding affinity. In particular, the presence of the amino moiety at the C7 position of α-cyperone is beneficial for binding to sigmia-1 receptor. The molecular mechanism of compound 8 involved in the high binding affinity to sigma-1 receptor is under investigation. We first synthesized α-cyperone 1 by following the previously reported synthetic routes.15-19 In brief, azeotropic imination of (+)-dihydrocarvone and (R)-(+)-1-phenylethylamine followed by alkylation with a slight excess of ethyl vinyl ketone (EVK) in THF at 40 .deg. C produced the Micheal adduct. The resulting adduct was hydrolyzed and then treated with sodium methoxide at room temperature to give an easily separable mixture of α-cyperone 1 and its side product. Flash chromatography resulted in pure α-cyperone 1 in a 30% yield from (+)-dihydrocarvone.

  15. Ovarian steroid hormones modulate the expression of progesterone receptors and histone acetylation patterns in uterine leiomyoma cells.

    Science.gov (United States)

    Sant'Anna, Gabriela Dos Santos; Brum, Ilma Simoni; Branchini, Gisele; Pizzolato, Lolita Schneider; Capp, Edison; Corleta, Helena von Eye

    2017-08-01

    Uterine leiomyomas are the most common benign smooth muscle cell tumors in women. Estrogen (E2), progesterone (P4) and environmental factors play important roles in the development of these tumors. New treatments, such as mifepristone, have been proposed. We evaluated the gene expression of total (PRT) and B (PRB) progesterone receptors, and the histone acetyltransferase (HAT) and deacetylase (HDAC) activity after treatment with E2, P4 and mifepristone (RU486) in primary cell cultures from uterine leiomyoma and normal myometrium. Compared to myometrium, uterine leiomyoma cells showed an increase in PRT mRNA expression when treated with E2, and increase in PRB mRNA expression when treated with E2 and P4. Treatment with mifepristone had no significant impact on mRNA expression in these cells. The HDAC activity was higher in uterine leiomyoma compared to myometrial cells after treatment with E2 and E2 + P4 + mifepristone. HAT activity was barely detectable. Our results suggest that ovarian steroid hormones modulate PR, and mifepristone was unable to decrease PRT and PRB mRNA. The higher activity of HDAC leiomyoma cells could be involved in transcriptional repression of genes implicated in normal myometrium cell function, contributing to the maintenance and growth of uterine leiomyoma.

  16. C/EBPβ LIP and c-Jun synergize to regulate expression of the murine progesterone receptor.

    Science.gov (United States)

    Wang, Weizhong; Do, Han Ngoc; Aupperlee, Mark D; Durairaj, Srinivasan; Flynn, Emily E; Miksicek, Richard J; Haslam, Sandra Z; Schwartz, Richard C

    2018-06-02

    CCAAT/enhancer binding protein β (C/EBPβ) is required for murine mammary ductal morphogenesis and alveologenesis. Progesterone is critical for proliferation and alveologenesis in adult mammary glands, and there is a similar requirement for progesterone receptor isoform B (PRB) in alveologenesis. We examined C/EBPβ regulation of PR expression. All three C/EBPβ isoforms, including typically inhibitory LIP, transactivated the PR promoter. LIP, particularly, strongly synergized with c-Jun to drive PR transcription. Endogenous C/EBPβ and c-Jun stimulated a PR promoter-reporter and these two factors showed promoter occupancy on the endogenous PR gene. Additionally, LIP overexpression elevated endogenous PR protein expression. In pregnancy, both PRB and the relative abundance of LIP among C/EBPβ isoforms increase. Consistent with a role in PRB expression, in vivo C/EBPβ and PR isoform A expression showed mutually exclusive localization in mammary epithelium, while C/EBPβ and PRB largely co-localized. We suggest a critical role for C/EBPβ, particularly LIP, in PRB expression. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Identification of high- and low-affinity NGF receptors during development of the chicken central nervous system

    International Nuclear Information System (INIS)

    Escandon, E.; Chao, M.V.

    1990-01-01

    In order to study regulation of the nerve growth factor (NGF) receptor during embryogenesis in chick brain, we have used affinity crosslinking of tissues with 125 I-NGF. NGF interacts with high- and low-affinity receptors; high-affinity receptors are required for the majority of NGF's actions. Most measurements of receptor levels do not distinguish between high- and low-affinity forms of the receptor. We have used the lipophilic crosslinking agent HSAB to identify the high-affinity, functional receptor during development of the chicken central nervous system. A peak of expression during Embryonic Days 5-10 was detected in all regions of the chicken central nervous system, but, shortly after birth, only the cerebellar region displays significant levels of NGF receptor protein. The time course of expression confirms the dramatic regulation of the NGF receptor gene during defined embryonic periods. The detection of high-affinity NGF receptors in brain and neural retina provides strong evidence that NGF is involved in essential ontogenetic events in the development of the chicken central nervous system

  18. X-ray structures of progesterone receptor ligand binding domain in its agonist state reveal differing mechanisms for mixed profiles of 11beta-substituted steroids.

    NARCIS (Netherlands)

    Lusher, S.J.; Raaijmakers, H.C.A.; Vu-Pham, D.; Kazemier, B.; Bosch, R.; McGuire, R.; Azevedo, R.; Hamersma, H.; Dechering, K.; Oubrie, A.; Duin, M. van; Vlieg, J. de

    2012-01-01

    We present here the x-ray structures of the progesterone receptor (PR) in complex with two mixed profile PR modulators whose functional activity results from two differing molecular mechanisms. The structure of Asoprisnil bound to the agonist state of PR demonstrates the contribution of the ligand

  19. GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

    DEFF Research Database (Denmark)

    Bartoi, Tudor; Rigbolt, Kristoffer T G; Du, Dan

    2010-01-01

    lines that allow a straightforward biochemical isolation of GABA(B) receptors. The transgenic mice express GABA(B1) isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice...... and wild-type control animals revealed two novel components of the GABA(B1) complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABA(B) receptors via the GABA(B2) subunit. In transfected hippocampal neurons, potassium...

  20. Free energy calculations offer insights into the influence of receptor flexibility on ligand-receptor binding affinities.

    Science.gov (United States)

    Dolenc, Jožica; Riniker, Sereina; Gaspari, Roberto; Daura, Xavier; van Gunsteren, Wilfred F

    2011-08-01

    Docking algorithms for computer-aided drug discovery and design often ignore or restrain the flexibility of the receptor, which may lead to a loss of accuracy of the relative free enthalpies of binding. In order to evaluate the contribution of receptor flexibility to relative binding free enthalpies, two host-guest systems have been examined: inclusion complexes of α-cyclodextrin (αCD) with 1-chlorobenzene (ClBn), 1-bromobenzene (BrBn) and toluene (MeBn), and complexes of DNA with the minor-groove binding ligands netropsin (Net) and distamycin (Dist). Molecular dynamics simulations and free energy calculations reveal that restraining of the flexibility of the receptor can have a significant influence on the estimated relative ligand-receptor binding affinities as well as on the predicted structures of the biomolecular complexes. The influence is particularly pronounced in the case of flexible receptors such as DNA, where a 50% contribution of DNA flexibility towards the relative ligand-DNA binding affinities is observed. The differences in the free enthalpy of binding do not arise only from the changes in ligand-DNA interactions but also from changes in ligand-solvent interactions as well as from the loss of DNA configurational entropy upon restraining.

  1. High affinity soluble ILT2 receptor: a potent inhibitor of CD8(+) T cell activation.

    Science.gov (United States)

    Moysey, Ruth K; Li, Yi; Paston, Samantha J; Baston, Emma E; Sami, Malkit S; Cameron, Brian J; Gavarret, Jessie; Todorov, Penio; Vuidepot, Annelise; Dunn, Steven M; Pumphrey, Nicholas J; Adams, Katherine J; Yuan, Fang; Dennis, Rebecca E; Sutton, Deborah H; Johnson, Andy D; Brewer, Joanna E; Ashfield, Rebecca; Lissin, Nikolai M; Jakobsen, Bent K

    2010-12-01

    Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

  2. Agonist-induced affinity alterations of a central nervous system. cap alpha. -bungarotoxin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Lukas, R.J.; Bennett, E.L.

    1979-01-01

    The ability of cholinergic agonists to block the specific interaction of ..cap alpha..-bungarotoxin (..cap alpha..-Bgt) with membrane-bound sites derived from rat brain is enhanced when membranes are preincubated with agonist. Thus, pretreatment of ..cap alpha..-Bgt receptors with agonist (but not antagonist) causes transformation of sites to a high-affinity form toward agonist. This change in receptor state occurs with a half-time on the order of minutes, and is fully reversible on dilution of agonist. The results are consistent with the identity of ..cap alpha..-Bgt binding sites as true central nicotinic acetylcholine receptors. Furthermore, this agonist-induced alteration in receptor state may represent an in vitro correlate of physiological desensitization. As determined from the effects of agonist on toxin binding isotherms, and on the rate of toxin binding to specific sites, agonist inhibition of toxin binding to the high-affinity state is non-competitive. This result suggests that there may exist discrete toxin-binding and agonist-binding sites on central toxin receptors.

  3. Conformational destabilization of Immunoglobulin G increases the low pH-binding affinity with the Neonatal Fc Receptor

    DEFF Research Database (Denmark)

    Walters, Benjamin T; Jensen, Pernille Foged; Larraillet, Vincent

    2016-01-01

    Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the ......H-dependent affinity in IgG-FcRn interactions and exemplify the important and often ignored role of intrinsic conformational dynamics in a protein ligand, to dictate affinity for biologically important receptors.......Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain...... the diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and Fc...

  4. Progesterone receptor membrane component 1 as the mediator of the inhibitory effect of progestins on cytokine-induced matrix metalloproteinase 9 activity in vitro.

    Science.gov (United States)

    Allen, Terrence K; Feng, Liping; Grotegut, Chad A; Murtha, Amy P

    2014-02-01

    Progesterone (P4) and the progestin, 17α-hydroxyprogesterone caproate, are clinically used to prevent preterm births (PTBs); however, their mechanism of action remains unclear. Cytokine-induced matrix metalloproteinase 9 (MMP-9) activity plays a key role in preterm premature rupture of the membranes and PTB. We demonstrated that the primary chorion cells and the HTR8/SVneo cells (cytotrophoblast cell line) do not express the classical progesterone receptor (PGR) but instead a novel progesterone receptor, progesterone receptor membrane component 1 (PGRMC1), whose role remains unclear. Using HTR8/SVneo cells in culture, we further demonstrated that 6 hours pretreatment with medroxyprogesterone acetate (MPA) and dexamethasone (Dex) but not P4 or 17α-hydroxyprogesterone hexanoate significantly attenuated tumor necrosis factor α-induced MMP-9 activity after a 24-hour incubation period. The inhibitory effect of MPA, but not Dex, was attenuated when PGRMC1 expression was successfully reduced by PGRMC1 small interfering RNA. Our findings highlight a possible novel role of PGRMC1 in mediating the effects of MPA and in modulating cytokine-induced MMP-9 activity in cytotrophoblast cells in vitro.

  5. Design and Application of Synthetic Receptors for Recognition of Methylated Lysine and Supramolecular Affinity Labeling

    Science.gov (United States)

    Gober, Isaiah Nathaniel

    This dissertation involves the design and synthesis of new synthetic receptors and their application in the molecular recognition of methylated lysine and their use as tools for chemical biology. The dissertation is divided into four parts. The first section focuses on the development of a novel labeling method that is based on ligand-directed affinity labeling principles. In this labeling method, a synthetic receptor that binds to trimethyl lysine (Kme3) is attached through a linker to an electrophilic tag group that can react with a nucleophilic amine in a histone peptide. This affinity labeling probe, which we called CX4-ONBD, is equipped with an electrophilic tag that allows for turn-on fluorescence labeling of Kme3 histone peitdes. We show that the probe gives a pronounced turn-on fluorescence response when it is incubated with a histone peptide that contains Kme3 and a nearby reactive lysine. This probe also displays >5-fold selectivity in covalent labeling over an unmethylated lysine peptide. This represents the first time a synthetic receptor has been used for affinity labeling purposes, and it also expands on the chemical toolkit that is available for sensing PTMs like lysine methylation. In the second section, the supramolecular affinity labeling method that was optimized using CX4-ONBD was applied to the development of a real-time assay for measuring enzymatic activity. More specifically, the probe was used to create a turn-on fluorescence assay for histone deacetylase (HDAC) activity and for inhibitor screening and IC50 determination. Most commercial kits for HDAC activity have limited substrate scope, and other common methods used for characterizing enzymatic activity often require chromatographic separation and are therefore not high-throughput. This small molecule receptor-mediated affinity labeling strategy allowed for facile readout of HDAC activity and inhibition. Overall, this application of supramolecular affinity labeling expands on the

  6. Simulating the influence of plasma protein on measured receptor affinity in biochemical assays reveals the utility of Schild analysis for estimating compound affinity for plasma proteins.

    Science.gov (United States)

    Blakeley, D; Sykes, D A; Ensor, P; Bertran, E; Aston, P J; Charlton, S J

    2015-11-01

    Plasma protein binding (PPB) influences the free fraction of drug available to bind to its target and is therefore an important consideration in drug discovery. While traditional methods for assessing PPB (e.g. rapid equilibrium dialysis) are suitable for comparing compounds with relatively weak PPB, they are not able to accurately discriminate between highly bound compounds (typically >99.5%). The aim of the present work was to use mathematical modelling to explore the potential utility of receptor binding and cellular functional assays to estimate the affinity of compounds for plasma proteins. Plasma proteins are routinely added to in vitro assays, so a secondary goal was to investigate the effect of plasma proteins on observed ligand-receptor interactions. Using the principle of conservation of mass and the law of mass action, a cubic equation was derived describing the ligand-receptor complex [LR] in the presence of plasma protein at equilibrium. The model demonstrates the profound influence of PPB on in vitro assays and identifies the utility of Schild analysis, which is usually applied to determine receptor-antagonist affinities, for calculating affinity at plasma proteins (termed KP ). We have also extended this analysis to functional effects using operational modelling and demonstrate that these approaches can also be applied to cell-based assay systems. These mathematical models can potentially be used in conjunction with experimental data to estimate drug-plasma protein affinities in the earliest phases of drug discovery programmes. © 2015 The British Pharmacological Society.

  7. Investigations into the binding affinities of different human 5-HT4 receptor splice variants.

    Science.gov (United States)

    Irving, Helen R; Tochon-Danguy, Nathalie; Chinkwo, Kenneth A; Li, Jian G; Grabbe, Carmen; Shapiro, Marina; Pouton, Colin W; Coupar, Ian M

    2010-01-01

    This study examined whether the drug-receptor-binding sites of 5 selected human 5-HT(4) receptor splice variants [h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g)] display preferential affinities towards agonists. The agonists selected on the basis of chemical diversity and clinical relevance were: 5-HT4 benzamides, renzapride, zacopride and prucalopride; the benzimidazolones, DAU 6236 and BIMU 1; the aromatic ketone, RS67333, and the indole carbazimidamide tegaserod. The rank order of affinities ranging across the splice variants was: tegaserod (pKi: 7.38-7.91) > or = Y-36912 (pKi: 7.03-7.85) = BIMU 1 (pKi: 6.92-7.78) > or = DAU 6236 (pKi: 6.79-7.99) > or = 5-HT (pKi: 5.82-7.29) > or = 5-MeOT (pKi: 5.64-6.83) > or = renzapride (pKi: 4.85-5.56). We obtained affinity values for the 5-HT4(b), (d) and (g) variants for RS67333 (pKi: 7:48-8.29), prucalopride (pKi: 6.86-7.37) and zacopride (pKi: 5.88-7.0). These results indicate that the ligands interact with the same conserved site in each splice variant. Some splice variants have a higher affinity for certain agonists and the direction of selectivity followed a common trend of lowest affinity at the (d) variant. However, this trend was not evident in functional experiments. Our findings suggest that it may be possible to design splice variant selective ligands, which may be of relevance for experimental drugs but may be difficult to develop clinically. 2010 S. Karger AG, Basel.

  8. Computational estimation of rainbow trout estrogen receptor binding affinities for environmental estrogens

    International Nuclear Information System (INIS)

    Shyu, Conrad; Cavileer, Timothy D.; Nagler, James J.; Ytreberg, F. Marty

    2011-01-01

    Environmental estrogens have been the subject of intense research due to their documented detrimental effects on the health of fish and wildlife and their potential to negatively impact humans. A complete understanding of how these compounds affect health is complicated because environmental estrogens are a structurally heterogeneous group of compounds. In this work, computational molecular dynamics simulations were utilized to predict the binding affinity of different compounds using rainbow trout (Oncorhynchus mykiss) estrogen receptors (ERs) as a model. Specifically, this study presents a comparison of the binding affinity of the natural ligand estradiol-17β to the four rainbow trout ER isoforms with that of three known environmental estrogens 17α-ethinylestradiol, bisphenol A, and raloxifene. Two additional compounds, atrazine and testosterone, that are known to be very weak or non-binders to ERs were tested. The binding affinity of these compounds to the human ERα subtype is also included for comparison. The results of this study suggest that, when compared to estradiol-17β, bisphenol A binds less strongly to all four receptors, 17α-ethinylestradiol binds more strongly, and raloxifene has a high affinity for the α subtype only. The results also show that atrazine and testosterone are weak or non-binders to the ERs. All of the results are in excellent qualitative agreement with the known in vivo estrogenicity of these compounds in the rainbow trout and other fishes. Computational estimation of binding affinities could be a valuable tool for predicting the impact of environmental estrogens in fish and other animals.

  9. Progesterone receptor variation and risk of ovarian cancer is limited to the invasive endometrioid subtype: results from the Ovarian Cancer Association Consortium pooled analysis

    DEFF Research Database (Denmark)

    Pearce, C.L.; Wu, A.H.; Gayther, S.A.

    2008-01-01

    There is evidence that progesterone plays a role in the aetiology of invasive epithelial ovarian cancer. Therefore, genes involved in pathways that regulate progesterone may be candidates for susceptibility to this disease. Previous studies have suggested that genetic variants in the progesterone...... receptor gene (PGR) may be associated with ovarian cancer risk, although results have been inconsistent. We have established an international consortium to pool resources and data from many ovarian cancer case-control studies in an effort to identify variants that influence risk. In this study, three PGR...... single nucleotide polymorphisms (SNPs), for which previous data have suggested they affect ovarian cancer risk, were examined. These were +331 C/T (rs10895068), PROGINS (rs1042838), and a 3' variant (rs608995). A total of 4788 ovarian cancer cases and 7614 controls from 12 case-control studies were...

  10. Abundance and Localization of Progesterone Receptor Isoforms in Endometrium in Women With and Without Endometriosis and in Peritoneal and Ovarian Endometriotic Implants.

    Science.gov (United States)

    Bedaiwy, Mohamed A; Dahoud, Wissam; Skomorovska-Prokvolit, Yelena; Yi, Lijuan; Liu, James H; Falcone, Tommaso; Hurd, William W; Mesiano, Sam

    2015-09-01

    Several studies suggest that resistance to progesterone may contribute to the pathophysiology of endometriosis. Progesterone mediates its biological activity via the 2 progesterone receptor (PR) isoforms (PR-A and PR-B). Effects of progesterone are determined by the PR-A:PR-B ratio such that a PR-B-dominant state promotes progesterone signaling, whereas a PR-A-dominant state decreases progesterone responsiveness. Our objective was to compare the abundance and cellular localization of the PR isoforms in endometrium and endometriotic lesions from women with and without peritoneal and ovarian endometriosis. This in vitro study was conducted in a tertiary care facility. Reproductive-age women with surgically diagnosed endometriosis (n = 18) and asymptomatic control individuals (n = 20) were prospectively recruited at the late proliferative and the early secretory phases. At laparoscopy, samples of eutopic endometrium, peritoneal and ovarian endometriosis, and disease-free peritoneum were obtained for subsequent immunohistochemical and immunoblot analysis of PR-B and total PR localization and PR-A and PR-B abundance, respectively. The PR-A and PR-B were detected in eutopic endometrium and in peritoneal and ovarian endometriosis but not in disease-free peritoneum from patients with and without endometriosis. In peritoneal endometriosis, PR-A was the predominant isoform detected, whereas both receptors were detected in ovarian endometriosis and eutopic endometrium. In eutopic endometrium, levels of PR-A were significantly elevated in women with endometriosis compared with women without disease, regardless of menstrual phase. The PR-A levels were significantly elevated in ovarian endometriosis compared with peritoneal endometriosis. Endometriotic lesions and eutopic endometrium from women with endometriosis are uniform in a PR-A-dominant state. The data suggest that menstrual efflux of a PR-A-dominant endometrial tissue into the peritoneal cavity may play a role in the

  11. Expression and localization of the progesterone receptor in mouse and human reproductive organs

    DEFF Research Database (Denmark)

    Teilmann, Stefan Cuoni; Clement, Christian Alexandro; Thorup, Jørgen

    2006-01-01

    examined, primary granulosa cell cilia lacked PR staining. SDS-PAGE and western blotting analysis of tissues from oviduct, uterus, and ovary confirmed antibody specificity, and identified two bands corresponding to the PR isoforms PR-A and PR-B. Upon hCG stimulation, a new band cross-reacting with anti......-PR emerged above the PR-A form in oviduct fractions, suggesting LH-induced phosphorylation of PR-A. We suggest that ciliary PR in the oviduct plays a role in progesterone signaling after ovulation, possibly via non-genomic events. These novel findings warrant further studies of oviduct and postovulatory...... were unstained or faintly stained. Pubertal female mice were further studied by confocal laser scanning microscopy and western blotting before and after injection with FSH and LH followed by human chorionic gonadotropin (hCG) injection after a 48-h period. PR immunolocalization to the oviduct cilia...

  12. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    International Nuclear Information System (INIS)

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-01-01

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 0 C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17β-[ 3 H]estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins

  13. Evidence that the angiotensin at 2-receptor agonist compound 21 is also a low affinity thromboxane TXA2-receptor antagonist

    DEFF Research Database (Denmark)

    Fredgart, M.; Leurgans, T.; Stenelo, M.

    2015-01-01

    Objective: The objective of this study was to test whether Compound 21 (C21), a high-affinity, non-peptide angiotensinAT2-receptor agonist, is also an antagonist of thromboxane A2 (TXA2) receptors thus reducing both vasoconstriction and platelet aggregation. Design and method: Binding of C21...... to the TXA2 receptor was determined by TBXA2R Arrestin Biosensor Assay. Mouse mesenteric arteries were mounted in wire myographs, and responses to increasing concentrations of C21 (1nM- 10muM) were recorded during submaximal contractions with 0.1muM U46619 (TXA2 analogue) or 1muMphenylephrine. To control for......AT2-receptor specificity, arteries were pre-incubated with the AT2-receptor antagonist PD123319 (10muM), or mesenteric arteries from AT2-receptor knock-out (AT2R-/y) mice were used. An inhibitory effect of C21 (100nM - 10muM) on U46619 (0,3muM) induced platelet aggregation was examined in whole human...

  14. Influence of Dexamethasone on Some Reproductive Hormones and Uterine Progesterone Receptor Localization in Pregnant Yankasa Sheep in Semiarid Zones of Nigeria.

    Science.gov (United States)

    Yahi, Dauda; Ojo, Nicholas Adetayo; Mshelia, Gideon Dauda

    2017-01-01

    Dexamethasone is widely used in both veterinary and human medical practices. However, it seems to cause some deleterious effects on pregnancy probably by causing changes in the reproductive hormone levels and their corresponding receptor concentrations. This study investigated the effects of dexamethasone on these parameters. Twenty healthy adult Yankasa sheep comprising 18 ewes and 2 rams were used for this study. Pregnancies were achieved by natural mating after estrus synchronization. Dexamethasone was administered at 0.25 mg/kg body weight on days 1, 3, and 5 during first trimester; days 51, 53, and 55 during second trimester; and days 101, 103, and 105 during the third trimester. Blood samples were collected biweekly for hormonal assay. Uterine biopsies were harvested through caesarean section for immunohistochemical analysis. Results showed that dexamethasone significantly ( p progesterone concentrations and caused abortion in Yankasa sheep but had no significant ( p > 0.05) effect on estrogen, while progesterone receptors (PR) were upregulated. The abortion could probably be due to decreased progesterone concentrations as a consequence of the adverse effects on placenta. The PR upregulation may be a compensatory mechanism to increase progesterone sensitivity. It was concluded that dexamethasone should not be used in advanced pregnancy in Yankasa sheep.

  15. Transient expression of progesterone receptor and cathepsin-l in human granulosa cells during the periovulatory period.

    Science.gov (United States)

    García, Víctor; Kohen, Paulina; Maldonado, Carola; Sierralta, Walter; Muñoz, Alex; Villarroel, Claudio; Strauss, Jerome F; Devoto, Luigi

    2012-03-01

    To study in vivo the progesterone receptor (PR) expression levels in human granulosa cells (GCs) during the periovulatory period and the affect of the protein kinase A (PKA) pathway on PR expression and cathepsin-L expression-activation. Experimental study. University research unit. Twenty-five women of reproductive age. Follicular fluid and GCs obtained from spontaneous cycles before and during the normal luteinizing hormone surge, and samples obtained 36 hours after human chorionic gonadotropin (hCG) administration in patients undergoing in vitro fertilization. To determine PR, cathepsin-L messenger RNA (mRNA) analysis via real-time polymerase chain reaction, and protein of PR, cathepsin-L, and PKA in human GCs. The Western blot analysis revealed that bands of PR (isoform A) were the most abundant and that mRNA (PR-A and PR-B) have a temporal pattern of expression throughout the periovulatory period. The protein levels of PR and cathepsin-L were up-regulated by hCG. The abundance of PR was diminished in the presence of PKA inhibitor, and cathepsin-L with PR receptor antagonist. The transient expression of PR in human GCs of the preovulatory follicle suggests that PR and its ligand play a role in the activation of cathepsin-L, which is presumably involved in the degradation of the follicular extracellular matrix during human ovulation. Copyright © 2012 American Society for Reproductive Medicine. All rights reserved.

  16. Ligand Binding Affinities of Arctigenin and Its Demethylated Metabolites to Estrogen Receptor Alpha

    Directory of Open Access Journals (Sweden)

    Masao Hattori

    2013-01-01

    Full Text Available Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (−-arctigenin, the aglycone of arctiin, was demethylated to (−-dihydroxyenterolactone (DHENL by Eubacterium (E. sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (−-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (−-arctigenin using a ligand binding screen assay method. The IC50 value of (2R,3R-2-(4-hydroxy-3-methoxybenzyl-3-(3,4-dihydroxybenzyl-butyrolactone was 7.9 × 10−4 M.

  17. Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

    Science.gov (United States)

    Taft, William C.; Delorenzo, Robert J.

    1984-05-01

    Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

  18. Parturition induction in ewes by a progesterone receptor blocker, aglepristone, and subsequent neonatal survival: Preliminary results.

    Science.gov (United States)

    Özalp, R G; Yavuz, A; Orman, A; Seker, I; Udum Küçükşen, D; Rişvanlı, A; Demiral, Ö O; Wehrend, A

    2017-01-01

    The clinical effects of aglepristone treatment to induce parturition in ewes and their newborns were reported. Three experimental groups were defined: group AG5 (n = 5), group AG10 (n = 5), and group CG (n = 5) in which ewes were injected twice with 5, 10 mg/kg of aglepristone, and saline solution of ewes, respectively. Different parameters associated with parturition in ewes and their newborns were investigated. Serum progesterone, oxytocin, and free and conjugated total estrogens were measured after treatments until parturition. No statistical difference was found from first aglepristone administration to onset of lambing between AG5 and AG10 (23.90 ± 6.20, 40.00 ± 6.71 hours). Parturition induction in two groups shortened the gestational length significantly compared with the control group (P = 0.003). Dystocia was observed in two ewes in group AG10. The placental weight showed statistically significant difference only between the AG10 and CG (P = 0.039), but no difference was observed in the placental expulsion period between the groups. Decrease in food consumption 24 to 36 hours after parturition in all ewes and skin necrosis in an ewe in group AG5 were observed. Progesterone concentration was significantly lower in AG5 than that in ewes in group AG10 and CG (P < 0.05). No difference was observed in concentrations of free total estrogens and oxytocin between groups. The body temperature of lambs was significantly different between AG10 and CG groups both right after (P = 0.011) and 12 hours after parturition (P = 0.014). The lambs in CG had the highest mean birth weight (4.29 ± 0.28 kg), which was significantly different from the induced groups. No significant difference of blood pH and blood gases values between groups was identified both at birth and 12 hours after parturition for lambs. Significant differences could clearly be observed in total protein and blood urea nitrogen and total protein findings 12 hours after parturition

  19. High Affinity IgE-Fc Receptor alpha and gamma Subunit Interactions

    International Nuclear Information System (INIS)

    Rashid, A.; Housden, J. E. M.; Sabban, S.; Helm, B.

    2014-01-01

    Objective: To explore the relationships between the subunits (alpha, beta and gamma) of the high affinity IgE receptor (Fc and RI) and its ability to mediate transmembrane signaling. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. Methodology: The approach employed was to create a chimera (human alpha-gamma-gamma) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc and RIalpha) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. Results: The RBLs cell lines transfected with the huFc and RIalpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc and RIalpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc and RIalpha subunit expression on the surface of transfected cells as seen by flowcytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. Conclusion: In the present study the grounds for non-expression of huFc and RIalpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes. (author)

  20. Testosterone stimulates progesterone production and STAR, P450 cholesterol side-chain cleavage and LH receptor mRNAs expression in hen (Gallus domesticus) granulosa cells.

    Science.gov (United States)

    Rangel, P L; Rodríguez, A; Rojas, S; Sharp, P J; Gutierrez, C G

    2009-12-01

    The chicken ovary is organized into a hierarchy of yellow yolky follicles that ovulate on successive days. Active or passive immunization of laying hens against testosterone blocks ovulation without affecting follicle development. Testosterone may play a role in pre-ovulatory follicle maturation by stimulating granulosa progesterone production. We assessed whether this stimulus is dose-related and depends on the maturity of the donor follicle, and if it does so by stimulating granulosa cell STAR, P450 cholesterol side-chain cleavage (P450scc), and LH receptor (LHCGR) mRNAs expression. Progesterone production by granulosa cells from F1, F3, and F4 follicles, cultured for 3 h without testosterone was greater in cells collected 11-14 h than 1-4 h after ovulation. These differences in progesterone production were less pronounced after granulosa cells had been cultured for 24 h. Culture of granulosa cells for 3 or 24 h with testosterone (1-100 ng/ml) stimulated progesterone production in cells collected from F4, F3, or F1 follicles 1-4, or 11-14 h after ovulation. Testosterone (0-4000 ng/ml) alone or in combination with LH (0-100 ng/ml) increased progesterone production by F1 granulosa cells, collected 1-4 and 11-14 h after ovulation and cultured for 3 h. Finally, testosterone (10 or 100 ng/ml) increased STAR, P450scc, and LHCGR mRNAs, when added to 3 h cultures of F1 granulosa cells. In conclusion, testosterone stimulates granulosa cell progesterone production in hen pre-ovulatory hierarchical follicles irrespective of maturational state, acting alone or additively with LH. We propose that testosterone promotes granulosa cell maturation to facilitate the pre-ovulatory release of LH.

  1. Progesterone receptor blockade in human breast cancer cells decreases cell cycle progression through G2/M by repressing G2/M genes.

    Science.gov (United States)

    Clare, Susan E; Gupta, Akash; Choi, MiRan; Ranjan, Manish; Lee, Oukseub; Wang, Jun; Ivancic, David Z; Kim, J Julie; Khan, Seema A

    2016-05-23

    The synthesis of specific, potent progesterone antagonists adds potential agents to the breast cancer prevention and treatment armamentarium. The identification of individuals who will benefit from these agents will be a critical factor for their clinical success. We utilized telapristone acetate (TPA; CDB-4124) to understand the effects of progesterone receptor (PR) blockade on proliferation, apoptosis, promoter binding, cell cycle progression, and gene expression. We then identified a set of genes that overlap with human breast luteal-phase expressed genes and signify progesterone activity in both normal breast cells and breast cancer cell lines. TPA administration to T47D cells results in a 30 % decrease in cell number at 24 h, which is maintained over 72 h only in the presence of estradiol. Blockade of progesterone signaling by TPA for 24 h results in fewer cells in G2/M, attributable to decreased expression of genes that facilitate the G2/M transition. Gene expression data suggest that TPA affects several mechanisms that progesterone utilizes to control gene expression, including specific post-translational modifications, and nucleosomal organization and higher order chromatin structure, which regulate access of PR to its DNA binding sites. By comparing genes induced by the progestin R5020 in T47D cells with those increased in the luteal-phase normal breast, we have identified a set of genes that predict functional progesterone signaling in tissue. These data will facilitate an understanding of the ways in which drugs such as TPA may be utilized for the prevention, and possibly the therapy, of human breast cancer.

  2. Progesterone receptor blockade in human breast cancer cells decreases cell cycle progression through G2/M by repressing G2/M genes

    International Nuclear Information System (INIS)

    Clare, Susan E.; Gupta, Akash; Choi, MiRan; Ranjan, Manish; Lee, Oukseub; Wang, Jun; Ivancic, David Z.; Kim, J. Julie; Khan, Seema A.

    2016-01-01

    The synthesis of specific, potent progesterone antagonists adds potential agents to the breast cancer prevention and treatment armamentarium. The identification of individuals who will benefit from these agents will be a critical factor for their clinical success. We utilized telapristone acetate (TPA; CDB-4124) to understand the effects of progesterone receptor (PR) blockade on proliferation, apoptosis, promoter binding, cell cycle progression, and gene expression. We then identified a set of genes that overlap with human breast luteal-phase expressed genes and signify progesterone activity in both normal breast cells and breast cancer cell lines. TPA administration to T47D cells results in a 30 % decrease in cell number at 24 h, which is maintained over 72 h only in the presence of estradiol. Blockade of progesterone signaling by TPA for 24 h results in fewer cells in G2/M, attributable to decreased expression of genes that facilitate the G2/M transition. Gene expression data suggest that TPA affects several mechanisms that progesterone utilizes to control gene expression, including specific post-translational modifications, and nucleosomal organization and higher order chromatin structure, which regulate access of PR to its DNA binding sites. By comparing genes induced by the progestin R5020 in T47D cells with those increased in the luteal-phase normal breast, we have identified a set of genes that predict functional progesterone signaling in tissue. These data will facilitate an understanding of the ways in which drugs such as TPA may be utilized for the prevention, and possibly the therapy, of human breast cancer. The online version of this article (doi:10.1186/s12885-016-2355-5) contains supplementary material, which is available to authorized users

  3. Estrogen and progesterone receptors have distinct roles in the establishment of the hyperplastic phenotype in PR-A transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Simian, Marina; Bissell, Mina J.; Barcellos-Hoff, Mary Helen; Shyamala, Gopalan

    2009-05-11

    Expression of the A and B forms of progesterone receptor (PR) in an appropriate ratio is critical for mammary development. Mammary glands of PR-A transgenic mice, carrying an additional A form of PR as a transgene, exhibit morphological features associated with the development of mammary tumors. Our objective was to determine the roles of estrogen (E) and progesterone (P) in the genesis of mammary hyperplasias/preneoplasias in PR-A transgenics. We subjected PR-A mice to hormonal treatments and analyzed mammary glands for the presence of hyperplasias and used BrdU incorporation to measure proliferation. Quantitative image analysis was carried out to compare levels of latency-associated peptide and transforming growth factor beta 1 (TGF{beta}1) between PR-A and PR-B transgenics. Basement membrane disruption was examined by immunofluorescence and proteolytic activity by zymography. The hyperplastic phenotype of PR-A transgenics is inhibited by ovariectomy, and is reversed by treatment with E + P. Studies using the antiestrogen ICI 182,780 or antiprogestins RU486 or ZK 98,299 show that the increase in proliferation requires signaling through E/estrogen receptor alpha but is not sufficient to give rise to hyperplasias, whereas signaling through P/PR has little impact on proliferation but is essential for the manifestation of hyperplasias. Increased proliferation is correlated with decreased TGF{beta}1 activation in the PR-A transgenics. Analysis of basement membrane integrity showed loss of laminin-5, collagen III and collagen IV in mammary glands of PR-A mice, which is restored by ovariectomy. Examination of matrix metalloproteases (MMPs) showed that total levels of MMP-2 correlate with the steady-state levels of PR, and that areas of laminin-5 loss coincide with those of activation of MMP-2 in PR-A transgenics. Activation of MMP-2 is dependent on treatment with E and P in ovariectomized wild-type mice, but is achieved only by treatment with P in PR-A mice. These data

  4. Estrogen and progesterone receptor levels in nonneoplastic breast epithelium of breast cancer cases versus benign breast biopsy controls

    International Nuclear Information System (INIS)

    Woolcott, Christy G; SenGupta, Sandip K; Hanna, Wedad M; Aronson, Kristan J

    2008-01-01

    Previous studies and biological mechanisms of carcinogenesis suggest that the steroid receptor content of benign breast epithelium may be related to breast cancer risk. The objective in this study was to compare the levels of estrogen receptor-α (ER) and progesterone receptor (PR) in nonneoplastic breast epithelium between breast cancer cases and biopsy controls. Between 1995 and 1997 at two sites (Women's College Hospital in Toronto and Kingston General Hospital), 667 women who were scheduled for diagnostic excisional breast biopsies completed a questionnaire providing personal information and agreed to allow analysis of routinely resected tissue. Histological slides with nonneoplastic epithelium were available for 101 cancer cases and 200 biopsy controls in Toronto and for 105 cancer cases and 119 controls in Kingston. Nonneoplastic epithelium was examined with immunohistochemical assays to determine the percent of epithelial cells staining for ER and PR. Unconditional logistic regression was used to calculate odds ratios (OR) stratified by study site. The ER content of nonneoplastic tissue was higher in cases than biopsy controls in unadjusted analyses; after adjustment for age, however, a weak association remained in only one of the study sites. After adjustment for age, the PR content of nonneoplastic tissue was slightly lower in breast cancer cases than controls in one study site. Furthermore, this inverse association was confined to women with PR negative breast cancer in comparison to the controls. No interaction between ER and PR content of nonneoplastic tissue was observed in relation to the odds of having breast cancer. The results of this study are consistent with only a slight indication of increased ER levels in nonneoplastic tissue in breast cancer cases relative to controls. This study contributes to the understanding of breast cancer by examining both ER and PR in nonneoplastic tissue. Limitations remain, however, such as the necessity of

  5. A double-labeling assay for simultaneous estimation and characterization of estrogen and progesterone receptors using radioiodinated estradiol and tritiaded Org 2058

    International Nuclear Information System (INIS)

    Ronchi, E.; Granata, G.; Brivio, M.

    1986-01-01

    Estrogen (ER) and progesterone receptors (PgR) appear to be a perequisite to elicit a biologic response by a hormone-target organ. Current methodologies for analysis of these proteins (e.g:dextran-coated charcoal, DCC) in single-label assay (SLA) require relatively large amounts of tissue material, time and laboriousness. Therefore, the authors have developed for breast cancer tissue an improved dual-label assay (DLA) for simultaneous titration (by DCC) and/or characterization (by sedimentation properties) of ER and PgR on the same sample, using *H1*H2*H5I-E*L2 and *H3H-Org 2058 as tracers. The interaction of *H1*H2*H5I-E*L2 with ER and plasma proteins in comparison to *H3H-E*L2 was studied in terms of specificity, time course, affinity binding and sedimentation pattern. *H1*H2*H5I-E*L2 bound the same molecular forms displayed by *H3H-E*L2 (9 and 3S) but with lower titers (about 1.3-fold), irrespective of the technique used, and did not bind to sex hormone-binding globulin. Simultaneous detection of *H1*H2*H5I and *H3H was achieved by use of a gamma counter plus a beta counter sequentially. ER and PgR titrations with DCC in DLA were in good agreement with those obtained with SLA, in terms of titers and Ka values. An analogous results was obtained with sucrose density gradient (SDG) analysis. Both the DLA methods were highly reproducible (CV<8.0%). Between the rotors available for SDG, the vertical one was preferable because of the larger number of samples processed and of less purturbation of sedimenting receptor molecules. Furthermore, a biochemical applications of the method is described. In conclusion, the DLA procedure, by simplifying ER and PgR estimation, makes it possible to study, even on small tumor biopsies, the molecular properties of these proteins in relation to the clinical response of the disease

  6. Diffusion Profiling via a Histogram Approach Distinguishes Low-grade from High-grade Meningiomas, Can Reflect the Respective Proliferative Potential and Progesterone Receptor Status.

    Science.gov (United States)

    Gihr, Georg Alexander; Horvath-Rizea, Diana; Garnov, Nikita; Kohlhof-Meinecke, Patricia; Ganslandt, Oliver; Henkes, Hans; Meyer, Hans Jonas; Hoffmann, Karl-Titus; Surov, Alexey; Schob, Stefan

    2018-02-01

    Presurgical grading, estimation of growth kinetics, and other prognostic factors are becoming increasingly important for selecting the best therapeutic approach for meningioma patients. Diffusion-weighted imaging (DWI) provides microstructural information and reflects tumor biology. A novel DWI approach, histogram profiling of apparent diffusion coefficient (ADC) volumes, provides more distinct information than conventional DWI. Therefore, our study investigated whether ADC histogram profiling distinguishes low-grade from high-grade lesions and reflects Ki-67 expression and progesterone receptor status. Pretreatment ADC volumes of 37 meningioma patients (28 low-grade, 9 high-grade) were used for histogram profiling. WHO grade, Ki-67 expression, and progesterone receptor status were evaluated. Comparative and correlative statistics investigating the association between histogram profiling and neuropathology were performed. The entire ADC profile (p10, p25, p75, p90, mean, median) was significantly lower in high-grade versus low-grade meningiomas. The lower percentiles, mean, and modus showed significant correlations with Ki-67 expression. Skewness and entropy of the ADC volumes were significantly associated with progesterone receptor status and Ki-67 expression. ROC analysis revealed entropy to be the most accurate parameter distinguishing low-grade from high-grade meningiomas. ADC histogram profiling provides a distinct set of parameters, which help differentiate low-grade versus high-grade meningiomas. Also, histogram metrics correlate significantly with histological surrogates of the respective proliferative potential. More specifically, entropy revealed to be the most promising imaging biomarker for presurgical grading. Both, entropy and skewness were significantly associated with progesterone receptor status and Ki-67 expression and therefore should be investigated further as predictors for prognostically relevant tumor biological features. Since absolute ADC

  7. Effect of long-term treatment with steroid hormones or tamoxifen on the progesterone receptor and androgen receptor in the endometrium of ovariectomized cynomolgus macaques

    Directory of Open Access Journals (Sweden)

    Cline J Mark

    2003-02-01

    Full Text Available Abstract The progesterone receptor (PR and androgen receptor (AR belong to the nuclear receptor superfamily. Two isoforms of PR (A and B have been identified with different functions. The expression of AR, each isoform of PR and their involvement in long-term effects on the endometrium after hormonal replacement therapy (HRT or tamoxifen (TAM treatment is not known. The aims of this study were to determine PR(A+B, PRB and AR distribution by immunohistochemistry in the macaque (Macaca fascicularis endometrium. Ovariectomized (OVX animals were orally treated continuously for 35 months with either conjugated equine estrogens (CEE; medroxyprogesterone acetate (MPA; the combination of CEE/MPA; or TAM. Treatment with CEE/MPA tended to down-regulate PR in the superficial glands, but increased it in the stroma. TAM treatment increased both the PR and PRB levels in the stroma. Overall, less than 20% of the cells were positive for the PRB isoform and less variation was observed after steroid treatment. AR was found in the stroma, mainly distributed in the basal layer of the endometrium in the OVX and steroid treated groups, but was absent in the TAM treated group. No AR was found in the glandular epithelium. The present data show that long-term hormone treatment affects the PR level, and also the ratio between PRA and PRB in the endometrium.

  8. Development of the correlation study between mammographic appearances and expression of estrogen receptor, progesterone receptor and C-erbB-2 in patients with breast cancer

    International Nuclear Information System (INIS)

    Liu Jie; Liu Peifang

    2012-01-01

    Breast cancer is one of the most common female malignant tumors. The occurrence and development of breast cancer are often accompanied by abnormal gene expression. It has been well accepted that estrogen receptor (ER), progesterone receptor (PR) and C-erbB-2 (epidermal growth factor receptor-2) are the main reference indexes for clinical endocrine therapy and prognosis prediction. Oncogene expression reflects the malignant biological behavior of breast cancer from different perspectives. Those aggressive behaviors cause a variety of changes in histopathology and thus in imaging. Therefore, the imaging features of breast cancer may indirectly demonstrate the status of ER, PR and C-erbB-2 to a certain extent. Until now, because of its simple operation,low cost and high accuracy, mammography is still the preferred method in breast cancer screening and diagnosis, and has been proposed as a noninvasive technique for prediction of the expression of ER, PR and C-erbB-2. It has potential clinical value in preoperative assessment and non-surgical treatment for those who are not able to perform immunohistochemistry. This review summarized previous related studies and explored mammography as a noninvasive predictor for ER, PR and C-erbB-2. (authors)

  9. High-affinity binding of [3H]estradiol-17 beta by an estrogen receptor in the liver of the turtle

    International Nuclear Information System (INIS)

    Ho, S.M.; Fehrer, S.; Yu, M.; Liang, L.C.; Press, D.

    1988-01-01

    Specific [3H]estradiol-17 beta ([3H]E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds [3H]E2 with high affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of [3H]E2 binding activity in both cytosolic and nuclear fractions. The exchange between [3H]E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species

  10. Molecular Affinity of Mabolo Extracts to an Octopamine Receptor of a Fruit Fly

    Directory of Open Access Journals (Sweden)

    Francoise Neil D. Dacanay

    2017-10-01

    Full Text Available Essential oils extracted from plants are composed of volatile organic compounds that can affect insect behavior. Identifying the active components of the essential oils to their biochemical target is necessary to design novel biopesticides. In this study, essential oils extracted from Diospyros discolor (Willd. were analyzed using gas chromatography mass spectroscopy (GC-MS to create an untargeted metabolite profile. Subsequently, a conformational ensemble of the Drosophila melanogaster octopamine receptor in mushroom bodies (OAMB was created from a molecular dynamics simulation to resemble a flexible receptor for docking studies. GC-MS analysis revealed the presence of several metabolites, i.e. mostly aromatic esters. Interestingly, these aromatic esters were found to exhibit relatively higher binding affinities to OAMB than the receptor’s natural agonist, octopamine. The molecular origin of this observed enhanced affinity is the π -stacking interaction between the aromatic moieties of the residues and ligands. This strategy, computational inspection in tandem with untargeted metabolomics, may provide insights in screening the essential oils as potential OAMB inhibitors.

  11. NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

    Science.gov (United States)

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

    2002-07-01

    Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

  12. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    International Nuclear Information System (INIS)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-01-01

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K d 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K d 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy

  13. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  14. Decreased Progesterone Receptor B/A Ratio in Endometrial Cells by Tumor Necrosis Factor-Alpha and Peritoneal Fluid from Patients with Endometriosis.

    Science.gov (United States)

    Chae, Uisoo; Min, Jin Young; Kim, Sung Hoon; Ihm, Hyo Jin; Oh, Young Sang; Park, So Yun; Chae, Hee Dong; Kim, Chung Hoon; Kang, Byung Moon

    2016-11-01

    Progesterone resistance is thought to be a major factor that contributes to progression of endometriosis. However, it is not clear what causes progesterone resistance in endometriosis. This study aimed to assess whether cytokines or peritoneal fluid can affect progesterone receptor (PR) expression in endometrial cells and to verify whether PR expression is reduced in endometriosis. The PR-B/A ratio was measured via real-time polymerase chain reaction after in vitro culture, in which endometrial cells were treated with either tumor necrosis factor-alpha (TNF-α), interleukin-1 beta, or peritoneal fluid obtained from women with advanced-stage endometriosis. Immunohistochemistry was performed to compare PR-B expression between eutopic and ectopic endometrial tissues from women with and without advanced-stage endometriosis. The PR-B/A ratio was significantly decreased by treatment with either TNF-α (p=0.011) or peritoneal fluid from women with advanced-stage endometriosis (p=0.027). Immunoreactivity of PR-B expression was significantly lower during the secretory phase than during the proliferative phase in endometrial tissues from control subjects (pendometriosis compared with eutopic endometrium tissues from control subjects. Progesterone resistance in endometriosis may be caused by proinflammatory conditions in the pelvic peritoneal microenvironment.

  15. Diagnostic value of progesterone receptor, p16, p53 and pHH3 expression in uterine atypical leiomyoma.

    Science.gov (United States)

    Liang, Yun; Zhang, Xiaofei; Chen, Xiaoduan; Lü, Weiguo

    2015-01-01

    The differential diagnosis between atypical leiomyoma and leiomyosarcoma may be hard based on morphological criterion at times. It would be helpful to find out biomarkers that can be used to distinguish them. The aim of the study was to investigate the diagnostic value of progesterone receptor (PR), p16, p53 and pHH3 expression in a series of uterine smooth muscle tumors. Immunohistochemical expression of PR, p16, p53 and pHH3 was investigated on 32 atypical leiomyomas, 15 leiomyosarcomas and 15 usual leomyomas. The difference in expression was compared between atypical leiomyoma and other groups. The expression of PR, p16, and pHH3 was found significantly different between atypical leiomyomas and leiomyosarcomas, but lack of significant difference between atypical leiomyomas and usual leiomyomas. There was no significant difference with regard to p53 distribution among these uterine smooth muscle tumors. High p16, pHH3 expression and low PR expression preferred the diagnosis of leiomyosarcoma. The panel of antibodies used in this study is a useful complementary analysis in the assessment of problematic uterine smooth muscle tumors.

  16. Progesterone radioimmunoassay

    International Nuclear Information System (INIS)

    Allen, R.M.; Redshaw, M.R.

    1981-01-01

    This patent claims a radioimmunoassay for progesterone, which comprises contacting, in an acidic medium a sample of liquid with a predetermined amount of antibodies raised against a progesterone-protein complex, the protein being attached to the 11-position of progesterone by means of a bridging group and with a predetermined amount of a progesterone derivative having an iodinatable group attached to its 3-position by means of a bridging group, the iodinatable group being iodinated with one or more atom(s) of a radioisotope of iodine, separating the steroid bound in the resulting antibody-antigen complex from the free steroid and measuring the radioactivity of the free steroid component or of the antibody-antigen complex. Sufficient sensitivity has been achieved to enable a progesterone assay to be carried out directly on a sample of biological fluid, such as serum, plasma, urine or milk. (U.K.)

  17. Stromal progesterone receptors mediate induction of Indian Hedgehog (IHH) in uterine epithelium and its downstream targets in uterine stroma.

    Science.gov (United States)

    Simon, Liz; Spiewak, Kerry A; Ekman, Gail C; Kim, Jaeyeon; Lydon, John P; Bagchi, Milan K; Bagchi, Indrani C; DeMayo, Francesco J; Cooke, Paul S

    2009-08-01

    Uterine receptivity to embryo implantation depends on appropriate progesterone (P4) and estrogen stimulation. P4 rapidly stimulates production of the morphogen Indian hedgehog (IHH) in murine uterine epithelium as well as downstream molecules in the hedgehog pathway such as Patched homolog 1 (PTCH1) and nuclear receptor subfamily 2, group F, member 2 (NR2F2) in uterine stroma. Studies using IHH-null mice indicate that IHH is obligatory for the normal P4 response in the uterus. To determine whether IHH induction in uterine epithelium is mediated through P4 receptor (PR) in epithelium (E) and/or stroma (S), we produced tissue recombinants using uteri from neonatal PR knockout (ko) mice and wild-type (wt) mice containing PR in S and/or E or lacking PR altogether using a tissue recombinant methodology and assessed their response to P4. In tissue recombinants containing wt-S (wt-S + wt-E and wt-S + ko-E), P4 induced Ihh mRNA expression at 6 h that was 6-fold greater than in oil-treated controls (P Ihh mRNA expression was unaffected by P4 in ko-S + ko-E and ko-S + wt-E grafts despite epithelial PR expression in the latter. Nr2f2 and Ptch1 mRNA expression was similar in that it was stimulated by P4 only in recombinants containing stromal PR. These results indicate that stromal PR is both necessary and sufficient for P4 stimulation of epithelial IHH as well as downstream events such as PTCH1 and NR2F2 increases in stroma.

  18. Body weight and incidence of breast cancer defined by estrogen and progesterone receptor status--a meta-analysis.

    Science.gov (United States)

    Suzuki, Reiko; Orsini, Nicola; Saji, Shigehira; Key, Timothy J; Wolk, Alicja

    2009-02-01

    Epidemiological evidence indicates that the association between body weight and breast cancer risk may differ across menopausal status as well as the estrogen receptor (ER) and progesterone receptor (PR) tumor status. To date, no meta-analysis has been conducted to assess the association between body weight and ER/PR defined breast cancer risk, taking into account menopausal status and study design. We searched MEDLINE for relevant studies published from January 1, 1970 through December 31, 2007. Summarized risk estimates with 95% confidence intervals (CIs) were calculated using a random-effects model. The summarized results of 9 cohorts and 22 case-control studies comparing the highest versus the reference categories of relative body weight showed that the risk for ER+PR+ tumors was 20% lower (95% CI=-30% to -8%) among premenopausal (2,643 cases) and 82% higher (95% CI=55-114%) among postmenopausal (5,469 cases) women. The dose-response meta-analysis of ER+PR+ tumors showed that each 5-unit increase in body mass index (BMI, kg/m2) was associated with a 33% increased risk among postmenopausal women (95% CI=20-48%) and 10% decreased risk among premenopausal women (95% CI=-18% to -1%). No associations were observed for ER-PR- or ER+PR- tumors. For discordant tumors ER+PR- (pre) and ER-PR+ (pre/post) the number of cases were too small (weight and breast cancer risk is critically dependent on the tumor's ER/PR status and the woman's menopausal status. Body weight control is the effective strategy for preventing ER+PR+ tumors after menopause. Copyright (c) 2008 Wiley-Liss, Inc.

  19. Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

    International Nuclear Information System (INIS)

    Gil, D.W.; Wolfe, B.B.

    1986-01-01

    Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands [ 3 H]quinuclidinyl benzilate or [ 3 H]PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of [ 3 H]quinuclidinyl benzilate in a biphasic manner

  20. Progesterone impairs social recognition in male rats.

    Science.gov (United States)

    Bychowski, Meaghan E; Auger, Catherine J

    2012-04-01

    The influence of progesterone in the brain and on the behavior of females is fairly well understood. However, less is known about the effect of progesterone in the male system. In male rats, receptors for progesterone are present in virtually all vasopressin (AVP) immunoreactive cells in the bed nucleus of the stria terminalis (BST) and the medial amygdala (MeA). This colocalization functions to regulate AVP expression, as progesterone and/or progestin receptors (PR)s suppress AVP expression in these same extrahypothalamic regions in the brain. These data suggest that progesterone may influence AVP-dependent behavior. While AVP is implicated in numerous behavioral and physiological functions in rodents, AVP appears essential for social recognition of conspecifics. Therefore, we examined the effects of progesterone on social recognition. We report that progesterone plays an important role in modulating social recognition in the male brain, as progesterone treatment leads to a significant impairment of social recognition in male rats. Moreover, progesterone appears to act on PRs to impair social recognition, as progesterone impairment of social recognition is blocked by a PR antagonist, RU-486. Social recognition is also impaired by a specific progestin agonist, R5020. Interestingly, we show that progesterone does not interfere with either general memory or olfactory processes, suggesting that progesterone seems critically important to social recognition memory. These data provide strong evidence that physiological levels of progesterone can have an important impact on social behavior in male rats. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Characterization of the somatogenic receptor in rat liver. Hydrodynamic properties and affinity cross-linking

    International Nuclear Information System (INIS)

    Husman, B.; Haldosen, L.A.; Andersson, G.; Gustafsson, J.A.

    1988-01-01

    Rat liver somatogenic receptors have been characterized by gel permeation chromatography, sucrose density gradients in H 2 O and D 2 O, and affinity cross-linking using 125 I-bovine growth hormone (bGH) as a specific somatogenic receptor ligand. Cross-linking of 125 I-bovine growth hormone to a Triton X-100-treated low density fraction isolated from livers of late pregnant rats followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions showed three major binders with Mr 95,000, 86,000, and 43,000 and a minor binder of Mr 55,000, after correction for bound ligand assuming a 1:1 binding ratio of ligand-receptor. The Mr 86,000, 55,000, and 43,000 species were recovered in the detergent-soluble supernatant after high-speed centrifugation, whereas the Mr 95,000 species remained Triton X-100 insoluble. Detergent-soluble 125 I-bGH-receptor complexes were further analyzed by sedimentation into sucrose density gradients. The sedimentation coefficient was S20,w = 5.2 S and the partial specific volume v = 0.72 ml/g. Gel permeation chromatography on a Sepharose S-400 column indicated a Stokes radius of 61 A for the 125 I-bGH-receptor-Triton X-100 complex. Based on these figures, the molecular weight of the complex was calculated as 131,100. The molecular weight of the ligand-free receptor-Triton X-100 complex was calculated as Mr 109,100. Affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 61 A peak from Sephacryl S-400 chromatography (cf. above) showed two binding entities, one major and one minor with Mr values 86,000 and 43,000, respectively, in the absence of reductant. When electrophoresis was run in the presence of reductant the Mr 43,000 species was the major binding entity

  2. Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity.

    Science.gov (United States)

    Jin, Rongsheng; Rummel, Andreas; Binz, Thomas; Brunger, Axel T

    2006-12-21

    Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng kg(-1), they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. The molecular details of the toxin-cell recognition have been elusive. Here we report the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15 A resolution. On binding, a helix is induced in the luminal domain which binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. Our results provide the basis for the rational development of preventive vaccines or inhibitors against these neurotoxins.

  3. The prognostic value of age for invasive lobular breast cancer depending on estrogen receptor and progesterone receptor-defined subtypes: A NCDB analysis.

    Science.gov (United States)

    Liu, Jieqiong; Chen, Kai; Mao, Kai; Su, Fengxi; Liu, Qiang; Jacobs, Lisa K

    2016-02-02

    We aimed to assess the effect of age on survival according to estrogen receptor (ER) and progesterone receptor (PR)-defined lobular breast cancer subtype in a wide age range. 43,230 invasive lobular breast cancer women without comorbidities diagnosed between 2004 and 2011 in the National Cancer Database (NCDB) were analyzed. The effects of age on overall survival (OS) among different age groups were evaluated by log-rank test and Cox proportional model. Multivariate analysis showed that patients diagnosed at both young ( 0.1); and in ER-PR+ subgroup, the HRs were similar in patients younger than 70 (P > 0.1); thus, the plots of HRs in these three subtypes remained steady until the age of 60 or 70. Our findings identified that the effect of age on OS in lobular breast cancer varied with ER/PR-defined subtypes. Personalized treatment strategies should be developed to improve outcomes of breast cancer patients with different ages and ER/PR statuses.

  4. The progesterone receptor antagonist, onapristone has differential effects on the timing and control of the luteolytic mechanism depending on timing of administration in sheep.

    Science.gov (United States)

    Mann, G E; Wathes, D C; Robinson, R S

    2013-08-25

    Cyclic ewes were treated with control vehicle or progesterone receptor antagonist (onapristone; 100mg i.m. twice daily) during either early (day 3-5) or late (day 12-14) luteal phase and plasma samples collected for hormone analysis and to determine endogenous and oxytocin induced PGF2α release. On day 14 and 17, ewes were euthanised and reproductive tracts collected for ovarian morphology and endometrium for oxytoxin and steroid hormone receptor analysis. Early treatment increased LH, but not progesterone or oestradiol, while late treatment elevated all three hormones. Early treatment delayed the up-regulation of endometrial oxytocin receptors and responsiveness to oxytocin challenge, delaying luteolysis. Late treatment advanced development of oxytocin receptors and responsiveness to oxytocin though not timing of luteolysis. Patterns of hormone receptor mRNA were differentially disrupted by treatments. Results provide mechanistic insight into hormonal control of the oestrous cycle and identify the ability of the luteolytic mechanism to dissociate from functional luteolysis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain.

    Science.gov (United States)

    Lai, H; Carino, M A

    1992-07-01

    Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.

  6. Influence of Dexamethasone on Some Reproductive Hormones and Uterine Progesterone Receptor Localization in Pregnant Yankasa Sheep in Semiarid Zones of Nigeria

    Directory of Open Access Journals (Sweden)

    Dauda Yahi

    2017-01-01

    Full Text Available Dexamethasone is widely used in both veterinary and human medical practices. However, it seems to cause some deleterious effects on pregnancy probably by causing changes in the reproductive hormone levels and their corresponding receptor concentrations. This study investigated the effects of dexamethasone on these parameters. Twenty healthy adult Yankasa sheep comprising 18 ewes and 2 rams were used for this study. Pregnancies were achieved by natural mating after estrus synchronization. Dexamethasone was administered at 0.25 mg/kg body weight on days 1, 3, and 5 during first trimester; days 51, 53, and 55 during second trimester; and days 101, 103, and 105 during the third trimester. Blood samples were collected biweekly for hormonal assay. Uterine biopsies were harvested through caesarean section for immunohistochemical analysis. Results showed that dexamethasone significantly (p0.05 effect on estrogen, while progesterone receptors (PR were upregulated. The abortion could probably be due to decreased progesterone concentrations as a consequence of the adverse effects on placenta. The PR upregulation may be a compensatory mechanism to increase progesterone sensitivity. It was concluded that dexamethasone should not be used in advanced pregnancy in Yankasa sheep.

  7. Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular Leydig cells

    International Nuclear Information System (INIS)

    Stalker, A.; Hermo, L.; Antakly, T.

    1989-01-01

    The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor

  8. Progesterone increases ex vivo testosterone production and decreases the expression of progestin receptors and steroidogenic enzymes in the fathead minnow (Pimephales promelas) ovary.

    Science.gov (United States)

    Chishti, Yasmin Z; Feswick, April; Martyniuk, Christopher J

    2014-04-01

    Progesterone (P4) is a metabolic precursor for a number of steroids, including estrogens and androgens. P4 also has diverse roles within the vertebrate ovary that include oocyte growth and development. The objectives of this study were to measure the effects of P4 on testosterone (T) and 17β-estradiol (E2) production in the fathead minnow (FHM) ovary and on the mRNA abundance of transcripts involved in steroidogenesis and steroid receptor signaling. Ovary explants were treated with P4 (10(-6)M) for 6 and 12h. P4 administration significantly increased T production ∼3-fold at both 6 and 12h, whereas E2 production was not affected, consistent with the hypothesis that excess P4 is not converted to terminal estrogens in the mature ovary. Nuclear progesterone receptor mRNA was decreased at 6h and membrane progesterone receptor gamma-2 mRNA was significantly down-regulated at both 6 and 12h; however there was no change in membrane progesterone receptor alpha or beta mRNA levels. Androgen receptor (ar) and estrogen receptor 2a (esr2a) mRNA were significantly reduced at 6h with P4 treatment, but there was no change in esr2b mRNA at either time point. Transcripts for enzymes in the steroid pathway (star, hsd11b2) were significantly lower at 6h compared to controls, whereas cyp17a and cyp19a mRNA abundance did not change with treatments at either time point. These data suggest that P4 incubation can lead to increased T production in the FHM ovary without a concomitant change in E2, and that the membrane bound progestin receptors are differentially regulated by P4 in the teleost ovary. As environmental progestins have received increased attention due to their suspected role as endocrine disruptors, mechanistic data on the role of exogenous P4 treatments in the male and female gonad is warranted. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Estrogen Receptor Binding Affinity of Food Contact Material Components Estimated by QSAR.

    Science.gov (United States)

    Sosnovcová, Jitka; Rucki, Marián; Bendová, Hana

    2016-09-01

    The presented work characterized components of food contact materials (FCM) with potential to bind to estrogen receptor (ER) and cause adverse effects in the human organism. The QSAR Toolbox, software application designed to identify and fill toxicological data gaps for chemical hazard assessment, was used. Estrogen receptors are much less of a lock-and-key interaction than highly specific ones. The ER is nonspecific enough to permit binding with a diverse array of chemical structures. There are three primary ER binding subpockets, each with different requirements for hydrogen bonding. More than 900 compounds approved as of FCM components were evaluated for their potential to bind on ER. All evaluated chemicals were subcategorized to five groups with respect to the binding potential to ER: very strong, strong, moderate, weak binder, and no binder to ER. In total 46 compounds were characterized as potential disturbers of estrogen receptor. Among the group of selected chemicals, compounds with high and even very high affinity to the ER binding subpockets were found. These compounds may act as gene activators and cause adverse effects in the organism, particularly during pregnancy and breast-feeding. It should be considered to carry out further in vitro or in vivo tests to confirm their potential to disturb the regulation of physiological processes in humans by abnormal ER signaling and subsequently remove these chemicals from the list of approved food contact materials. Copyright© by the National Institute of Public Health, Prague 2016

  10. Specific, high affinity receptors for insulin-like growth factor II in the rat kidney glomerulus

    International Nuclear Information System (INIS)

    Haskell, J.F.; Pillion, D.J.; Meezan, E.

    1988-01-01

    Rat renal glomeruli were isolated by a technique involving kidney perfusion with a solution containing magnetic iron oxide particles, followed by homogenization, sieving, and concentration over a strong magnet. Isolated glomeruli were treated with 1% Triton X-100 to solubilize plasma membrane components, while insoluble basement membrane components were removed by centrifugation. [ 125 I]Insulin-like growth factor II (IGF-II) binding to this preparation was competitively inhibited by increasing amounts of unlabeled IGF-II, with 50% inhibition at an IGF-II concentration of 1 ng/ml. [ 125 I]IGF-II was covalently cross-linked with disuccinimidyl suberate to its receptor in rat renal glomeruli and a specific high mol wt (255,000) band could be identified on autoradiograms of dodecyl sulfate-polyacrylamide gels. [ 125 I]IGF-II binding and cross-linking to this band was inhibited by a polyclonal antibody against the type II IGF receptor. These results demonstrate for the first time that the isolated rat renal glomerulus contains a high affinity receptor for IGF-II

  11. Estrogen and progesterone receptors in human decidua after RU486 treatment.

    Science.gov (United States)

    Shi, W L; Wang, J D; Fu, Y; Zhu, P D

    1993-07-01

    To examine RU486 action on decidua at the level of cellular estrogen receptor (ER) and P receptor (PR). Controlled basic study for contragestion mechanism of mifepristone. Normal human volunteers in an academic research environment. Sixty women with 6 to 7 weeks of gestation who voluntarily requested termination of pregnancy were recruited and randomly divided into three groups. A single dose of 200 mg RU486 was orally administered to the two treatment groups 12 and 24 hours, respectively, before surgical interruption of pregnancies. Placebo was used for control group. Decidual tissues were collected right after operation. Immunocytochemical reactions of PR and ER in decidua after RU486 treatment were compared with the control subjects. The differences of the reaction in decidual area with or without trophoblast invasion were noted. RU486 treatment increased PR and ER staining in vessel and stroma of decidua without trophoblast invasion (decidua parietalis) but not in decidua with trophoblast invasion (decidua capsularis or basalis). Chi-squared analysis indicated a significant increase in the number of ER-positive samples after RU486 treatment. The decidua parietalis was the primary target site of RU486. The lack of RU486 effect on decidua capsularis implied that trophoblast invasion prevented against antiprogestin impact.

  12. TIMP1 overexpression mediates resistance of MCF-7 human breast cancer cells to fulvestrant and down-regulates progesterone receptor expression

    DEFF Research Database (Denmark)

    Bjerre, Christina; Vinther, Lena; Belling, Kirstine C.

    2013-01-01

    is associated with endocrine sensitivity. We established a panel of 11 MCF-7 subclones with a wide range of TIMP1 mRNA and protein expression levels. Cells with high expression of TIMP1 versus low TIMP1 displayed significantly reduced sensitivity to the antiestrogen fulvestrant (ICI 182,780, Faslodex®), while......, the effects of fulvestrant, 4-hydroxytamoxifen, or estrogen on estrogen receptor expression were not associated with TIMP1 levels. Gene expression analyses revealed associations between expression of TIMP1 and genes involved in metabolic pathways, epidermal growth factor receptor 1/cancer signaling pathways......, and cell cycle. Gene and protein expression analyses showed no general defects in estrogen receptor signaling except from lack of progesterone receptor expression and estrogen inducibility in clones with high TIMP1. The present study suggests a relation between high expression level of TIMP1 and loss...

  13. Enhancement of GABAergic transmission by zolpidem, an imidazopyridine with preferential affinity for type I benzodiazepine receptors.

    Science.gov (United States)

    Biggio, G; Concas, A; Corda, M G; Serra, M

    1989-02-28

    The effect of zolpidem, an imidazopyridine derivative with high affinity at the type I benzodiazepine recognition site, on the function of the GABAA/ionophore receptor complex was studied in vitro. Zolpidem, mimicking the action of diazepam, increased [3H]GABA binding, enhanced muscimol-stimulated 36Cl- uptake and reduced [35S]TBPS binding in rat cortical membrane preparations. Zolpidem was less effective than diazepam on the above parameters. Zolpidem induced a lower increase of [3H]GABA binding (23 vs. 35%) and muscimol-stimulated 36Cl- uptake (22 vs. 40%) and a smaller decrease of [35S]TBPS binding (47 vs. 77%) than diazepam. The finding that zolpidem enhanced the function of GABAergic synapses with an efficacy qualitatively and quantitatively different from that of diazepam suggests that this compound is a partial agonist at the benzodiazepine recognition site. Thus, our results are consistent with the view that the biochemical and pharmacological profile of a benzodiazepine recognition site ligand reflects its efficacy to enhance GABAergic transmission. Whether the preferential affinity of zolpidem at the type I site is involved in its atypical biochemical and pharmacological profile remains to be clarified.

  14. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

    Science.gov (United States)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. The ancestral retinoic acid receptor was a low-affinity sensor triggering neuronal differentiation

    KAUST Repository

    Handberg-Thorsager, Mette

    2018-02-22

    Retinoic acid (RA) is an important intercellular signaling molecule in vertebrate development, with a well-established role in the regulation of hox genes during hindbrain patterning and in neurogenesis. However, the evolutionary origin of the RA signaling pathway remains elusive. To elucidate the evolution of the RA signaling system, we characterized RA metabolism and signaling in the marine annelid Platynereis dumerilii, a powerful model for evolution, development, and neurobiology. Binding assays and crystal structure analyses show that the annelid retinoic acid receptor (RAR) binds RA and activates transcription just as vertebrate RARs, yet with a different ligand-binding pocket and lower binding affinity, suggesting a permissive rather than instructive role of RA signaling. RAR knockdown and RA treatment of swimming annelid larvae further reveal that the RA signal is locally received in the medial neuroectoderm, where it controls neurogenesis and axon outgrowth, whereas the spatial colinear hox gene expression in the neuroectoderm remains unaffected. These findings suggest that one early role of the new RAR in bilaterian evolution was to control the spatially restricted onset of motor and interneuron differentiation in the developing ventral nerve cord and to indicate that the regulation of hox-controlled anterior-posterior patterning arose only at the base of the chordates, concomitant with a high-affinity RAR needed for the interpretation of a complex RA gradient.

  16. High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

    International Nuclear Information System (INIS)

    Lach, E.; Trifilieff, A.; Landry, Y.; Gies, J.P.

    1991-01-01

    The binding of the radiolabeled bombesin analogue [ 125 I-Tyr 4 ]bombesin to guinea-pig lung membranes was investigated. Binding of [ 125 I-Tyr 4 ]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25C indicated the presence of a single class of non-interacting binding sites for bombesin (B max = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K D = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [ 125 I-Tyr 4 ]bombesin, neuromedin B and neuromedin C inhibited the binding of [ 125 I-Tyr 4 ]bombesin in an order of potencies as follows: [ 125 I-Tyr 4 ]bombesin > bombesin ≥ neuromedin C much-gt neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B

  17. The ancestral retinoic acid receptor was a low-affinity sensor triggering neuronal differentiation

    Science.gov (United States)

    Handberg-Thorsager, Mette; Gutierrez-Mazariegos, Juliana; Arold, Stefan T.; Kumar Nadendla, Eswar; Bertucci, Paola Y.; Germain, Pierre; Tomançak, Pavel; Pierzchalski, Keely; Jones, Jace W.; Albalat, Ricard; Kane, Maureen A.; Bourguet, William; Laudet, Vincent; Arendt, Detlev; Schubert, Michael

    2018-01-01

    Retinoic acid (RA) is an important intercellular signaling molecule in vertebrate development, with a well-established role in the regulation of hox genes during hindbrain patterning and in neurogenesis. However, the evolutionary origin of the RA signaling pathway remains elusive. To elucidate the evolution of the RA signaling system, we characterized RA metabolism and signaling in the marine annelid Platynereis dumerilii, a powerful model for evolution, development, and neurobiology. Binding assays and crystal structure analyses show that the annelid retinoic acid receptor (RAR) binds RA and activates transcription just as vertebrate RARs, yet with a different ligand-binding pocket and lower binding affinity, suggesting a permissive rather than instructive role of RA signaling. RAR knockdown and RA treatment of swimming annelid larvae further reveal that the RA signal is locally received in the medial neuroectoderm, where it controls neurogenesis and axon outgrowth, whereas the spatial colinear hox gene expression in the neuroectoderm remains unaffected. These findings suggest that one early role of the new RAR in bilaterian evolution was to control the spatially restricted onset of motor and interneuron differentiation in the developing ventral nerve cord and to indicate that the regulation of hox-controlled anterior-posterior patterning arose only at the base of the chordates, concomitant with a high-affinity RAR needed for the interpretation of a complex RA gradient. PMID:29492455

  18. Vasorelaxant potencies and receptor binding affinities of atrial natriuretic hormone (ANH) analogues

    International Nuclear Information System (INIS)

    Bush, E.N.; Green, E.M.; Artman, L.D.; Devine, E.M.; Sarin, V.; Rockway, T.W.; Connolly, P.J.; Kiso, Y.; Holleman, W.H.

    1986-01-01

    ANH (1-28) (α-rat ANP) produces hypotensive effects in vivo, presumably via interaction with specific receptors. Vasorelaxant potencies (pD 2 ) and intrinsic activities of ANH analogues were measured in histamine constricted rabbit aorta rings. Binding affinities (K/sub I/) of the compounds were studied in rabbit aorta renal cortex and adrenal, using the radio-ligand 125 I-Tyr 28 -ANH (1-28). Significant correlations (r 2 s in aorta, and the log D/sub I/s in each of the three tissues were observed for the following cyclic compounds, listed in order of potency: ANH (1-28) greater than or equal to ANH (6-28) greater than or equal to Met 12 -ANH (1-28) (α-human ANP) greater than or equal to cyclohexyl-Ala (Cha) 8 -ANH (5-28) > Lys 11 -ANH (5-28) = ANH (5-28) (atriopeptin III) = ANH (5-27) (atriopeptin II) = Cha 21 -ANH (5-28) greater than or equal to ANH (7-28) > Cha 15 -ANH (5-28) = Pro 10 -ANH (5-28) = ANH (5-25) (atriopeptin I) = Asn 13 -ANH (5-28) = Tyr 9 -ANH (5-28) > des-Gly 9 -ANH (5-28) > ANH (7-23) = Pro 10 -ANH (7-23) greater than or equal to (D)Ala 9 -ANH (7-23) > Pro 9 -ANH (7-13). In summary, the affinities of several ANH analogues for both vascular and nonvascular receptors agree with their vasorelaxant potencies

  19. Progesterone Test

    Science.gov (United States)

    ... replacement therapy, or help diagnose the cause of abnormal uterine bleeding When To Get Tested? At specific times during ... receiving progesterone replacement therapy; when a woman has abnormal uterine bleeding Sample Required? A blood sample drawn from a ...

  20. Estradiol upregulates progesterone receptor and orphanin FQ colocalization in arcuate nucleus neurons and opioid receptor-like receptor-1 expression in proopiomelanocortin neurons that project to the medial preoptic nucleus in the female rat

    Science.gov (United States)

    Sanathara, Nayna M.; Moreas, Justine; Mahavongtrakul, Matthew; Sinchak, Kevin

    2014-01-01

    Background Ovarian steroids regulate sexual receptivity in the female rat by acting on neurons that converge on proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARH) that project to the medial preoptic nucleus (MPN). Estradiol rapidly activates these neurons to release β-endorphin that activates MPN μ-opioid receptors (MOP) to inhibit lordosis. Lordosis is facilitated by the subsequent action of progesterone that deactivates the estradiol-induced MPN MOP activation. Orphanin FQ (OFQ/N; aka nociceptin) infusions into the ARH, like progesterone, deactivate MPN MOP and facilitate lordosis in estradiol-primed rats. OFQ/N reduces the activity of ARH β-endorphin neurons through post- and presynaptic mechanisms via its cognate receptor, ORL-1. Methods We tested the hypotheses that progesterone receptors (PR) are expressed in ARH OFQ/N neurons by immunohistochemistry and ORL-1 is expressed in POMC neurons that project to the MPN by combining Fluoro-Gold injection into the MPN and double-label fluorescent in situ hybridization (FISH). We also hypothesized that estradiol increases coexpression of PR-OFQ/N and ORL-1-POMC in ARH neurons of ovariectomized rats. Results The number of PR and OFQ/N immunopositive ARH neurons was increased as was their colocalization by estradiol treatment. FISH for ORL-1 and POMC mRNA revealed a subpopulation of ARH neurons that was triple-labeled indicating these neurons project to the MPN and coexpress ORL-1 and POMC mRNA. Estradiol was shown to upregulate ORL-1 and POMC expression in MPN-projecting ARH neurons. Conclusion Estradiol upregulates the ARH OFQ/N-ORL-1 system projecting to the MPN that regulates lordosis. PMID:24821192

  1. Development of 99mTc labelled somatostatin analogues with high affinity for somatostatin receptors

    International Nuclear Information System (INIS)

    Maina, T.; Nock, B.; Nicolopoulou, A.; Tsipra, C.; Poppe, M.; Chiotellis, E.

    2001-01-01

    A recent development in oncology involves the use of metabolically stabilized peptide hormone analogues labelled with metallic radionuclides for the diagnosis or therapy of malignant disease. This approach was successfully applied for the first time in the visualization of somatostatin positive tumours and their metastases with 111 In DTPA-octreotide. In an effort to obtain a 99m Tc somatostatin receptor affine radioligand we describe herein the synthesis, radiochemistry and preliminary biological evaluation of two novel 99m Tc labelled somatostatin analogues, N 4 -TOC and N 4 -RC-160. In these compounds a tetraamine bifunctional unit was covalently attached to the N-terminal (D)Phe 1 of the peptide chain using Boc-protection strategies. The peptide conjugates were purified by high performance liquid chromatography (HPLC) and characterized by UV/Vis and ES-MS spectroscopies. As revealed by HPLC, 99m Tc labelling was quantitative under mild conditions, leading to a single 99m Tc species in high specific activities. Affinity of 99m Tc N 4 -TOC for the somatostatin receptor, as determined by in vitro binding assays in rat brain cortex membranes, was found unaffected by the presence of the bulky metal chelate. The binding properties of 99m Tc N 4 -RC-160 could not be determined by this assay due to an extremely high non-specific binding of this radioligand, and will be shortly investigated by other methods. Tissue distribution in healthy mice revealed that 99m Tc N 4 -TOC is clearing mainly through the kidneys and the urinary tract whereas 99m Tc N 4 -RC-160 shows a high accumulation in the liver as a result of its lipophilicity. Analysis of urine samples by HPLC showed that 99m Tc N 4 -TOC is excreted integer from the body of mice, while 99m Tc N 4 -RC-160 is totally transformed to an unidentified hydrophilic metabolite in vivo. The location of this metabolism is currently investigated. In vivo blocking experiments using animals pre-treated with 50 μg octreotide

  2. Contributions of pocket depth and electrostatic interactions to affinity and selectivity of receptors for methylated lysine in water.

    Science.gov (United States)

    Beaver, Joshua E; Peacor, Brendan C; Bain, Julianne V; James, Lindsey I; Waters, Marcey L

    2015-03-21

    Dynamic combinatorial chemistry was used to generate a set of receptors for peptides containing methylated lysine (KMen, n = 0-3) and study the contribution of electrostatic effects and pocket depth to binding affinity and selectivity. We found that changing the location of a carboxylate resulted in an increase in preference for KMe2, presumably based on ability to form a salt bridge with KMe2. The number of charged groups on either the receptor or peptide guest systematically varied the binding affinities to all guests by approximately 1-1.5 kcal mol(-1), with little influence on selectivity. Lastly, formation of a deeper pocket led to both increased affinity and selectivity for KMe3 over the lower methylation states. From these studies, we identified that the tightest binder was a receptor with greater net charge, with a Kd of 0.2 μM, and the receptor with the highest selectivity was the one with the deepest pocket, providing 14-fold selectivity between KMe3 and KMe2 and a Kd for KMe3 of 0.3 μM. This work provides key insights into approaches to improve binding affinity and selectivity in water, while also demonstrating the versatility of dynamic combinatorial chemistry for rapidly exploring the impact of subtle changes in receptor functionality on molecular recognition in water.

  3. Progesterone Receptor Membrane Component 1 (PGRMC1 in cell division: its role in bovine granulosa cells mitosis

    Directory of Open Access Journals (Sweden)

    Laura Terzaghi

    2015-07-01

    Full Text Available The present studies were aimed to assess Progesterone Receptor Membrane Component-1 (PGRMC1 role in regulating bovine granulosa cells (bGC mitosis. First, we performed immunofluorescence studies on in vitro cultured bGC collected from antral follicles, which showed that PGRMC1 localizes to the spindle apparatus in mitotic cells. Then, to evaluate PGRMC1 effect on cell proliferation we silenced its expression with RNA interference technique (RNAi. Quantitative RT-PCR and immunoblotting confirmed down-regulation of PGRMC1 expression, when compared to CTRL-RNAi treated bGC (p<0.05. After 72h of culture, PGRMC1 silencing determined a lower growth rate (p<0.05 and a higher percentage of cells arrested at G2/M phase as assessed by flowcytometry (p<0.05. Accordingly, live imaging studies revealed more aberrant mitosis and a delayed M-phase in PGRMC1-RNAi treated cells compared to CTRL-RNAi group (p<0.05. These data confirmed that PGRMC1 is directly involved in bGC mitosis and ongoing preliminary studies are aimed to elucidate its putative mechanisms of action. Since PGRMC1 is a membrane protein, we hypothesize its possible involvement in vesicular trafficking and endocytosis, which is in turn an important process to assure proper cell division. To assess this hypothesis, we have preliminarily conducted immunofluorescence and in situ proximity ligation assay experiments that showed PGRMC1 co-localization and direct interaction with clathrin. This is important since clathrin is an essential protein for both endosomes formation, and cell division acting directly on the spindle apparatus. Thus our studies set the stage for analysis aimed to further characterize PGRMC1’s mechanism of action in mitotic cell.

  4. Antiprogestin mifepristone inhibits the growth of cancer cells of reproductive and non-reproductive origin regardless of progesterone receptor expression

    International Nuclear Information System (INIS)

    Tieszen, Chelsea R; Goyeneche, Alicia A; Brandhagen, BreeAnn N; Ortbahn, Casey T; Telleria, Carlos M

    2011-01-01

    Mifepristone (MF) has been largely used in reproductive medicine due to its capacity to modulate the progesterone receptor (PR). The study of MF has been expanded to the field of oncology; yet it remains unclear whether the expression of PR is required for MF to act as an anti-cancer agent. Our laboratory has shown that MF is a potent inhibitor of ovarian cancer cell growth. In this study we questioned whether the growth inhibitory properties of MF observed in ovarian cancer cells would translate to other cancers of reproductive and non-reproductive origin and, importantly, whether its efficacy is related to the expression of cognate PR. Dose-response experiments were conducted with cancer cell lines of the nervous system, breast, prostate, ovary, and bone. Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and analysed for cell number and cell cycle traverse, and hypodiploid DNA content characteristic of apoptotic cell death. For all cell lines, expression of steroid hormone receptors upon treatment with vehicle or cytostatic doses of MF for 24 h was studied by Western blot, whereas the activity of the G1/S regulatory protein Cdk2 in both treatment groups was monitored in vitro by the capacity of Cdk2 to phosphorylate histone H1. MF growth inhibited all cancer cell lines regardless of tissue of origin and hormone responsiveness, and reduced the activity of Cdk2. Cancer cells in which MF induced G1 growth arrest were less susceptible to lethality in the presence of high concentrations of MF, when compared to cancer cells that did not accumulate in G1. While all cancer cell lines were growth inhibited by MF, only the breast cancer MCF-7 cells expressed cognate PR. Antiprogestin MF inhibits the growth of different cancer cell lines with a cytostatic effect at lower concentrations in association with a decline in the activity of the cell cycle regulatory protein Cdk2, and apoptotic lethality at higher doses in association with increased

  5. Antiprogestin mifepristone inhibits the growth of cancer cells of reproductive and non-reproductive origin regardless of progesterone receptor expression

    Directory of Open Access Journals (Sweden)

    Ortbahn Casey T

    2011-05-01

    Full Text Available Abstract Background Mifepristone (MF has been largely used in reproductive medicine due to its capacity to modulate the progesterone receptor (PR. The study of MF has been expanded to the field of oncology; yet it remains unclear whether the expression of PR is required for MF to act as an anti-cancer agent. Our laboratory has shown that MF is a potent inhibitor of ovarian cancer cell growth. In this study we questioned whether the growth inhibitory properties of MF observed in ovarian cancer cells would translate to other cancers of reproductive and non-reproductive origin and, importantly, whether its efficacy is related to the expression of cognate PR. Methods Dose-response experiments were conducted with cancer cell lines of the nervous system, breast, prostate, ovary, and bone. Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and analysed for cell number and cell cycle traverse, and hypodiploid DNA content characteristic of apoptotic cell death. For all cell lines, expression of steroid hormone receptors upon treatment with vehicle or cytostatic doses of MF for 24 h was studied by Western blot, whereas the activity of the G1/S regulatory protein Cdk2 in both treatment groups was monitored in vitro by the capacity of Cdk2 to phosphorylate histone H1. Results MF growth inhibited all cancer cell lines regardless of tissue of origin and hormone responsiveness, and reduced the activity of Cdk2. Cancer cells in which MF induced G1 growth arrest were less susceptible to lethality in the presence of high concentrations of MF, when compared to cancer cells that did not accumulate in G1. While all cancer cell lines were growth inhibited by MF, only the breast cancer MCF-7 cells expressed cognate PR. Conclusions Antiprogestin MF inhibits the growth of different cancer cell lines with a cytostatic effect at lower concentrations in association with a decline in the activity of the cell cycle regulatory protein Cdk2, and

  6. Affinity selection-mass spectrometry and its emerging application to the high throughput screening of G protein-coupled receptors.

    Science.gov (United States)

    Whitehurst, Charles E; Annis, D Allen

    2008-07-01

    Advances in combinatorial chemistry and genomics have inspired the development of novel affinity selection-based screening techniques that rely on mass spectrometry to identify compounds that preferentially bind to a protein target. Of the many affinity selection-mass spectrometry techniques so far documented, only a few solution-based implementations that separate target-ligand complexes away from unbound ligands persist today as routine high throughput screening platforms. Because affinity selection-mass spectrometry techniques do not rely on radioactive or fluorescent reporters or enzyme activities, they can complement traditional biochemical and cell-based screening assays and enable scientists to screen targets that may not be easily amenable to other methods. In addition, by employing mass spectrometry for ligand detection, these techniques enable high throughput screening of massive library collections of pooled compound mixtures, vastly increasing the chemical space that a target can encounter during screening. Of all drug targets, G protein coupled receptors yield the highest percentage of therapeutically effective drugs. In this manuscript, we present the emerging application of affinity selection-mass spectrometry to the high throughput screening of G protein coupled receptors. We also review how affinity selection-mass spectrometry can be used as an analytical tool to guide receptor purification, and further used after screening to characterize target-ligand binding interactions, enabling the classification of orthosteric and allosteric binders.

  7. Functional characterization of the high affinity IgG Receptor : making heads and tails of FcγRI

    NARCIS (Netherlands)

    van der Poel, C.E.

    2011-01-01

    This thesis focuses on human FcγRI, a high affinity receptor for antibodies of the IgG isotype. IgG is the most abundant antibody type in blood and all currently FDA approved therapeutic antibodies are of the IgG isotype. FcγRI, a member of the activating Fcγ receptors, exists as a complex of a

  8. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Directory of Open Access Journals (Sweden)

    Béatrice Marquèze-Pouey

    Full Text Available Signaling mediated by the epidermal growth factor (EGF is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer. In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  9. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Science.gov (United States)

    Marquèze-Pouey, Béatrice; Mailfert, Sébastien; Rouger, Vincent; Goaillard, Jean-Marc; Marguet, Didier

    2014-01-01

    Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  10. Progesterone receptor (PR) polyproline domain (PPD) mediates inhibition of epidermal growth factor receptor (EGFR) signaling in non-small cell lung cancer cells.

    Science.gov (United States)

    Kawprasertsri, Sornsawan; Pietras, Richard J; Marquez-Garban, Diana C; Boonyaratanakornkit, Viroj

    2016-05-01

    Recent evidence has suggested a possible role for progesterone receptor (PR) in the progression of non-small cell lung cancer (NSCLC). However, little is known concerning roles of PR in NSCLC. PR contains a polyproline domain (PPD), which directly binds to the SH3 domain of signaling molecules. Because PPD-SH3 interactions are essential for EGFR signaling, we hypothesized that the presence of PR-PPD interfered with EGFR-mediated signaling and cell proliferation. We examined the role of PR-PPD in cell proliferation and signaling by stably expressing PR-B, or PR-B with disrupting mutations in the PPD (PR-BΔSH3), from a tetracycline-regulated promoter in A549 NSCLC cells. PR-B dose-dependently inhibited cell growth in the absence of ligand, and progestin (R5020) treatment further suppressed the growth. Treatment with RU486 abolished PR-B- and R5020-mediated inhibition of cell proliferation. Expression of PR-BΔSH3 and treatment with R5020 or RU486 had no effect on cell proliferation. Furthermore, PR-B expression but not PR-BΔSH3 expression reduced EGF-induced A549 proliferation and activation of ERK1/2, in the absence of ligand. Taken together, our data demonstrated the significance of PR extranuclear signaling through PPD interactions in EGFR-mediated proliferation and signaling in NSCLC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Are basophil histamine release and high affinity IgE receptor expression involved in asymptomatic skin sensitization?

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Assing, K; Jensen, Lone Hummelshøj

    2006-01-01

    Immunoglobulin (Ig)E-sensitized persons with positive skin prick test, but no allergy symptoms, are classified as being asymptomatic skin sensitized (AS). The allergic type 1 disease is dependant on IgE binding to the high affinity IgE-receptor (FcepsilonRI) expressed on basophils and mast cells....

  12. α-Estrogen and Progesterone Receptors Modulate Kisspeptin Effects on Prolactin: Role in Estradiol-Induced Prolactin Surge in Female Rats.

    Science.gov (United States)

    Aquino, Nayara S S; Araujo-Lopes, Roberta; Henriques, Patricia C; Lopes, Felipe E F; Gusmao, Daniela O; Coimbra, Candido C; Franci, Celso R; Reis, Adelina M; Szawka, Raphael E

    2017-06-01

    Kisspeptin (Kp) regulates prolactin (PRL) in an estradiol-dependent manner. We investigated the interaction between ovarian steroid receptors and Kp in the control of PRL secretion. Intracerebroventricular injections of Kp-10 or Kp-234 were performed in ovariectomized (OVX) rats under different hormonal treatments. Kp-10 increased PRL release and decreased 3,4-dihydroxyphenylacetic acid levels in the median eminence (ME) of OVX rats treated with estradiol (OVX+E), which was prevented by tamoxifen. Whereas these effects of Kp-10 were absent in OVX rats, they were replicated in OVX rats treated with selective agonist of estrogen receptor (ER)α, propylpyrazole triol, but not of ERβ, diarylpropionitrile. Furthermore, the Kp-10-induced increase in PRL was two times higher in OVX+E rats also treated with progesterone (OVX+EP), which was associated with a reduced expression of both tyrosine hydroxylase (TH) and Ser40-phosphorylated TH in the ME. Kp-10 also reduced dopamine levels in the ME of OVX+EP rats, an effect blocked by the progesterone receptor (PR) antagonist RU486. We also determined the effect of Kp antagonism with Kp-234 on the estradiol-induced surges of PRL and luteinizing hormone (LH), using tail-tip blood sampling combined with ultrasensitive enzyme-linked immunosorbent assay. Kp-234 impaired the early phase of the PRL surge and prevented the LH surge in OVX+E rats. Thus, we provide evidence that Kp stimulation of PRL release requires ERα and is potentiated by progesterone via PR activation. Moreover, alongside its essential role in the LH surge, Kp seems to play a role in the peak phase of the estradiol-induced PRL surge. Copyright © 2017 Endocrine Society.

  13. 99mTc(CO)3-DTMA bombesin conjugates having high affinity for the GRP receptor

    International Nuclear Information System (INIS)

    Lane, Stephanie R.; Veerendra, Bhadrasetty; Rold, Tammy L.; Sieckman, Gary L.; Hoffman, Timothy J.; Jurisson, Silvia S.; Smith, Charles J.

    2008-01-01

    Introduction: Targeted diagnosis of specific human cancer types continues to be of significant interest in nuclear medicine. 99m Tc is ideally suited as a diagnostic radiometal for in vivo tumor targeting due to its ideal physical characteristics and diverse labeling chemistries in numerous oxidation states. Methods: In this study, we report a synthetic approach toward design of a new tridentate amine ligand for the organometallic aqua-ion [ 99m Tc(H 2 O) 3 (CO) 3 ] + . The new chelating ligand framework, 2-(N,N'-Bis(tert-butoxycarbonyl)diethylenetriamine) acetic acid (DTMA), was synthesized from a diethylenetriamine precursor and fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy ( 1 H and 13 C). DTMA was conjugated to H 2 N-(X)-BBN(7-14)NH 2 , where X=an amino acid or aliphatic pharmacokinetic modifier and BBN=bombesin peptide, by means of solid phase peptide synthesis. DTMA-(X)-BBN(7-14)NH 2 conjugates were purified by reversed-phase high-performance chromatography and characterized by electrospray-ionization mass spectrometry. Results: The new conjugates were radiolabeled with [ 99m Tc(H 2 O) 3 (CO) 3 ] + produced via Isolink radiolabeling kits to produce [ 99m Tc(CO) 3 -DTMA-(X)-BBN(7-14)NH 2 ]. Radiolabeled conjugates were purified by reversed-phase high-performance chromatography. Effective receptor binding behavior was evaluated in vitro and in vivo. Conclusions: [ 99m Tc(CO) 3 -DTMA-(X)-BBN(7-14)NH 2 ] conjugates displayed very high affinity for the gastrin releasing peptide receptor in vitro and in vivo. Therefore, these conjugates hold some propensity to be investigated as molecular imaging agents that specifically target human cancers uniquely expressing the gastrin releasing peptide receptor subtypes

  14. Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection

    DEFF Research Database (Denmark)

    Tramm, Trine; Hennig, Guido; Kyndi, Marianne

    2013-01-01

    Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal...... tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α (ESR1), progesterone receptor (PGR) and human epidermal growth factor...... receptor 2 (ERBB2), by comparing gene expression from whole slide and tumor-enriched sections, and correlating gene expression from whole slide sections with corresponding immunohistochemistry. Gene expression, based on mRNA extracted from a training set (36 paraffin blocks) and two validation sets (133...

  15. Biphasic regulation of development of the high-affinity saxitoxin receptor by innervation in rat skeletal muscle

    International Nuclear Information System (INIS)

    Sherman, S.J.; Catterall, W.A.

    1982-01-01

    Specific binding of 3 H-saxitoxin (STX) was used to quantitate the density of voltage-sensitive sodium channels in developing rat skeletal muscle. In adult triceps surae, a single class of sites with a KD . 2.9 nM and a density of 21 fmol/mg wet wt was detected. The density of these high-affinity sites increased from 2.0 fmol/mg wet wt to the adult value in linear fashion during days 2-25 after birth. Denervation of the triceps surae at day 11 or 17 reduced final saxitoxin receptor site density to 10.4 or 9.2 fmol/mg wet wt, respectively, without changing KD. Denervation of the triceps surae at day 5 did not alter the subsequent development of saxitoxin receptor sites during days 5-9 and accelerated the increase of saxitoxin receptor sites during days 9-13. After day 13, saxitoxin receptor development abruptly ceased and the density of saxitoxin receptor sites declined to 11 fmol/wg wet wt. These results show that the regulation of high-affinity saxitoxin receptor site density by innervation is biphasic. During the first phase, which is independent of continuing innervation, the saxitoxin receptor density increases to 47-57% of the adult level. After day 11, the second phase of development, which is dependent on continuing innervation, gives rise to the adult density of saxitoxin receptors

  16. The presence of a membrane-bound progesterone receptor sensitizes the estradiol-induced effect on the proliferation of human breast cancer cells.

    Science.gov (United States)

    Neubauer, Hans; Yang, Yang; Seeger, Harald; Fehm, Tanja; Cahill, Michael A; Tong, Xiaowen; Ruan, Xiangyan; Mueck, Alfred O

    2011-08-01

    Breast cancer risk is still an important topic regarding hormone therapy as well as oral contraception. Evidence that progestogens may play a crucial role is accumulating. Progesterone receptor membrane component 1 (PGRMC1) expressed in breast cancer may be important in tumorigenesis and thus may increase breast cancer risk. The aim of this project was to investigate the influence of different estradiol (E2) concentrations and the addition of two progestogens on MCF-7 breast cancer cells overexpressing PGRMC1. MCF-7 cells were stably transfected with PGRMC1 expression plasmid (MCF-7/PGRMC1-3HA [WT-12]). To test the effects of E2 and progestogens on cell proliferation, MCF-7 and WT-12 cells were stimulated with different concentrations of E2 (10 and 10 M) alone and in combination with progesterone and medroxyprogesterone acetate (each 10 M). E2 elicited a concentration-dependent proliferative effect on both cell lines, which was much more pronounced in WT-12 cells (50% vs 200%). This effect could be completely abrogated by the addition of the E2 antagonist fulvestrant. Addition of progesterone had no influence on the E2-induced effect, whereas medroxy-progesterone acetate enhanced the E2-induced effect at a low E2 concentration, which was, again, more pronounced in the WT-12 cells. The figures were between 20% and 40% in MCF-7 and between 60% and 250% in WT-12 cells. Overexpression of PGRMC1 sensitizes the proliferative response of the MCF-7 breast cancer cell line to estradiol. The effect of progestogens on breast cancer tumorigenesis may depend on the specific progestogen used for hormone therapy or oral contraception.

  17. Expression of progesterone receptor membrane component-2 within the immature rat ovary and its role in regulating mitosis and apoptosis of spontaneously immortalized granulosa cells.

    Science.gov (United States)

    Griffin, Daniel; Liu, Xiufang; Pru, Cindy; Pru, James K; Peluso, John J

    2014-08-01

    Progesterone receptor membrane component 2 (Pgrmc2) mRNA was detected in the immature rat ovary. By 48 h after eCG, Pgrmc2 mRNA levels decreased by 40% and were maintained at 48 h post-hCG. Immunohistochemical studies detected PGRMC2 in oocytes and ovarian surface epithelial, interstitial, thecal, granulosa, and luteal cells. PGRMC2 was also present in spontaneously immortalized granulosa cells, localizing to the cytoplasm of interphase cells and apparently to the mitotic spindle of cells in metaphase. Interestingly, PGRMC2 levels appeared to decrease during the G1 stage of the cell cycle. Moreover, overexpression of PGRMC2 suppressed entry into the cell cycle, possibly by binding the p58 form of cyclin dependent kinase 11b. Conversely, Pgrmc2 small interfering RNA (siRNA) treatment increased the percentage of cells in G1 and M stage but did not increase the number of cells, which was likely due to an increase in apoptosis. Depleting PGRMC2 did not inhibit cellular (3)H-progesterone binding, but attenuated the ability of progesterone to suppress mitosis and apoptosis. Taken together these studies suggest that PGRMC2 affects granulosa cell mitosis by acting at two specific stages of the cell cycle. First, PGRMC2 regulates the progression from the G0 into the G1 stage of the cell cycle. Second, PGRMC2 appears to localize to the mitotic spindle, where it likely promotes the final stages of mitosis. Finally, siRNA knockdown studies indicate that PGRMC2 is required for progesterone to slow the rate of granulosa cell mitosis and apoptosis. These findings support a role for PGRMC2 in ovarian follicle development. © 2014 by the Society for the Study of Reproduction, Inc.

  18. Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

    Science.gov (United States)

    Nakanishi, Takeshi; Maru, Takamitsu; Tahara, Kazuhiro; Sanada, Hideaki; Umetsu, Mitsuo; Asano, Ryutaro; Kumagai, Izumi

    2013-02-01

    We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

  19. Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

    International Nuclear Information System (INIS)

    Chiba, S.; Shibuya, K.; Miyazono, K.; Tojo, A.; Oka, Y.; Miyagawa, K.; Takaku, F.

    1990-01-01

    The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of 125 I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB- 125 I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein

  20. Differences in receptor binding affinity of several phytocannabinoids do not explain their effects on neural cell cultures.

    Science.gov (United States)

    Rosenthaler, Sarah; Pöhn, Birgit; Kolmanz, Caroline; Huu, Chi Nguyen; Krewenka, Christopher; Huber, Alexandra; Kranner, Barbara; Rausch, Wolf-Dieter; Moldzio, Rudolf

    2014-01-01

    Phytocannabinoids are potential candidates for neurodegenerative disease treatment. Nonetheless, the exact mode of action of major phytocannabinoids has to be elucidated, but both, receptor and non-receptor mediated effects are discussed. Focusing on the often presumed structure-affinity-relationship, Ki values of phytocannabinoids cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), THC acid (THCA) and THC to human CB1 and CB2 receptors were detected by using competitive inhibition between radioligand [(3)H]CP-55,940 and the phytocannabinoids. The resulting Ki values to CB1 range from 23.5 nM (THCA) to 14711 nM (CBDV), whereas Ki values to CB2 range from 8.5 nM (THC) to 574.2 nM (CBDV). To study the relationship between binding affinity and effects on neurons, we investigated possible CB1 related cytotoxic properties in murine mesencephalic primary cell cultures and N18TG2 neuroblastoma cell line. Most of the phytocannabinoids did not affect the number of dopaminergic neurons in primary cultures, whereas propidium iodide and resazurin formation assays revealed cytotoxic properties of CBN, CBDV and CBG. However, THC showed positive effects on N18TG2 cell viability at a concentration of 10 μM, whereas CBC and THCA also displayed slightly positive activities. These findings are not linked to the receptor binding affinity therewith pointing to another mechanism than a receptor mediated one. [Corrected] Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D2 receptor

    International Nuclear Information System (INIS)

    Borgundvaag, B.; George, S.R.

    1985-01-01

    The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of [ 3 H]-ATP to [ 3 H]-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC 50 values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC 50 values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D 2 dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity. 12 references, 4 figures, 1 table

  2. DOTA-derivatives of octreotide dicarba-analogues with high affinity for somatostatin sst2,5 receptors

    Science.gov (United States)

    Pratesi, Alessandro; Ginanneschi, Mauro; Lumini, Marco; Papini, Anna M.; Novellino, Ettore; Brancaccio, Diego; Carotenuto, Alfonso

    2017-02-01

    In vivo somatostatin receptor scintigraphy is a valuable method for the visualization of human endocrine tumours and their metastases. In fact, peptide ligands of somatostatin receptors (sst’s) conjugated with chelating agents are in clinical use. We have recently developed octreotide dicarba-analogues, which show interesting binding profiles at sst’s. In this context, it was mandatory to explore the possibility that our analogues could maintain their activity also upon conjugation with DOTA. In this paper, we report and discuss the synthesis, binding affinity and conformational preferences of three DOTA-conjugated dicarba-analogues of octreotide. Interestingly, two conjugated analogues exhibited nanomolar affinities on sst2 and sst5 somatostatin receptor subtypes.

  3. Expression of progesterone receptor protein in the ovine uterus during the estrous cycle: Effects of nutrition, arginine and FSH.

    Science.gov (United States)

    Grazul-Bilska, Anna T; Thammasiri, Jiratti; Kraisoon, Aree; Reyaz, Arshi; Bass, Casie S; Kaminski, Samantha L; Navanukraw, Chainarong; Redmer, Dale A

    2018-03-01

    To evaluate expression of progesterone receptor (PGR) AB in follicle stimulating hormone (FSH)-treated or non-treated sheep administered with arginine (Arg) or saline (Sal) fed a control (C), excess (O) or restricted (U) diet, uterine tissues were collected at the early, mid and/or late luteal phases. In exp. 1, ewes from each diet were randomly assigned to one of two treatments, Arg or Sal administration three times daily from day 0 of the first estrous cycle until uterine tissue collection. In exp. 2, ewes were injected twice daily with FSH on days 13-15 of the first estrous cycle. Uterine tissues were immunostained to detect PGR followed by image analysis. PGR were detected in luminal epithelium (LE), endometrial glands (EG), endometrial stroma (ES), myometrium (Myo), and endometrial and myometrial blood vessels. The percentage of PR-positive cells and/or intensity of staining were affected by phase of the estrous cycle, plane of nutrition, and/or FSH but not by Arg. In exp. 1, percentage of PGR-positive cells in LE and EG but not in ES and Myo was greater at the early and mid than late luteal phase, was not affected by plane of nutrition, and was similar in LE and EG. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in LE, EG and Myo, and was the greatest in LE, less in EG, and least in ES and Myo. In exp. 2, percentage of PGR-positive cells in LE, EG, ES and Myo was affected by phase of the estrous cycle, but not by plane of nutrition; was greater at the early than mid luteal phase; and was greatest in LE and EG, less in luminal (superficial) ES and Myo and least in deep ES. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in all compartments but ES, and was the greatest in LE and luminal EG, less in deep EG, and least in ES and Myo. Comparison of data for FSH (superovulated) and Sal-treated (non-superovulated) ewes demonstrated that FSH affected PR expression in all evaluated

  4. Depletion of calcium stores contributes to progesterone-induced attenuation of calcium signaling of G protein-coupled receptors

    Czech Academy of Sciences Publication Activity Database

    Gehrig-Burger, K.; Slaninová, Jiřina; Gimpl, G.

    2010-01-01

    Roč. 67, č. 16 (2010), s. 2815-2824 ISSN 1420-682X Institutional research plan: CEZ:AV0Z40550506 Keywords : non-genomic effect * oxytocin * bradykinin * progesterone Subject RIV: CC - Organic Chemistry Impact factor: 7.047, year: 2010

  5. Early signs of pathological cognitive aging in mice lacking high-affinity nicotinic receptors.

    Directory of Open Access Journals (Sweden)

    Eleni eKonsolaki

    2016-04-01

    Full Text Available In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. Α deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-, which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioural signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviours, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm and extend the hypothesis that β2-/- animals exhibit age-related cognitive impairments, manifested in both spatial learning and recognition memory tasks. In addition, we reveal deficits in spontaneous behaviour and habituation processes earlier in life. To our knowledge, this is the first study to perform an extensive behavioural examination of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioural changes to global dementia due to the combined effect of the neuropathology and aging.

  6. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    International Nuclear Information System (INIS)

    Nye, J.S.

    1988-01-01

    The mechanism by which delta 9 tetrahydrocannabinol (delta 9 THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5'-Trimethylammonium-delta 8 THC (TMA) is a positively charged analog of delta- 8 THC modified on the 5' carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of [ 3 H]-5'-trimethylammonium-delta- 8 THC ([ 3 H]TMA) to rat neuronal membranes. [ 3 H]TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of [ 3 H]TMA binding activity of approximately 60,000 daltons apparent molecular weight

  7. Sex-specific effects of daily gavage with a mixed progesterone and glucocorticoid receptor antagonist on hypoxic ventilatory response in newborn rats.

    Science.gov (United States)

    Fournier, Stéphanie; Doan, Van Diep; Joseph, Vincent

    2012-01-01

    We tested the hypothesis that daily gavage with mifepristone, a mixed progesterone/glucocorticoid receptor antagonist would alter hypoxic ventilatory response (HVR) in newborn male and female rats. Rats were treated with mifepristone (40µg/g/day), or vehicle between postnatal days 3-12, and used at 10-12 days of age to record baseline ventilatory and metabolic values using whole body plethysmography. HVR was tested by exposing the animals to 14% and 12% O(2) for 20 minutes each. HVR was enhanced by mifepristone treatment, mainly due to an effect on tidal volume that remained higher in mifepristone treated rats during both levels of hypoxic exposure. This effect was sex-specific being apparent only in male rats. In Vehicle treated rats, HVR was higher in females than in males, which was also due to a higher tidal volume in hypoxia (at 14 and 12% O(2)). We conclude that the activity of the progesterone and/or glucocorticoid receptors modulates respiratory control in rat pups, and that these effects are different in males and females.

  8. Activation of adenosine low-affinity A3 receptors inhibits the enteric short interplexus neural circuit triggered by histamine.

    Science.gov (United States)

    Bozarov, Andrey; Wang, Yu-Zhong; Yu, Jun Ge; Wunderlich, Jacqueline; Hassanain, Hamdy H; Alhaj, Mazin; Cooke, Helen J; Grants, Iveta; Ren, Tianhua; Christofi, Fievos L

    2009-12-01

    We tested the novel hypothesis that endogenous adenosine (eADO) activates low-affinity A3 receptors in a model of neurogenic diarrhea in the guinea pig colon. Dimaprit activation of H2 receptors was used to trigger a cyclic coordinated response of contraction and Cl(-) secretion. Contraction-relaxation was monitored by sonomicrometry (via intracrystal distance) simultaneously with short-circuit current (I(sc), Cl(-) secretion). The short interplexus reflex coordinated response was attenuated or abolished by antagonists at H2 (cimetidine), 5-hydroxytryptamine 4 receptor (RS39604), neurokinin-1 receptor (GR82334), or nicotinic (mecamylamine) receptors. The A1 agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) abolished coordinated responses, and A1 antagonists could restore normal responses. A1-selective antagonists alone [8-cyclopentyltheophylline (CPT), 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX), or 8-cyclopentyl-N(3)-[3-(4-(fluorosulfonyl)benzoyloxy)propyl]-xanthine (FSCPX)] caused a concentration-dependent augmentation of crypt cell secretion or contraction and acted at nanomolar concentrations. The A3 agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) abolished coordinated responses and the A3 antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191) could restore and further augment responses. The IB-MECA effect was resistant to knockdown of adenosine A1 receptor with the irreversible antagonist FSCPX; the IC(50) for IB-MECA was 0.8 microM. MRS1191 alone could augment or unmask coordinated responses to dimaprit, and IB-MECA suppressed them. MRS1191 augmented distension-evoked reflex I(sc) responses. Adenosine deaminase mimicked actions of adenosine receptor antagonists. A3 receptor immunoreactivity was differentially expressed in enteric neurons of different parts of colon. After tetrodotoxin, IB-MECA caused circular muscle relaxation. The data support the novel concept that

  9. Progesterone receptor membrane component-1 (PGRMC1) is the mediator of progesterone's antiapoptotic action in spontaneously immortalized granulosa cells as revealed by PGRMC1 small interfering ribonucleic acid treatment and functional analysis of PGRMC1 mutations.

    Science.gov (United States)

    Peluso, John J; Romak, Jonathan; Liu, Xiufang

    2008-02-01

    Progesterone (P4) receptor membrane component-1 (PGRMC1) and its binding partner, plasminogen activator inhibitor 1 RNA binding protein (PAIRBP1) are thought to form a complex that functions as membrane receptor for P4. The present investigations confirm PGRMC1's role in this membrane receptor complex by demonstrating that depleting PGMRC1 with PGRMC1 small interfering RNA results in a 60% decline in [(3)H]P4 binding and the loss of P4's antiapoptotic action. Studies conducted on partially purified GFP-PGRMC1 fusion protein indicate that [(3)H]P4 specifically binds to PGRMC1 at a single site with an apparent K(d) of about 35 nm. In addition, experiments using various deletion mutations reveal that the entire PGRMC1 molecule is required for maximal [(3)H]P4 binding and P4 responsiveness. Analysis of the binding data also suggests that the P4 binding site is within a segment of PGRMC1 that is composed of the transmembrane domain and the initial segment of the C terminus. Interestingly, PAIRBP1 appears to bind to the C terminus between amino acids 70-130, which is distal to the putative P4 binding site. Taken together, these data provide compelling evidence that PGRMC1 is the P4 binding protein that mediates P4's antiapoptotic action. Moreover, the deletion mutation studies indicate that each domain of PGRMC1 plays an essential role in modulating PGRMC1's capacity to both bind and respond to P4. Additional studies are required to more precisely delineate the role of each PGRMC1 domain in transducing P4's antiapoptotic action.

  10. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Science.gov (United States)

    2011-01-01

    Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of

  11. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Directory of Open Access Journals (Sweden)

    Person Alexandra M

    2011-11-01

    Full Text Available Abstract Background Along with high affinity binding of epibatidine (Kd1≈10 pM to α4β2 nicotinic acetylcholine receptor (nAChR, low affinity binding of epibatidine (Kd2≈1-10 nM to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after

  12. Effects of local anesthetics on cholinergic agonist binding affinity of central nervous system. cap alpha. -bungarotoxin receptors

    Energy Technology Data Exchange (ETDEWEB)

    Lukas, R.L.; Bennett, E.L.

    1979-12-01

    In general, pharmacological effects of local anesthetics may be attributed to their ability to reversibly block the propagation of nerve and muscle action potentials. At physiologically potent concentrations, local anesthetics (LA) also act as noncompetitive antagonists of the physiological response of post-synaptic nicotinic acetylcholine receptors (nAChR) to cholinergic agonists, and increase agonist binding affinities of nAChR from electric organ. It is postulated that the primary site of LA action on nAChR function is at the receptor-coupled ionophore. Furthermore, LA-nAChR ionophore interactions are thought to accelerate physiological desensitization of nAChR, manifest biochemically as increased affinity of nAChR for agonist. Specific receptors for ..cap alpha..-bungarotoxin (..cap alpha..-Bgt), a potent competitive antagonist at nAChR sites in the periphery, have been detected in rat central nervous system membrane preparations. The affinity of these central ..cap alpha..-Bgt receptors (..cap alpha..-BgtR) for cholinergic agonists is found to increase on exposure to agonist. Nevertheless, on the basis of inconsistent pharmacological and physiological results, uncertainty remains regarding the relationship between ..cap alpha..-BgtR and authentic nAChR in the CNS, despite a wide body of biochemical and histological evidence consistent with their identity. Reasoning that if CNS ..cap alpha..-BgtR are true in nAChR, coupled to functional ion channels, LA might be expected to cause biochemically measurable increases in ..cap alpha..-BgtR affinity for cholinergic agonists, we have undertaken a study of the effects of LA on the ability of acetylcholine (ACh) to inhibit interaction of ..cap alpha..-BgtR with /sup 3/H-labeled ..cap alpha..-Bgt.

  13. Affinity column for purification of the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor

    International Nuclear Information System (INIS)

    Venton, D.L.; Arora, S.K.; Kim, S.O.; Lim, C.T.; Le Breton, G.C.

    1987-01-01

    The TXA 2 /PGH 2 receptor antagonist, 13-azaprostanoic acid (13-APA), was synthesized and used as the immobilized ligand in the affinity column purification of the 13-APA/U46619 binding component in human platelets. Diazo coupling of the ligand to the phenol of this tyr-gly-gly-NH-(CO)-O-Sepharose gave the affinity column material. Isolated platelet membranes were solubilized with detergent, applied directly to the affinity column and the eluate collected as 6 x 70 ml fractions. For each fraction, protein concentration and specific 3 H-13-APA/numberH-U46619 binding were determined. The majority of the applied protein (>98%) eluted in fraction number1. However, the specific 13-APA/U46619 binding per mg of protein was localized in fractions number4 and number5, representing approximately a 500-fold purification of this binding component. These results suggest that the platelet TXA 2 /PGH 2 receptor protein is retarded by this column, and that starting from crude, solubilized platelet membranes, a single pass through the column provides a 500-fold purification of the receptor

  14. Occupation of low-affinity cholecystokinin (CCK) receptors by CCK activates signal transduction and stimulates amylase secretion in pancreatic acinar cells.

    Science.gov (United States)

    Vinayek, R; Patto, R J; Menozzi, D; Gregory, J; Mrozinski, J E; Jensen, R T; Gardner, J D

    1993-03-10

    Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

  15. An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

    Directory of Open Access Journals (Sweden)

    Marcela V Maus

    2016-01-01

    Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

  16. 14-O-Methylmorphine: A Novel Selective Mu-Opioid Receptor Agonist with High Efficacy and Affinity.

    Science.gov (United States)

    Zádor, Ferenc; Balogh, Mihály; Váradi, András; Zádori, Zoltán S; Király, Kornél; Szűcs, Edina; Varga, Bence; Lázár, Bernadette; Hosztafi, Sándor; Riba, Pál; Benyhe, Sándor; Fürst, Susanna; Al-Khrasani, Mahmoud

    2017-11-05

    14-O-methyl (14-O-Me) group in morphine-6-O-sulfate (M6SU) or oxymorphone has been reported to be essential for enhanced affinity, potency and antinociceptive effect of these opioids. Herein we report on the pharmacological properties (potency, affinity and efficacy) of the new compound, 14-O-methylmorphine (14-O-MeM) in in vitro. Additionally, we also investigated the antinociceptive effect of the novel compound, as well as its inhibitory action on gastrointestinal transit in in vivo. The potency and efficacy of test compound were measured by [ 35 S]GTPγS binding, isolated mouse vas deferens (MVD) and rat vas deferens (RVD) assays. The affinity of 14-O-MeM for opioid receptors was assessed by radioligand binding and MVD assays. The antinociceptive and gastrointestinal effects of the novel compound were evaluated in the rat tail-flick test and charcoal meal test, respectively. Morphine, DAMGO, Ile 5,6 deltorphin II, deltorphin II and U-69593 were used as reference compounds. 14-O-MeM showed higher efficacy (E max ) and potency (EC 50 ) than morphine in MVD, RVD or [ 35 S]GTPγS binding. In addition, 14-O-MeM compared to morphine showed higher affinity for μ-opioid receptor (MOR). In vivo, in rat tail-flick test 14-O-MeM proved to be stronger antinociceptive agent than morphine after peripheral or central administration. Additionally, both compounds inhibited the gastrointestinal peristalsis. However, when the antinociceptive and antitransit doses for each test compound are compared, 14-O-MeM proved to have slightly more favorable pharmacological profile. Our results affirm that 14-O-MeM, an opioid of high efficacy and affinity for MOR can be considered as a novel analgesic agent of potential clinical value. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Affinity of the enantiomers of. alpha. - and. beta. -cyclazocine for binding to the phencyclidine and. mu. opioid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Todd, S.L.; Balster, R.L.; Martin, B.R. (Virginia Commonwealth Univ., Richmond (USA))

    1990-01-01

    The enantiomers in the {alpha} and {beta} series of cyclazocine were evaluated for their ability to bind to phencyclidine (PCP) and {mu}-opioid receptors in order to determine their receptor selectivity. The affinity of (-)-{beta}-cyclazocine for the PCP receptor was 1.5 greater than PCP itself. In contrast, (-)-{alpha}-cyclazocine, (+)-{alpha}-cyclazocine, and (+)-{beta}-cyclazocine were 3-, 5- and 138-fold less potent than PCP, respectively. Scatchard analysis of saturable binding of ({sup 3}H)Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) also exhibited a homogeneous population of binding sites with an apparent K{sub D} of 1.9 nM and an estimated Bmax of 117 pM. (3H)Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) binding studies revealed that (-)-{alpha}-cyclazocine (K{sub D} = 0.48 nM) was 31-, 1020- and 12,600-fold more potent than (-)-{beta}-cyclazocine, (+)-{alpha}-cyclazocine and (+)-{beta}-cyclazocine, respectively, for binding to the {mu}-opioid receptor. These data show that, although (-)-{beta}-cyclazocine is a potent PCP receptor ligand consistent with its potent PCP-like discriminative stimulus effects, it shows little selectivity for PCP receptor since it also potently displaces {mu}-opioid binding. However, these cyclazocine isomers, due to their extraordinary degree of stereoselectivity, may be useful in characterizing the structural requirements for benzomorphans having activity at the PCP receptor.

  18. Susceptibility to endotoxin induced uveitis is not reduced in mice deficient in BLT1, the high affinity leukotriene B4 receptor

    OpenAIRE

    Smith, J R; Subbarao, K; Franc, D T; Haribabu, B; Rosenbaum, J T

    2004-01-01

    Aim: To investigate the role of arachidonic acid derived chemotactic factor, LTB4, in the development of endotoxin induced uveitis (EIU), using mice deficient in the BLT1 gene which encodes the high affinity LTB4 receptor.

  19. Pesquisa de estrógeno e progesterona no epitélio das pregas vocais de mulheres por imunohistoquímica Immunohistochemical searching for estrogen and progesterone receptors in women vocal fold epithelia

    Directory of Open Access Journals (Sweden)

    Oswaldo Angel Bellido Rios

    2008-08-01

    Full Text Available A laringe é extremamente sensível a mudanças endocrinológicas. A maioria das alterações da mucosa das pregas vocais é causada por modificações do conteúdo líquido das pregas vocais e das suas modificações epiteliais. O estrógeno e a progesterona interferem e modificam esse conteúdo líquido das pregas vocais. O objetivo deste trabalho é verificar a presença de receptores de estrógeno e progesterona no epitélio das pregas vocais de mulheres. MATERIAL E MÉTODO: Estudo de casos prospectivos. Foram realizados exames de imunohistoquímica para receptores de estrógeno e progesterona em 19 espécimes de epitélio de pregas vocais que não apresentavam quaisquer indícios de afecção, inclusive inflamatória. Foram descartados casos de pacientes com idade superior a 40 anos e inferior a 15 anos. RESULTADOS: Foram encontrados receptores para progesterona em 18 de 19 pacientes. Os receptores de progesterona estão localizados tanto no núcleo quanto no citoplasma e principalmente na camada basal. Não houve nenhum caso de receptores de estrógeno nas pregas vocais. CONCLUSÃO: O epitélio das pregas vocais apresenta receptores para progesterona, tanto no citoplasma quanto no núcleo. Não foram encontrados receptores para estrógeno no epitélio das pregas vocais estudadas.Larynx is extremely sensitive to endocrinologic changes. Most vocal fold mucosa alterations are caused by changes in vocal fold liquid content and its epithelial changes. Estrogen and progesterone interfere and change this liquid content in the vocal folds. Our goal with the present paper is to study the presence of estrogen and progesterone receptors on vocal fold epithelium in 19 vocal fold epithelium specimens that did not present any indication of disease, especially inflammatory disease. We discarded those cases of patients above 40 years of age and those below 15. RESULTS: we found progesterone receptors in 18 of the 19 patients. The progesterone receptors

  20. Abnormal excessive per vagina (PV) bleeding on Esmya-selective progesterone receptor modulator (SPRM) in a symptomatic patient with uterine fibroid.

    Science.gov (United States)

    Matytsina-Quinlan, Lyubov; Matytsina, Laura

    2015-05-14

    A woman in her late 40s presented with excessive per vagina (PV) bleeding and uterine fibroid. She reported excessive PV bleeding after starting Esmya; she was brought in by ambulance to the emergency department with profuse bleeding. Abnormal uterine bleeding (AUB) developed after selective progesterone receptor modulator (SPRM) administration in this symptomatic patient with uterine fibroid. The drug was withheld and surgical treatment considered. Progressive deterioration of PV bleeding after receiving SPRM led to an urgent laparoscopic total hysterectomy, which had to be postponed due to severe anaemia. Surgery took place regardless because the excessive bleeding continued. Histology revealed a 6 cm submucosal uterine fibroid (SMUF) and adenomyosis. Physicians prescribing SPRMs to stop PV bleeding should be aware of potential AUB, which could lead to urgent hysterectomy. The mechanism of action of SPRMs is not clearly understood. Awareness of the side effects of Esmya, such as AUB, must be kept in mind when administering SPRMs. 2015 BMJ Publishing Group Ltd.

  1. Affinities and densities of high-affinity [3H]muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

    International Nuclear Information System (INIS)

    Butterworth, R.F.; Lavoie, J.; Giguere, J.F.; Pomier-Layrargues, G.

    1988-01-01

    The integrity of GABA-A receptors and of central benzodiazepine receptors was evaluated in membrane preparations from prefrontal cortex and caudate nuclei obtained at autopsy from nine cirrhotic patients who died in hepatic coma and an equal number of age-matched control subjects. Histopathological studies revealed Alzheimer Type II astrocytosis in all cases in the cirrhotic group; controls were free from neurological, psychiatric or hepatic diseases. Binding to GABA-A receptors was studied using [ 3 H]muscimol as radioligand. The integrity of central benzodiazepine receptors was evaluated using [ 3 H]flunitrazepam and [ 3 H]Ro15-1788. Data from saturation binding assays was analyzed by Scatchard plot. No modifications of either affinities (Kd) or densities (Bmax) of [ 3 H]muscimol of central benzodiazepine binding sites were observed. These findings do not support recent suggestions that alterations of either high-affinity GABA or benzodiazepine receptors play a significant role in the pathogenesis of hepatic encephalopathy

  2. Whole transcriptome analysis for T cell receptor-affinity and IRF4-regulated clonal expansion of T cells

    Directory of Open Access Journals (Sweden)

    Wei Shi

    2014-12-01

    Full Text Available Clonal population expansion of T cells during an immune response is dependent on the affinity of the T cell receptor (TCR for its antigen [1]. However, there is little understanding of how this process is controlled transcriptionally. We found that the transcription factor IRF4 was induced in a manner dependent on TCR-affinity and was critical for the clonal expansion and maintenance of effector function of antigen-specific CD8+ T cells. We performed a genome-wide expression profiling experiment using RNA sequencing technology (RNA-seq to interrogate global expression changes when IRF4 was deleted in CD8+ T cells activated with either a low or high affinity peptide ligand. This allowed us not only to determine IRF4-dependent transcriptional changes but also to identify transcripts dependent on TCR-affinity [2]. Here we describe in detail the analyses of the RNA-seq data, including quality control, read mapping, quantification, normalization and assessment of differential gene expression. The RNA-seq data can be accessed from Gene Expression Omnibus database (accession number GSE49929.

  3. Photoaffinity labeling of mammalian α1-adrenergic receptors: identification of the ligand binding subunit with a high affinity radioiodinated probe

    International Nuclear Information System (INIS)

    Leeb-Lundberg, L.M.F.; Dickinson, K.E.J.; Heald, S.L.

    1984-01-01

    A description is given of the synthesised and characterization of a novel high affinity radioiodinated α 1 -adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido-3-[ 125 I]iodophenyl)pentanoyl]-1-piperazinyl] quinazoline. In the absence of light, this ligand binds with high affinity (K/sub d/ = 130 pm) in a reverisble and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an α 1 -adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical α 1 -adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at M/sub 4/ = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the M/sub r/ = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the α 1 -adrenergic receptor

  4. Interleukin (IL)-1 in rat parturition: IL-1 receptors 1 and 2 and accessory proteins abundance in pregnant rat uterus at term - regulation by progesterone.

    Science.gov (United States)

    Ishiguro, Tomohito; Takeda, Jun; Fang, Xin; Bronson, Heather; Olson, David M

    2016-07-01

    The role of interleukin-1 (IL-1), a pro-inflammatory cytokine, in parturition is typically noted by changes in its concentrations. Studying the expression of its receptor family, IL-1 receptor (IL-1R) 1, IL-1R2, IL-1R accessory protein (IL-1RAcP), and its predominantly brain isoform, IL-1RAcPb, during late gestation in the uterus in the Long-Evans rat is another. We assessed changes in their mRNA and protein relative abundance in the uterus and compared IL-1RAcP and IL-1RAcPb mRNA abundance in uterus, cervix, ovaries, placenta, and whole blood of Long-Evans rats during late gestation or in RU486 and progesterone-treated dams using quantitative real-time PCR and western immunoblotting. IL-1R1, IL-1RAcP, and IL-1RAcPb mRNA abundance significantly increased in the uterus at delivery whereas IL-1R2 mRNA abundance significantly decreased. IL-1R1 protein increased at term and IL-1R2 protein decreased at term compared to nonpregnant uteri. IL1-RAcPb mRNA abundance was less than IL-1RAcP, but in the lower uterine segment it was the highest of all tissues examined. RU486 stimulated preterm delivery and an increase in IL-1R1 mRNA abundance whereas progesterone administration extended pregnancy and suppressed the increase in IL-1R1. These data suggest that changes in uterine sensitivity to IL-1 occur during late gestation and suggest another level of regulation for the control of delivery. The roles for IL-1RAcP and IL-1RAcPb need to be determined, but may relate to different intracellular signaling pathways. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  5. Polimorfismo do gene dos receptores de progesterona e o aborto espontâneo de repetição Progesterone receptor gene polymorphism and recurrent spontaneous abortion

    Directory of Open Access Journals (Sweden)

    Évelyn Traina

    2010-05-01

    Full Text Available OBJETIVO: investigar se polimorfismos dos genes que codificam o receptor de progesterona (PROGINS estão relacionados à ocorrência de aborto espontâneo de repetição (AER. MÉTODOS: em estudo caso-controle, foram selecionados 85 pacientes com antecedente de pelo menos três abortos precoces sem etiologia definida (Grupo Caso e 157 mulheres com história de pelo menos duas gestações de termo sem intercorrências e sem passado de abortamento (Grupo Controle. Realizada coleta de 10 mL de sangue por punção venosa periférica e extração de DNA pela técnica DTAB/CTAB. As genotipagens foram feitas por reação em cadeia de polimerase (PCR, nas condições de ciclagem específica para o polimorfismo em estudo, seguida de amplificação em gel de agarose a 2%. A visualização das bandas foi feita sob luz ultravioleta e os géis foram fotografados. As diferenças genotípicas e alélicas entre os dois grupos para o polimorfismo PROGINS foram calculadas pelo teste de χ2, adotando-se como nível de significância valores de pPURPOSE: to assess a possible association between polymorphism of the progesterone receptor gene (PROGINS and recurrent spontaneous abortion (RSA. METHODS: in this case-control study, 85 women with at least three previous spontaneous abortions without an identifiable cause (RSA Group and 157 women with at least two previous term pregnancies without pathologies and no previous miscarriage (Control Group were selected. An amount of 10 mL of peripheral blood was collected by venipuncture and genomic DNA was extracted by the DTAB/CTAB method, followed by the polymerase chain reaction (PCR under specific conditions for this polymorphism and by amplification by 2% agarose gel electrophoresis. The bands were visualized with an ultraviolet light transilluminator and the gels were photographed. Differences in the PROGINS genotype and allele frequencies between groups were analyzed by the χ2 test, with the level of significance set

  6. Progesterone as a bone-trophic hormone.

    Science.gov (United States)

    Prior, J C

    1990-05-01

    Experimental, epidemiological, and clinical data indicate that progesterone is active in bone metabolism. Progesterone appears to act directly on bone by engaging an osteoblast receptor or indirectly through competition for a glucocorticoid osteoblast receptor. Progesterone seems to promote bone formation and/or increase bone turnover. It is possible, through estrogen-stimulated increased progesterone binding to the osteoblast receptor, that progesterone plays a role in the coupling of bone resorption with bone formation. A model of the interdependent actions of progesterone and estrogen on appropriately-"ready" cells in each bone multicellular unit can be tied into the integrated secretions of these hormones within the ovulatory cycle. Figure 5 is an illustration of this concept. It shows the phases of the bone remodeling cycle in parallel with temporal changes in gonadal steroids across a stylized ovulatory cycle. Increasing estrogen production before ovulation may reverse the resorption occurring in a "sensitive" bone multicellular unit while gonadal steroid levels are low at the time of menstrual flow. The bone remodeling unit would then be ready to begin a phase of formation as progesterone levels peaked in the midluteal phase. From this perspective, the normal ovulatory cycle looks like a natural bone-activating, coherence cycle. Critical analysis of the reviewed data indicate that progesterone meets the necessary criteria to play a causal role in mineral metabolism. This review provides the preliminary basis for further molecular, genetic, experimental, and clinical investigation of the role(s) of progesterone in bone remodeling. Much further data are needed about the interrelationships between gonadal steroids and the "life cycle" of bone. Feldman et al., however, may have been prophetic when he commented; "If this anti-glucocorticoid effect of progesterone also holds true in bone, then postmenopausal osteoporosis may be, in part, a progesterone deficiency

  7. Hypermethylation pattern of ESR and PgR genes and lacking estrogen and progesterone receptors in human breast cancer tumors: ER/PR subtypes.

    Science.gov (United States)

    Pirouzpanah, Saeed; Taleban, Forough-Azam; Mehdipour, Parvin; Sabour, Siamak; Atri, Morteza

    2018-02-14

    The option of endocrine therapy in breast cancer remains conventionally promising. We aimed to investigate how accurately the pattern of hypermethylation at estrogen receptor (ESR) and progesterone receptor (PgR) genes may associate with relative expression and protein status of ER, PR and the combinative phenotype of ER/PR. In this consecutive case-series, we enrolled 139 primary diagnosed breast cancer. Methylation specific PCR was used to assess the methylation status (individual test). Tumor mRNA expression levels were evaluated using real-time RT-PCR. Immunohistochemistry data was used to present hormonal receptor status of a tumor (as test reference). Methylation at ESR1 was comparably frequent in ER-breast tumors (83.0%, PPR- conditions (Cramer's V= 0.44, PPR (77.1%, PPR expressions (55.6%, PPR- (64.4%, PPR-, the hypermethylation of PgRb seem another epigenetic signalling variable actively associate with methylated ESR1 to show lack of ER+/PR+ tumors in breast cancer.

  8. Physiology, production and action of progesterone.

    Science.gov (United States)

    Taraborrelli, Stefania

    2015-11-01

    The aim of this article is to review the physiology of progesterone and focus on its physiological actions on tissues such as endometrium, uterus, mammary gland, cardiovascular system, central nervous system and bones. In the last decades, the interest of researchers has focused on the role of progesterone in genomic and non-genomic receptor mechanisms. We searched PubMed up to December 2014 for publications on progesterone/steroidogenesis. A better understanding of the biological genomic and non-genomic receptor mechanisms could enable us in the near future to obtain a more comprehensive knowledge of the safety and efficacy of this agent during hormone replacement therapy (natural progesterone), in vitro fertilization (water-soluble subcutaneous progesterone), in traumatic brain injury, Alzheimer's disease and diabetic neuropathy, even though further clinical studies are needed to prove its usefulness. © 2015 Nordic Federation of Societies of Obstetrics and Gynecology.

  9. Progesterone radioimmunoassay

    International Nuclear Information System (INIS)

    Stroufova, A.; Kozlova, J.

    1976-01-01

    RIA methods of determining progestorone using the SORIN kit made in Italy and the NEN kit made in Great Britain were compared. Plasma extraction, the initial sample size for examination and recovery, the range of calibration curves of the two kits, the variation coefficient, the values of the blank sample and the values of progesterone determined in the normal menstrual cycle are discussed in detail. Variation coefficient: Sorin (calibration curve 9 to 11%, biological material 9.2%), NEN (calibration curve 12%, biological material 27.8%); determinations of progesterone levels during a normal menstrual cycle were 10 to 15% higher with the NEN kit; tritiated samples were measured with a 30 to 37% efficiency using the SORIN kit and 15 to 18% efficiency using the NEN kit. The turbidity and later sediment which formed during the determination of steroid hormones in biological materials after the addition of the scintillation solution did not reduce the efficiency of measurement. Priority is given to the SORIN kit. (L.O.)

  10. Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking

    International Nuclear Information System (INIS)

    Sinnett-Smith, J.; Zachary, I.; Rozengurt, E.

    1988-01-01

    We have previously identified by chemical cross-linking a cell surface protein in Swiss 3T3 cells of apparent Mr 75,000-85,000, which may represent a major component of the receptor for peptides of the bombesin family in these cells. Because bombesin-like peptides may interact with other cell surface molecules, it was important to establish the correlation between receptor binding and functions of this complex and further characterize the Mr 75,000-85,000 cross-linked protein. Detailed time courses carried out at different temperatures demonstrated that the Mr 75,000-85,000 affinity-labelled band was the earliest cross-linked complex detected in Swiss 3T3 cells incubated with 125I-labelled gastrin-releasing peptide (125I-GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000-85,000 cross-linked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit 125I-GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 125I-GRP binding and the affinity labelling of the Mr 75,000-85,000 protein. We also show that the cross-linked complex is a glycoprotein. First, solubilized affinity labelled Mr 75,000-85,000 complex applied to wheat germ lectin-sepharose columns was eluted by addition of 0.3 M N-acetyl-D-glucosamine. Second, treatment with endo-beta-N-acetylglucosaminidase F reduced the apparent molecular weight of the affinity-labelled band from 75,000-85,000 to 43,000, indicating the presence of N-linked oligosaccharide groups

  11. Affinity purification and partial characterization of the zonulin/zonula occludens toxin (Zot) receptor from human brain.

    Science.gov (United States)

    Lu, R; Wang, W; Uzzau, S; Vigorito, R; Zielke, H R; Fasano, A

    2000-01-01

    The intercellular tight junctions (TJs) of endothelial cells represent the limiting structure for the permeability of the blood-brain barrier (BBB). Although the BBB has been recognized as being the interface between the bloodstream and the brain, little is known about its regulation. Zonulin and its prokaryotic analogue, zonula occludens toxin (Zot) elaborated by Vibrio cholerae, both modulate intercellular TJs by binding to a specific surface receptor with subsequent activation of an intracellular signaling pathway involving phospholipase C and protein kinase C activation and actin polymerization. Affinity column purification revealed that human brain plasma membrane preparations contain two Zot binding proteins of approximately 55 and approximately 45 kDa. Structural and kinetic studies, including saturation and competitive assays, identified the 55-kDa protein as tubulin, whereas the 45-kDa protein represents the zonulin/Zot receptor. Biochemical characterization provided evidence that this receptor is a glycoprotein containing multiple sialic acid residues. Comparison of the N-terminal sequence of the zonulin/Zot receptor with other protein sequences by BLAST analysis revealed a striking similarity with MRP-8, a 14-kDa member of the S-100 family of calcium binding proteins. The discovery and characterization of this receptor from human brain may significantly contribute to our knowledge on the pathophysiological regulation of the BBB.

  12. A pharmacological profile of the high-affinity GluK5 kainate receptor

    DEFF Research Database (Denmark)

    Møllerud, Stine; Kastrup, Jette Sandholm Jensen; Pickering, Darryl S

    2016-01-01

    -hydroxyisoxazol-4-yl)propionate (ATPA), dihydrokainate and (2 S,4 R)−4-methyl-glutamate (SYM2081) have higher affinity at GluK3 compared to GluK5. Since some studies have indicated that GluK5 is associated with various diseases in the central nervous system (e.g. schizophrenia, temporal lobe epilepsy, bipolar...

  13. Synthesis of new isoxazoline-based acidic amino acids and investigation of their affinity and selectivity profile at ionotropic glutamate receptors

    DEFF Research Database (Denmark)

    Pinto, Andrea; Conti, Paola; Grazioso, Giovanni

    2011-01-01

    The synthesis of four new isoxazoline-based amino acids being analogues of previously described glutamate receptor ligands is reported and their affinity for ionotropic glutamate receptors is analyzed in comparison with that of selected model compounds. Molecular modelling investigations have been...

  14. Effect of the low-affinity, noncompetitive N-methyl-D-aspartate receptor antagonist dextromethorphan on visceral perception in healthy volunteers

    NARCIS (Netherlands)

    Kuiken, S. D.; Lei, A.; Tytgat, G. N. J.; Holman, R.; Boeckxstaens, G. E. E.

    2002-01-01

    Background: The use of N-methyl-d-aspartate (NMDA) receptor antagonists may hold promise for the treatment of pain of visceral origin, in particular in conditions characterized by visceral hypersensitivity. Aim: To study the effect of dextromethorphan, a low affinity, non-competitive NMDA receptor

  15. Mechanisms of anaphylaxis in human low-affinity IgG receptor locus knock-in mice.

    Science.gov (United States)

    Gillis, Caitlin M; Jönsson, Friederike; Mancardi, David A; Tu, Naxin; Beutier, Héloïse; Van Rooijen, Nico; Macdonald, Lynn E; Murphy, Andrew J; Bruhns, Pierre

    2017-04-01

    Anaphylaxis can proceed through distinct IgE- or IgG-dependent pathways, which have been investigated in various mouse models. We developed a novel mouse strain in which the human low-affinity IgG receptor locus, comprising both activating (hFcγRIIA, hFcγRIIIA, and hFcγRIIIB) and inhibitory (hFcγRIIB) hFcγR genes, has been inserted into the equivalent murine locus, corresponding to a locus swap. We sought to determine the capabilities of hFcγRs to induce systemic anaphylaxis and identify the cell types and mediators involved. hFcγR expression on mouse and human cells was compared to validate the model. Passive systemic anaphylaxis was induced by injection of heat-aggregated human intravenous immunoglobulin and active systemic anaphylaxis after immunization and challenge. Anaphylaxis severity was evaluated based on hypothermia and mortality. The contribution of receptors, mediators, or cell types was assessed based on receptor blockade or depletion. The human-to-mouse low-affinity FcγR locus swap engendered hFcγRIIA/IIB/IIIA/IIIB expression in mice comparable with that seen in human subjects. Knock-in mice were susceptible to passive and active anaphylaxis, accompanied by downregulation of both activating and inhibitory hFcγR expression on specific myeloid cells. The contribution of hFcγRIIA was predominant. Depletion of neutrophils protected against hypothermia and mortality. Basophils contributed to a lesser extent. Anaphylaxis was inhibited by platelet-activating factor receptor or histamine receptor 1 blockade. Low-affinity FcγR locus-switched mice represent an unprecedented model of cognate hFcγR expression. Importantly, IgG-related anaphylaxis proceeds within a native context of activating and inhibitory hFcγRs, indicating that, despite robust hFcγRIIB expression, activating signals can dominate to initiate a severe anaphylactic reaction. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights

  16. Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

    International Nuclear Information System (INIS)

    Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi; Arai, Kenichi; Yokota, Takashi

    1990-01-01

    Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125 I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the α and β subunits of the GM-CSF receptor, respectively

  17. IMPROVED TUMOR CELL KILLING BY TRAIL REQUIRES SELECTIVE AND HIGH AFFINITY RECEPTOR ACTIVATION

    NARCIS (Netherlands)

    Szegezdi, Eva; van der Sloot, Almer M.; Alessandro, Natoni; Mahalingam, Devalingam; Cool, Robbert H.; Munoz, Ines G.; Montoya, Guillermo; Quax, Wim J.; Luis Serrano, Steven de Jong; Samali, Afshin; Wallach, D; Kovalenko, A; Feldman, M

    2011-01-01

    Apoptosis can be activated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a wide range of tumor cells, but not in non-transformed cells. TRAIL interaction with receptors DR4 or DR5 induces apoptosis, whereas DcR1, DcR2 and osteoprotegerin are decoy receptors for TRAIL. TRAIL

  18. Structure and affinity of two bicyclic glutamate analogues at AMPA and kainate receptors

    DEFF Research Database (Denmark)

    Møllerud, Stine; Pinto, Andrea; Marconi, Laura

    2017-01-01

    Ionotropic glutamate receptors (iGluRs) are involved in most of the fast excitatory synaptic transmission in the central nervous system. These receptors are important for learning and memory formation, but are also involved in the development of diseases such as Alzheimer’s disease, epilepsy...

  19. [3H]-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and [3H] ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

    International Nuclear Information System (INIS)

    Branchek, T.; Adham, N.; Macchi, M.; Kao, H.T.; Hartig, P.R.

    1990-01-01

    The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to [3H]ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding the serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both [3H]DOB and [3H]ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate [Gpp(NH)p] to this system caused a rightward shift and steepening of agonist competition curves for [3H] ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity [3H]DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that [3H]DOB and [3H]ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein

  20. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

    International Nuclear Information System (INIS)

    Bayne, M.L.; Applebaum, J.; Chicchi, G.G.; Hayes, N.S.; Green, B.G.; Cascieri, M.A.

    1988-01-01

    Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe -1 , Val 1 , Asn 2 , Gln 3 , His 4 , Ser 8 , His 9 , Glu 12 , Tyr 15 , Leu 16 ]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln 3 , Ala 4 ] IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr 15 , Leu 16 ] IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, [Gln 3 , Ala 4 , Tyr 15 ,Leu 16 ]IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I

  1. Influence of Estradiol-17beta on Progesterone and Estrogen Receptor mRNA Expression in Porcine Follicular Granulosa Cells during Short-Term, In Vitro Real-Time Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Sylwia Ciesiółka

    2016-01-01

    Full Text Available Progesterone (P4 and estradiol (E2 play a significant role in mammalian reproduction. Our study demonstrated that separated porcine cumulus cells (CCs and/or granulosa cells (GCs might proliferate in vitro during short-term, real-time primary culture. The GCs were analyzed according to gene expression of the progesterone receptor (nuclear form (pgr, progesterone receptor membrane component 1 (pgrmc1, and estrogen-related receptor beta 3 (esrrb3 in relation to two housekeeping genes: actb and pbgd. GCs were cultivated in medium with the E2. Both pgr/actb and pgr/pbgd revealed higher expression between 24 and 168 h of IVC of prolonged E2 treatment and at 48 h of IVC after acute E2 administration. The pgrmc1/actb and pgrmc1/pbgd displayed increased expression after prolonged E2 treatment between 24 and 120 h of IVC. The highest level of esrrb3/actb at 120 and 144 h, as well as esrrb3/pbgd at 120 h, in untreated controls as compared to the hormone-stimulated group, was observed. We suggest that E2 significantly influences the upregulation of pgr, pgrmc1, and esrrb3 expression in porcine GCs during real-time cell proliferation. Since esrrb3 expression is stimulated by E2 in both an acute and prolonged manner, estradiol may be recognized as a potential estrogen receptor agonist in GCs.

  2. A soluble form of the high affinity IgE receptor, Fc-epsilon-RI, circulates in human serum.

    Directory of Open Access Journals (Sweden)

    Eleonora Dehlink

    Full Text Available Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI, the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum.

  3. Solubilization and cleavage of human neutrophil (N) affinity-labeled receptors for leukotriene B4 (LTB4)

    International Nuclear Information System (INIS)

    Marotti, T.; Young, R.N.; Gifford, L.A.; Goldman, D.W.; Goetzl, E.J.

    1986-01-01

    LTB 4 chemotactic receptors in purified N plasma membranes (PMs) have been affinity-labeled with [ 3 H]-C-1 aminopropylamide-LTB 4 ([ 3 H]APA-LTB 4 ) by disuccinimidyl suberate (DSS) cross-linking. Intact Ns were pretreated with diisopropylfluorophosphate, suspended at 10 7 /ml in Hanks' solution-10 mM HEPES (pH 7.4), incubated for 30 min at 4 0 C with 30 nM [ 3 H]APA-LTB 4 and 25 min with 1 mM bis[2-(succinimidooxycarbonyloxy)-ethyl] sulfone, an impermeant analog of DSS, and sonified for 30 sec at 4 0 C. The 10,000 g supernatant of the sonicate was centrifuged at 40,000 g for 30 min at 4 0 C on a discontinuous gradient of 10-50 g % sucrose, from which a mean of 78% of the radiolabel was recovered with PM markers. The extent and specificity of labeling of intact N receptors were similar to those of receptors in PMs. The radioactively-labeled receptors appeared as a single band of 35-40 kd in sodium dodecyl sulfate (SDS) 10 g % polyacrylamide gel electrophoresis. Cleavage of radiolabeled receptors with 1 mg/ml of cyanogen bromide in 70% formic acid for 18 hr at room temperature or with 30 mM HCl under N 2 for 4 hr at 105 0 C converted a mean of 18-32% of the radioactivity to a band of 14 kd in SDS-15 g % PAGE. N receptors for LTB 4 , thus, are localized in the PM and can be isolated for structural studies

  4. Impact of maternal dietary exposure to endocrine-acting chemicals on progesterone receptor expression in microdissected hypothalamic medial preoptic areas of rat offspring

    International Nuclear Information System (INIS)

    Takagi, Hironori; Shibutani, Makoto; Lee, Kyoung-Youl; Masutomi, Naoya; Fujita, Haruka; Inoue, Kaoru; Mitsumori, Kunitoshi; Hirose, Masao

    2005-01-01

    We have previously examined the impact of perinatal exposure to ethinylestradiol (EE), methoxychlor (MXC), diisononyl phthalate (DINP), and genistein (GEN) in maternal diet on rat offspring, and found developmental and/or reproductive toxicity with 0.5 ppm EE, 1200 ppm MXC, and 20,000 ppm DINP. Although the toxicological profile with MXC was similar to the EE case, the population changes in pituitary hormone-producing cells totally differed between the two cases, changes being evident from 240 ppm with MXC. In the present study, to assess the impact of these agents on brain sexual differentiation, region-specific mRNA expression of estrogen receptors (ER) α and β, the progesterone receptor (PR), gonadotrophin-releasing hormone, steroid receptor coactivators (SRC)-1 and -2, and calbindin-D in microdissected hypothalamic medial preoptic areas (MPOAs) at postnatal day 10 was first analyzed in rats exposed to 0.5 ppm-EE from gestational day 15 by real-time RT-PCR. Sexually dimorphic expression of ERα and PR was noted with predominance in females and males, respectively, EE up-regulating SRC-1 in males and ERβ and PR in females. Next, we similarly examined expression changes of ERα and β, PR, and SRC-1 in animals exposed to MXC at 24, 240, and 1200 ppm, DINP at 4000 and 20,000 ppm, and GEN at 1000 ppm. MXC at 1200 ppm down- and up-regulated PR in males and females, respectively, and DINP at 20,000 ppm down-regulated PR in females, while GEN did not exert any clear effects. The results thus suggest that agents causing developmental and/or reproductive abnormalities in later life may affect hypothalamic PR expression during the exposure period in early life

  5. Progestogen treatments for cycle management in a sheep model of assisted conception affect the growth patterns, the expression of luteinizing hormone receptors, and the progesterone secretion of induced corpora lutea.

    Science.gov (United States)

    Letelier, Claudia; García-Fernández, Rosa Ana; Contreras-Solis, Ignacio; Sanchez, María Angeles; Garcia-Palencia, Pilar; Sanchez, Belen; Gonzalez-Bulnes, Antonio; Flores, Juana María

    2010-03-01

    To determine, in a sheep model, the effect of a short-term progestative treatment on growth dynamics and functionality of induced corpora lutea. Observational, model study. Public university. Sixty adult female sheep. Synchronization and induction of ovulation with progestogens and prostaglandin analogues; ovarian ultrasonography, blood sampling, and ovariectomy. Determination of pituitary function and morphologic characteristics, expression of luteinizing hormone (LH) receptors, and progesterone secretion of corpora lutea. The use of progestative pretreatments for assisted conception affect the growth patterns, the expression of LH receptors, and the progesterone secretion of induced corpora lutea. The current study indicates, in a sheep model, the existence of deleterious effects from progestogens on functionality of induced corpora lutea. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. Immunologic differentiation of two high-affinity neurotensin receptor isoforms in the developing rat brain.

    Science.gov (United States)

    Boudin, H; Lazaroff, B; Bachelet, C M; Pélaprat, D; Rostène, W; Beaudet, A

    2000-09-11

    Earlier studies have demonstrated overexpression of NT1 neurotensin receptors in rat brain during the first 2 weeks of life. To gain insight into this phenomenon, we investigated the identity and distribution of NT1 receptor proteins in the brain of 10-day-old rats by using two different NT1 antibodies: one (Abi3) directed against the third intracellular loop and the other (Abi4) against the C-terminus of the receptor. Immunoblot experiments that used Abi3 revealed the presence of two differentially glycosylated forms of the NT1 receptor in developing rat brain: one migrating at 54 and the other at 52 kDa. Whereas the 54-kDa form was expressed from birth to adulthood, the 52-kDa form was detected only at 10 and 15 days postnatal. Only the 52-kDa isoform was recognized by Abi4. By immunohistochemistry, both forms of the receptor were found to be predominantly expressed in cerebral cortex and dorsal hippocampus, in keeping with earlier radioligand binding and in situ hybridization data. However, whereas Abi4 immunoreactivity was mainly concentrated within nerve cell bodies and extensively colocalized with the Golgi marker alpha-mannosidase II, Abi3 immunoreactivity was predominantly located along neuronal processes. These results suggest that the transitorily expressed 52-kDa protein corresponds to an immature, incompletely glycosylated and largely intracellular form of the NT1 receptor and that the 54-kDa protein corresponds to a mature, fully glycosylated, and largely membrane-associated form. They also indicate that antibodies directed against different sequences of G-protein-coupled receptors may yield isoform-specific immunohistochemical labeling patterns in mammalian brain. Finally, the selective expression of the short form of the NT1 receptor early in development suggests that it may play a specific role in the establishment of neuronal circuitry. Copyright 2000 Wiley-Liss, Inc.

  7. A single base-pair change in 2009 H1N1 hemagglutinin increases human receptor affinity and leads to efficient airborne viral transmission in ferrets.

    Directory of Open Access Journals (Sweden)

    Akila Jayaraman

    2011-03-01

    Full Text Available The 2009 H1N1 influenza A virus continues to circulate among the human population as the predominant H1N1 subtype. Epidemiological studies and airborne transmission studies using the ferret model have shown that the transmission efficiency of 2009 H1N1 viruses is lower than that of previous seasonal strains and the 1918 pandemic H1N1 strain. We recently correlated this reduced transmission efficiency to the lower binding affinity of the 2009 H1N1 hemagglutinin (HA to α2→6 sialylated glycan receptors (human receptors. Here we report that a single point mutation (Ile219→Lys; a base pair change in the glycan receptor-binding site (RBS of a representative 2009 H1N1 influenza A virus, A/California/04/09 or CA04/09, quantitatively increases its human receptor-binding affinity. The increased human receptor-affinity is in the same range as that of the HA from highly transmissible seasonal and 1918 pandemic H1N1 viruses. Moreover, a 2009 H1N1 virus carrying this mutation in the RBS (generated using reverse genetics transmits efficiently in ferrets by respiratory droplets thereby reestablishing our previously observed correlation between human receptor-binding affinity and transmission efficiency. These findings are significant in the context of monitoring the evolution of the currently circulating 2009 H1N1 viruses.

  8. Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks

    Science.gov (United States)

    Schäffer, Lauge; Brissette, Renee E.; Spetzler, Jane C.; Pillutla, Renuka C.; Østergaard, Søren; Lennick, Michael; Brandt, Jakob; Fletcher, Paul W.; Danielsen, Gillian M.; Hsiao, Ku-Chuan; Andersen, Asser S.; Dedova, Olga; Ribel, Ulla; Hoeg-Jensen, Thomas; Hansen, Per Hertz; Blume, Arthur J.; Markussen, Jan; Goldstein, Neil I.

    2003-01-01

    Insulin is thought to elicit its effects by crosslinking the two extracellular α-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases. PMID:12684539

  9. Confirmation of 5p12 as a susceptibility locus for progesterone-receptor- positive, lower grade breast cancer

    NARCIS (Netherlands)

    R.L. Milne (Roger); E.L. Goode (Ellen); M. García-Closas (Montserrat); F.J. Couch (Fergus); G. Severi (Gianluca); R. Hein (Rebecca); Z. Fredericksen (Zachary); N. Malats (Núria); M.P. Zamora (Pilar); J.I.A. Perez (Jose Ignacio Arias); J. Benítez (Javier); T. Dörk (Thilo); P. Schürmann (Peter); J.H. Karstens (Johann); P. Hillemanns (Peter); A. Cox (Angela); I.W. Brock (Ian); K.S. Elliot (Katherine); S.S. Cross (Simon); S. Seal (Sheila); C. Turnbull (Clare); A. Renwick (Anthony); N. Rahman (Nazneen); C-Y. Shen (Chen-Yang); J-C. Yu (Jyh-Cherng); C.-S. Huang (Chiun-Sheng); M.-F. Hou (Ming-Feng); B.G. Nordestgaard (Børge); S.E. Bojesen (Stig); C. Lanng (Charlotte); G.G. Alnæs (Grethe); V. Kristensen (Vessela); A.-L. Børrensen-Dale (Anne-Lise); J.L. Hopper (John); G.S. Dite (Gillian); C. Apicella (Carmel); M.C. Southey (Melissa); D. Lambrechts (Diether); B.T. Yesilyurt (Betül); O.A.M. Floris; K. Leunen; S. Sangrajrang (Suleeporn); V. Gaborieau (Valerie); P. Brennan (Paul); J.D. McKay (James); J. Chang-Claude (Jenny); S. Wang-Gohrke (Shan); P. Radice (Paolo); P. Peterlongo (Paolo); S. Manoukian (Siranoush); M. Barile (Monica); G.G. Giles (Graham); L. Baglietto (Laura); E.M. John (Esther); A. Miron (Alexander); S.J. Chanock (Stephen); J. Lissowska (Jolanta); M.E. Sherman (Mark); J.D. Figueroa (Jonine); N.V. Bogdanova (Natalia); N.N. Antonenkova (Natalia); I.V. Zalutsky (Iosif); Y.I. Rogov (Yuri); P.A. Fasching (Peter); T. Bayer (T.); A.B. Ekici (Arif); M.W. Beckmann (Matthias); H. Brenner (Hermann); H. Müller (Heike); V. Arndt (Volker); C. Stegmaier (Christa); I.L. Andrulis (Irene); J.A. Knight (Julia); G. Glendon (Gord); A.M. Mulligan (Anna Marie); A. Mannermaa (Arto); V. Kataja (Vesa); V-M. Kosma (Veli-Matti); J. Hartikainen (Jaana); A. Meindl (Alfons); J. Heil (Joerg); C.R. Bartram (Claus); R.K. Schmutzler (Rita); G. Thomas (Gilles); R.N. Hoover (Robert); O. Fletcher (Olivia); L.J. Gibson (Lorna); I. dos Santos Silva (Isabel); J. Peto (Julian); S. Nickels (Stefan); D. Flesch-Janys (Dieter); H. Anton-Culver (Hoda); A. Ziogas (Argyrios); E.J. Sawyer (Elinor); I.P. Tomlinson (Ian); M. Kerin (Michael); N. Miller (Nicola); M.K. Schmidt (Marjanka); A. Broeks (Annegien); L.J. van 't Veer (Laura); R.A.E.M. Tollenaar (Rob); P.D.P. Pharoah (Paul); A.M. Dunning (Alison); K.A. Pooley (Karen); F. Marme (Federick); A. Schneeweiss (Andreas); C. Sohn (Christof); B. Burwinkel (Barbara); A. Jakubowska (Anna); J. Lubinski (Jan); K. Jaworska (Katarzyna); K. Durda (Katarzyna); D. Kang (Daehee); K-Y. Yoo (Keun-Young); D-Y. Noh (Dong-Young); S.-H. Ahn (Sei-Hyun); D. Hunter (David); S.E. Hankinson (Susan); P. Kraft (Peter); S. Lindstrom (Stephen); X. Chen (Xiaoqing); J. Beesley (Jonathan); U. Hamann (Ute); V. Harth (Volker); C. Justenhoven (Christina); R. Winqvist (Robert); K. Pykäs (Katri); A. Jukkola-Vuorinen (Arja); M. Grip (Mervi); M.J. Hooning (Maartje); A. Hollestelle (Antoinette); R.A. Oldenburg (Rogier); M.M.A. Tilanus-Linthorst (Madeleine); E.K. Khusnutdinova (Elza); M. Bermisheva (Marina); D. Prokofieva (Darya); A. Farahtdinova (Albina); J.E. Olson (Janet); X. Wang (Xing); M.K. Humphreys (Manjeet); Q. Wang (Qing); G. Chenevix-Trench (Georgia); D.F. Easton (Douglas)

    2011-01-01

    textabstractBackground: The single-nucleotide polymorphism (SNP) 5p12-rs10941679 has been found to be associated with risk of breast cancer, particularly estrogen receptor (ER)-positive disease. We aimed to further explore this association overall, and by tumor histopathology, in the Breast Cancer

  10. Structure-based Understanding of Binding Affinity and Mode of Estrogen Receptor α Agonists and Antagonists.

    Science.gov (United States)

    The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interact...

  11. Searsia species with affinity to the N-methyl-d-aspartic acid (NMDA) receptor

    DEFF Research Database (Denmark)

    Jäger, Anna; Knap, D.M.; Nielsen, Birgitte

    2012-01-01

    Species of Searsia are used in traditional medicine to treat epilepsy. Previous studies on S. dentata and S. pyroides have shown that this is likely mediated via the N-methyl-d-aspartic acid (NMDA) receptor. Ethanolic extracts of leaves of six Searsia species were tested in a binding assay...

  12. Relative binding affinity prediction of farnesoid X receptor in the D3R Grand Challenge 2 using FEP+

    Science.gov (United States)

    Schindler, Christina; Rippmann, Friedrich; Kuhn, Daniel

    2018-01-01

    Physics-based free energy simulations have increasingly become an important tool for predicting binding affinity and the recent introduction of automated protocols has also paved the way towards a more widespread use in the pharmaceutical industry. The D3R 2016 Grand Challenge 2 provided an opportunity to blindly test the commercial free energy calculation protocol FEP+ and assess its performance relative to other affinity prediction methods. The present D3R free energy prediction challenge was built around two experimental data sets involving inhibitors of farnesoid X receptor (FXR) which is a promising anticancer drug target. The FXR binding site is predominantly hydrophobic with few conserved interaction motifs and strong induced fit effects making it a challenging target for molecular modeling and drug design. For both data sets, we achieved reasonable prediction accuracy (RMSD ≈ 1.4 kcal/mol, rank 3-4 according to RMSD out of 20 submissions) comparable to that of state-of-the-art methods in the field. Our D3R results boosted our confidence in the method and strengthen our desire to expand its applications in future in-house drug design projects.

  13. Relative binding affinity prediction of farnesoid X receptor in the D3R Grand Challenge 2 using FEP.

    Science.gov (United States)

    Schindler, Christina; Rippmann, Friedrich; Kuhn, Daniel

    2018-01-01

    Physics-based free energy simulations have increasingly become an important tool for predicting binding affinity and the recent introduction of automated protocols has also paved the way towards a more widespread use in the pharmaceutical industry. The D3R 2016 Grand Challenge 2 provided an opportunity to blindly test the commercial free energy calculation protocol FEP+ and assess its performance relative to other affinity prediction methods. The present D3R free energy prediction challenge was built around two experimental data sets involving inhibitors of farnesoid X receptor (FXR) which is a promising anticancer drug target. The FXR binding site is predominantly hydrophobic with few conserved interaction motifs and strong induced fit effects making it a challenging target for molecular modeling and drug design. For both data sets, we achieved reasonable prediction accuracy (RMSD ≈ 1.4 kcal/mol, rank 3-4 according to RMSD out of 20 submissions) comparable to that of state-of-the-art methods in the field. Our D3R results boosted our confidence in the method and strengthen our desire to expand its applications in future in-house drug design projects.

  14. Stromal Clues in Endometrial Carcinoma: Loss of Expression of β-Catenin, Epithelial-Mesenchymal Transition Regulators, and Estrogen-Progesterone Receptor.

    Science.gov (United States)

    Senol, Serkan; Sayar, Ilyas; Ceyran, Ayse B; Ibiloglu, Ibrahim; Akalin, Ibrahim; Firat, Ugur; Kosemetin, Duygu; Engin Zerk, Pinar; Aydin, Abdullah

    2016-05-01

    Epithelial-stroma interactions in the endometrium are known to be responsible for physiological functions and emergence of several pathologic lesions. Periglandular stromal cells act on endometrial cells in a paracrine manner through sex hormones. In this study, we immunohistochemically evaluated the expression of epithelial-mesenchymal transition regulators (SNAIL/SLUG, TWIST, ZEB1), adhesion molecules (β-catenin and E-cadhenin), estrogen (ER)-progesterone (PR) receptor and their correlation with each other in 30 benign, 148 hyperplastic (EH), and 101 endometrioid-type endometrial carcinoma (EC) endometria. In the epithelial component, loss of expression in E-cadherin, ER and PR, and overexpression of TWIST and ZEB1 were significantly higher in EC than in EH (P<0.01). In the periglandular stromal component, β-catenin and SNAIL/SLUG expression were significantly higher in normal endometrium and simple without atypical EH compared with complex atypical EH and EC (P<0.01). In addition, periglandular stromal TWIST expression was significantly higher in EH group compared with EC (P<0.05). There was significantly negative correlation between β-catenin and ER, TWIST and ER, and TWIST and PR in hyperplastic and carcinomatous glandular epithelium, whereas there was a significantly positive correlation between β-catenin and SNAIL-SLUG, β-catenin and TWIST, β-catenin and ER, β-catenin and PR, SNAIL-SLUG and ER, SNAIL-SLUG and PR, TWIST and ER, TWIST and PR, in periglandular/cancer-associated stromal cells (P<0.01). In conclusion, the pattern of positive and negative correlations in the expression of epithelial-mesenchymal transition regulators (SNAIL-SLUG and TWIST), sex hormone receptors (ER and PR), and β-catenin between ECs and hyperplasia, as well as between epithelium and stroma herein, is suggestive of a significant role for these proteins and their underlying molecular processes in the development of endometrial carcinomas.

  15. Worse prognosis in breast cancer patients can be predicted by immunohistochemical analysis of positive MMP-2 and negative estrogen and progesterone receptors

    Directory of Open Access Journals (Sweden)

    Edneia A. S. Ramos

    Full Text Available Summary Introduction: Breast cancer is the most cause of death, and approximately 90% of these deaths are due to metastases. Matrix metalloproteinase-2 (MMP-2 gelatinase activity is able to degrade a major constituent of the tumor microenvironment, type IV collagen. Two well-established proteins used as markers in clinical practice for breast cancer are the receptors for estrogen (ER and progesterone (PR. Although the presence of these receptors has been associated with a better prognosis, loss of these proteins can occur during tumor progression, with subsequent resistance to hormone therapy. Objective: To study the correlation among MMP-2, ER, and PR, as well as the establishment of the metastatic process in primary breast tumors. Method: Breast cancer samples (n=44 were analyzed by immunohistochemistry for MMP-2, ER, and PR. Results: We observed that 90% of patients who had metastases and died showed positive staining for MMP-2 (p=0.0082 for both. Using Kaplan-Meier analysis, we found that negative ER patients who were also positive for MMP-2 had even worse disease-free survival (DFS and overall survival (OS (p= 0.012 and p=0.005, respectively. Similar results were found in PR-negative patients for DFS (a trend p=0.077 and OS (p=0.038. Conclusion: Regardless of our small sample size (n=44, the data obtained strongly suggest that MMP-2 in combination with already well-established markers could help to predict the emergence of metastases and death in patients with breast cancer.

  16. Progesterone receptor activates Msx2 expression by downregulating TNAP/Akp2 and activating the Bmp pathway in EpH4 mouse mammary epithelial cells.

    Directory of Open Access Journals (Sweden)

    Jodie M Fleming

    Full Text Available Previously we demonstrated that EpH4 mouse mammary epithelial cells induced the homeobox transcription factor Msx2 either when transfected with the progesterone receptor (PR or when treated with Bmp2/4. Msx2 upregulation was unaffected by Wnt inhibitors s-FRP or Dkk1, but was inhibited by the Bmp antagonist Noggin. We therefore hypothesized that PR signaling to Msx2 acts through the Bmp receptor pathway. Herein, we confirm that transcripts for Alk2/ActR1A, a non-canonical BmpR Type I, are upregulated in mammary epithelial cells overexpressing PR (EpH4-PR. Increased phosphorylation of Smads 1,5, 8, known substrates for Alk2 and other BmpR Type I proteins, was observed as was their translocation to the nucleus in EpH4-PR cells. Analysis also showed that Tissue Non-Specific Alkaline Phosphatase (TNAP/Akp2 was also found to be downregulated in EpH4-PR cells. When an Akp2 promoter-reporter construct containing a ½PRE site was transfected into EpH4-PR cells, its expression was downregulated. Moreover, siRNA mediated knockdown of Akp2 increased both Alk2 and Msx2 expression. Collectively these data suggest that PR inhibition of Akp2 results in increased Alk2 activity, increased phosphorylation of Smads 1,5,8, and ultimately upregulation of Msx2. These studies imply that re-activation of the Akp2 gene could be helpful in downregulating aberrant Msx2 expression in PR+ breast cancers.

  17. Changes in proliferating and apoptotic markers of leiomyoma following treatment with a selective progesterone receptor modulator or gonadotropin-releasing hormone agonist.

    Science.gov (United States)

    Yun, Bo Seong; Seong, Seok Ju; Cha, Dong Hyun; Kim, Ji Yeon; Kim, Mi-La; Shim, Jeong Yun; Park, Ji Eun

    2015-08-01

    To evaluate changes in proliferating and apoptotic markers of myoma tissue from patients treated with a selective progesterone receptor modulator (SPRM) or GnRH agonist by measuring expression of PDGF-A mRNA, IGF-1 mRNA, bcl-2 mRNA, and PCNA and caspase-3 protein. Between December 2013 and July 2014, women with symptomatic leiomyoma were divided into control (no treatment before surgery), SPRM (treatment with ulipristal acetate [SPRM] for 3 months before surgery), and GnRHa (treatment with leuprolide acetate [GnRH agonist] for 3 months before surgery) groups. Tissue specimens were collected from the myoma core and normal myometrium of all patients. The expression of mRNA and protein was assessed by quantitative real-time reverse transcriptase-polymerase chain reaction and Western blot. A total of 38 patients were enrolled (control group, n=14; SPRM group, n=13; GnRHa group, n=11). PDGF-A mRNA expression was lower in both the myoma core and normal myometrium tissues of the SPRM compared with the control group, but there was no difference between the control and GnRHa group. There were also no group differences in bcl-2 mRNA or IGF-1 mRNA expression. Both PCNA and caspase-3 protein expression were higher in the leiomyoma tissue of the SPRM compared with the control group, but there was no difference between the control and GnRHa groups in the expression of either protein. Both proliferation and apoptosis were increased in the leiomyoma of patients after SPRM treatment, but there was no change following GnRH agonist treatment, in vivo. However, PDGF-A mRNA was decreased after SPRM treatment, indicating a dual effect of progesterone on the regulation of growth factors. Furthermore, there was an increase in caspase-3 protein, but not bcl-2 mRNA, expression in the SPRM group suggesting that SPRM may exert its effects in pathways other than the bcl-2 apoptotic pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Genome-Wide Progesterone Receptor Binding: Cell Type-Specific and Shared Mechanisms in T47D Breast Cancer Cells and Primary Leiomyoma Cells

    Science.gov (United States)

    Huang, Lei; Owen, Jonas K.; Xie, Anna; Navarro, Antonia; Monsivais, Diana; Coon V, John S.; Kim, J. Julie; Dai, Yang; Bulun, Serdar E.

    2012-01-01

    Background Progesterone, via its nuclear receptor (PR), exerts an overall tumorigenic effect on both uterine fibroid (leiomyoma) and breast cancer tissues, whereas the antiprogestin RU486 inhibits growth of these tissues through an unknown mechanism. Here, we determined the interaction between common or cell-specific genome-wide binding sites of PR and mRNA expression in RU486-treated uterine leiomyoma and breast cancer cells. Principal Findings ChIP-sequencing revealed 31,457 and 7,034 PR-binding sites in breast cancer and uterine leiomyoma cells, respectively; 1,035 sites overlapped in both cell types. Based on the chromatin-PR interaction in both cell types, we statistically refined the consensus progesterone response element to G•ACA• • •TGT•C. We identified two striking differences between uterine leiomyoma and breast cancer cells. First, the cis-regulatory elements for HSF, TEF-1, and C/EBPα and β were statistically enriched at genomic RU486/PR-targets in uterine leiomyoma, whereas E2F, FOXO1, FOXA1, and FOXF sites were preferentially enriched in breast cancer cells. Second, 51.5% of RU486-regulated genes in breast cancer cells but only 6.6% of RU486-regulated genes in uterine leiomyoma cells contained a PR-binding site within 5 kb from their transcription start sites (TSSs), whereas 75.4% of RU486-regulated genes contained a PR-binding site farther than 50 kb from their TSSs in uterine leiomyoma cells. RU486 regulated only seven mRNAs in both cell types. Among these, adipophilin (PLIN2), a pro-differentiation gene, was induced via RU486 and PR via the same regulatory region in both cell types. Conclusions Our studies have identified molecular components in a RU486/PR-controlled gene network involved in the regulation of cell growth, cell migration, and extracellular matrix function. Tissue-specific and common patterns of genome-wide PR binding and gene regulation may determine the therapeutic effects of antiprogestins in uterine fibroids and

  19. Multiple affinity forms of the calcitonin gene-related peptide receptor in rat cerebellum

    International Nuclear Information System (INIS)

    Chatterjee, T.K.; Fisher, R.A.

    1991-01-01

    Binding of 125I-calcitonin gene-related peptide (125I-CGRP) to rat cerebellum membranes and the sensitivity to guanine nucleotides of binding were investigated. Cerebellum binding sites labeled by 125I-CGRP appear to be highly specific, inasmuch as CGRP inhibited binding with an IC50 of 100 pM but other peptides were inactive or much less active in displacing 125I-CGRP from these sites. 125I-CGRP binding sites in cerebellum membranes were saturable and of high affinity. Scatchard analysis of the saturation binding data revealed a homogeneous population of binding sites, with a KD of 224 ± 28 pM and Bmax of 131 ± 15 fmol/mg of protein. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) (100 microM), a single population of binding sites, with a KD of 464 ± 77 pM and Bmax of 100 ± 14 fmol/mg of protein, was observed. The kinetics of association of 125I-CGRP with cerebellum membranes were monophasic at all ligand concentrations tested. However, the observed association rate constant (kobs) was not dependent on [125I-CGRP] in a linear fashion in either the absence or the presence of GTP gamma S (100 microM). The kinetics of dissociation of 125I-CGRP from cerebellum membranes were multiexponential, with fast and slow dissociating components having rate constants of 0.34 ± 0.01 and 0.025 ± 0.001 min-1, respectively. The fast dissociating component represented 60 ± 2% of the total specific binding sites. Dissociation of 125I-CGRP from cerebellum sites was much faster in the presence of GTP gamma S (100 microM) but still exhibited dissociation from two affinity components. The rate constants for these components of dissociation were 0.67 ± 0.03 and 0.077 ± 0.007 min-1, with the faster dissociating component representing 66 ± 1% of the total specific binding sites

  20. Humoral immunity provides resident intestinal eosinophils access to luminal antigen via eosinophil-expressed low affinity Fc gamma receptors

    Science.gov (United States)

    Smith, Kalmia M.; Rahman, Raiann S.; Spencer, Lisa A.

    2016-01-01

    Eosinophils are native to the healthy gastrointestinal tract, and are associated with inflammatory diseases likely triggered by exposure to food allergens (e.g. food allergies and eosinophilic gastrointestinal disorders). In models of allergic respiratory diseases and in vitro studies, direct antigen engagement elicits eosinophil effector functions including degranulation and antigen presentation. However, it was not known whether intestinal tissue eosinophils that are separated from luminal food antigens by a columnar epithelium might similarly engage food antigens. Using an intestinal ligated loop model in mice, here we determined that resident intestinal eosinophils acquire antigen from the lumen of antigen-sensitized but not naïve mice in vivo. Antigen acquisition was immunoglobulin-dependent; intestinal eosinophils were unable to acquire antigen in sensitized immunoglobulin-deficient mice, and passive immunization with immune serum or antigen-specific IgG was sufficient to enable intestinal eosinophils in otherwise naïve mice to acquire antigen in vivo. Intestinal eosinophils expressed low affinity IgG receptors, and the activating receptor FcγRIII was necessary for immunoglobulin-mediated acquisition of antigens by isolated intestinal eosinophils in vitro. Our combined data suggest that intestinal eosinophils acquire lumen-derived food antigens in sensitized mice via FcγRIII antigen focusing, and may therefore participate in antigen-driven secondary immune responses to oral antigens. PMID:27683752

  1. Transmembrane adaptor proteins in the high-affinity IgE receptor signaling

    Czech Academy of Sciences Publication Activity Database

    Dráber, Petr; Hálová, Ivana; Levi-Schaffer, F.; Dráberová, Lubica

    2012-01-01

    Roč. 2, 11.1. (2012), s. 95 ISSN 1664-3224 R&D Projects: GA MŠk 1M0506; GA ČR GA301/09/1826; GA ČR GAP302/10/1759; GA AV ČR KAN200520701 Grant - others:AV ČR(CZ) M200520901 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : IgE receptor * LAT/LAT1 * LAX * NTAL/Lab/LAT2 * PAG/Cbp * mast cells * plasma membrane * transmembrane adaptor proteins Subject RIV: EB - Genetics ; Molecular Biology

  2. Pharmacokinetics and biodistribution of a radioiodine labeled peptidomimetic ligand for high-affinity nerve growth factor receptors

    Energy Technology Data Exchange (ETDEWEB)

    Jung, K. H.; Kim, D. H.; Paik, J. Y.; Koh, B. H.; Bae, J. S.; Choe, Y. S.; Lee, K. H.; Kim, B. T. [Samsung Medical Center, Seoul (Korea, Republic of)

    2005-07-01

    Some of the obstacles for the clinical application of whole nerve growth factor (NGF) may be overcome by utilizing small molecule mimetics. We thus investigated the in vivo pharmacokinetics and biodistribution of a small cyclic peptide derived from NGF-[C(92-96)] with high receptor binding affinity. I-125 C(92-96) was labeled with the Bolton-Hunter method, and binding to TrkA/IgG chimeric protein was confirmed on a polyacrylamide gel after cross-linking. Pharmacokinetic analysis was performed in normal ICR mice intravenously injected with 0.5 MBq I-125 C(92-96) containing varying doses of C(92-96). Biodistribution studies were done at 6 h after injection. Cross-linkage analysis confirmed binding of I-125 C(92-96) to the high affinity NGF receptor, TrkA. Intravenously injected I-125 C(92-96) was cleared from the blood in a biexponential manner with an early T1/2{alpha} of 5.2 min and late T1/2{beta} of 121.3 min. Log blood-concentration decreased over time with a k-slope of 0.0025, clearance of 11.8{+-}0.5 ml/min, T1/2 of 4.1{+-}0.4 hr, and volume of distribution of 69.7{+-}4.6 ml. The pattern of elimination from the blood remained essentially unchanged regardless of the dose of added C(92-96), with dose-proportionate increases in AUCs and peak concentrations consistent with linear pharmacokinetics. Biodistribution studies demonstrated high kidney activity suggesting renal excretion of I-125 C(92-96). There were moderate levels of accumulation in the spleen, lungs and liver, followed by the myocardium and skeletal muscle, whereas brain uptake was low (< 0.2 %ID/gm). Intravenously administered C(92-96) follows linear pharmacokinetics, and is cleared from the circulation at a rate comparable to whole NGF despite its substantially smaller size. Although intravenous C(92-96) does not adequately reach brain tissue, clinically relevant doses can achieve major organ accumulation levels that may be sufficient to elicit biologic responses through NGF receptors.

  3. Pharmacokinetics and biodistribution of a radioiodine labeled peptidomimetic ligand for high-affinity nerve growth factor receptors

    International Nuclear Information System (INIS)

    Jung, K. H.; Kim, D. H.; Paik, J. Y.; Koh, B. H.; Bae, J. S.; Choe, Y. S.; Lee, K. H.; Kim, B. T.

    2005-01-01

    Some of the obstacles for the clinical application of whole nerve growth factor (NGF) may be overcome by utilizing small molecule mimetics. We thus investigated the in vivo pharmacokinetics and biodistribution of a small cyclic peptide derived from NGF-[C(92-96)] with high receptor binding affinity. I-125 C(92-96) was labeled with the Bolton-Hunter method, and binding to TrkA/IgG chimeric protein was confirmed on a polyacrylamide gel after cross-linking. Pharmacokinetic analysis was performed in normal ICR mice intravenously injected with 0.5 MBq I-125 C(92-96) containing varying doses of C(92-96). Biodistribution studies were done at 6 h after injection. Cross-linkage analysis confirmed binding of I-125 C(92-96) to the high affinity NGF receptor, TrkA. Intravenously injected I-125 C(92-96) was cleared from the blood in a biexponential manner with an early T1/2α of 5.2 min and late T1/2β of 121.3 min. Log blood-concentration decreased over time with a k-slope of 0.0025, clearance of 11.8±0.5 ml/min, T1/2 of 4.1±0.4 hr, and volume of distribution of 69.7±4.6 ml. The pattern of elimination from the blood remained essentially unchanged regardless of the dose of added C(92-96), with dose-proportionate increases in AUCs and peak concentrations consistent with linear pharmacokinetics. Biodistribution studies demonstrated high kidney activity suggesting renal excretion of I-125 C(92-96). There were moderate levels of accumulation in the spleen, lungs and liver, followed by the myocardium and skeletal muscle, whereas brain uptake was low (< 0.2 %ID/gm). Intravenously administered C(92-96) follows linear pharmacokinetics, and is cleared from the circulation at a rate comparable to whole NGF despite its substantially smaller size. Although intravenous C(92-96) does not adequately reach brain tissue, clinically relevant doses can achieve major organ accumulation levels that may be sufficient to elicit biologic responses through NGF receptors

  4. [3H]naloxone as an opioid receptor label: Analysis of binding site heterogeneity and use for determination of opioid affinities of casomorphin analogues

    International Nuclear Information System (INIS)

    Schnittler, M.; Repke, H.; Liebmann, C.; Schrader, U.; Schulze, H.P.; Neubert, K.

    1990-01-01

    The nonselective antagonist [ 3 H]naloxone was used to identify opioid receptors in rat brain membranes. The multiple naloxone binding sites were related to different opioid receptors by means of selective opiod ligands as well as various β-casomorphin analogues. Analysis of binding site heterogeneity was performed using several computer curve fitting methods. The results indicate that structurally modified casomorphin peptides are able to discriminate between μ 1 and μ 2 binding sites. The affinities to the μ sites obtained with [ 3 H]naloxone as label are in a good agreement with those from experiments with the μ selective radioligand [ 3 H]DAGO. The μ 1 site affinities of these casomorphin derivatives are well correlated with their antinociceptive potencies. This finding suggests the mediation of the analgesic activity via the high-affinity μ 1 subtype. (author)

  5. Estrogen- and progesterone-receptor status in ECOG 2197: comparison of immunohistochemistry by local and central laboratories and quantitative reverse transcription polymerase chain reaction by central laboratory.

    Science.gov (United States)

    Badve, Sunil S; Baehner, Frederick L; Gray, Robert P; Childs, Barrett H; Maddala, Tara; Liu, Mei-Lan; Rowley, Steve C; Shak, Steven; Perez, Edith A; Perez, Edith D; Shulman, Lawrence J; Martino, Silvana; Davidson, Nancy E; Sledge, George W; Goldstein, Lori J; Sparano, Joseph A

    2008-05-20

    Central and local laboratory concordance for hormone receptor measurement is therapeutically important. This study compares estrogen receptor (ER) and progesterone receptor (PR) measured by local laboratory immunohistochemistry (IHC), central IHC, and central reverse-transcriptase polymerase chain reaction (RT-PCR) using a proprietary 21-gene assay. A case-control sample of 776 breast cancer patients from Eastern Cooperative Oncology Group (ECOG) study E2197 was evaluated. Central IHC Allred score for ER and PR was obtained using tissue microarrays and 1D5 ER antibody and 636 PR antibody. Quantitative RT-PCR for ER and PR in whole sections was performed using the 21-gene assay. For ER, the concordance between local and central IHC was 90% (95% CI, 88% to 92%), between local IHC and central RT-PCR was 91% (95% CI, 89% to 93%), and between central IHC and central RT-PCR was 93% (95% CI, 91% to 95%). For PR, the concordance between local IHC and central IHC was 84% (95% CI, 82% to 87%), between local IHC and central RT-PCR was 88% (95% CI, 85% to 90%), and between central IHC and central RT-PCR was 90% (95% CI, 88% to 92%). Although concordance was high, IHC ER-negative cases that were RT-PCR positive were more common than IHC ER-positive cases that were RT-PCR negative. In ER-positive patients, ER expression by central IHC Allred score was marginally associated with recurrence (P = .091), and ER expression by central RT-PCR was significantly associated with recurrence (P = .014). However, recurrence score, which incorporates additional genes/pathways, was a highly significant predictor of recurrence (P < .0001). There is a high degree of concordance among local IHC, central IHC, and central RT-PCR by the proprietary gene assay for ER and PR status. Although ER expression is marginally associated with relapse in ER-positive patients treated with chemohormonal therapy, recurrence score is a highly significant predictor of recurrence.

  6. The ketamine analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor.

    Directory of Open Access Journals (Sweden)

    Bryan L Roth

    Full Text Available In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as 'designer drugs' and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS-2-(ethylamino-2-(3-methoxyphenylcyclohexanone and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenylcyclohexanamine and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenylcyclohexyl]piperidine and 1-[1-(4-methoxyphenylcyclohexyl]piperidine, were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.

  7. Association of low-affinity FC gamma receptor 3B (FCGR3B) copy number variation with rheumatoid arthritis in Caucasian subjects

    NARCIS (Netherlands)

    Merriman, T.R.; Fanciulli, M.; Merriman, M.E.; Alizadeh, B.Z.; Koeleman, B.P.C.; Dalbeth, N.; Gow, P.; Harrison, A.A.; Highton, J.; Jones, P.B.; Stamp, L.K.; Steer, S.; Barrera, P.; Coenen, M.J.H.; Franke, B.; Vyse, T.; Aitman, T.; Radstake, T.; McKinney, C.

    2009-01-01

    Aim: There is increasing evidence that gene copy-number variation influences phenotypic variation. The low-affinity Fc receptor 3B (FCGR3B) is a copy-number polymorphic gene involved in the recruitment to sites of inflammation and activation of polymorphonuclear neutrophils (PMN). Given the

  8. Deficient p75 low-affinity neurotrophin receptor expression does alter the composition of cellular infiltrate in experimental autoimmune encephalomyelitis in C57BL/6 mice

    NARCIS (Netherlands)

    Kust, B; Mantingh-Otter, [No Value; Boddeke, E; Copray, S

    We have shown earlier that induction of experimental autoimmune encephalomyelitis (EAE)-a model for the human disease multiple sclerosis-in C5713L/6 wild-type mice resulted in the expression of the p75 low-affinity neurotrophin receptor (p75(NTR)) in endothelial cells in the CNS. In comparison to

  9. Expression of the low affinity neurotrophin receptor p75 in spinal motoneurons in a transgenic mouse model for amyotrophic lateral sclerosis

    NARCIS (Netherlands)

    Copray, JCVM; Jaarsma, D; Kust, BM; Bruggeman, RWG; Mantingh, [No Value; Brouwer, N; Boddeke, HWGM

    2003-01-01

    Amyotrophic lateral sclerosis is a lethal neurodegenerative disorder involving motoneuron loss in the cortex, brainstem and spinal cord, resulting in progressive paralysis. Aberrant neurotrophin signalling via the low affinity neurotrophin receptor p75 has been suggested to be involved in the

  10. The low-affinity neurotrophin receptor, p75, is upregulated in ganglioneuroblastoma/ganglioneuroma and reduces tumorigenicity of neuroblastoma cells in vivo

    NARCIS (Netherlands)

    Schulte, Johannes H.; Pentek, Falk; Hartmann, Wolfgang; Schramm, Alexander; Friedrichs, Nicolaus; Ora, Ingrid; Koster, Jan; Versteeg, Rogier; Kirfel, Jutta; Buettner, Reinhard; Eggert, Angelika

    2009-01-01

    Neuroblastoma, the most common extracranial tumor of childhood, is derived from neural crest progenitor cells that fail to differentiate along their predefined route to sympathetic neurons or sympatho-adrenergic adrenal cells. Although expression of the high-affinity neurotrophin receptors, TrkA and

  11. Plasma progesterone levels in progesterone treated cows

    International Nuclear Information System (INIS)

    Grosskopf, J.F.W.; Van Niekerk, C.H.; Morgenthal, J.C.

    1979-01-01

    A technique for the radioimmunoassay of progesterone in plasma is described. In one trial the oestrous cycles of four cycling cows and in another trial of one non-cycling cow and two cycling heifers were synchronized by the administration of progesterone. Each female received either 50 mg or 0,1 mg/kg of progesterone intramuscularly on alternate days in two courses of four and six injections respectively. Blood samples of the animals were collected either daily or two-daily before, over the entire period of treatment and for eight days after the last progesterone injection. The results of the progesterone assays are represented graphically for each individual cow or heifer. The plasma progesterone levels during treatment were maintained reasonably well at levels corresponding to those normally encountered during the luteal phase of the cycle. The progesterone levels, however, did not drop as rapidly as desired after the last injection but might have been influenced by a residual corpus luteum from a previous ovulation

  12. Embryonic exposure to the fungicide vinclozolin causes virilization of females and alteration of progesterone receptor expression in vivo: an experimental study in mice

    OpenAIRE

    Buckley, Jill; Willingham, Emily; Agras, Koray; Baskin, Laurence S

    2006-01-01

    Abstract Background Vinclozolin is a fungicide that has been reported to have anti-androgenic effects in rats. We have found that in utero exposure to natural or synthetic progesterones can induce hypospadias in mice, and that the synthetic progesterone medroxyprogesterone acetate (MPA) feminizes male and virilizes female genital tubercles. In the current work, we selected a relatively low dose of vinclozolin to examine its in utero effects on the development of the genital tubercle, both at ...

  13. Estradiol and progesterone regulate the expression of insulin-like growth factor-I receptor and insulin-like growth factor binding protein-2 in the hypothalamus of adult female rats.

    Science.gov (United States)

    Cardona-Gómez, G P; Chowen, J A; Garcia-Segura, L M

    2000-06-05

    Gonadal hormones interact with insulin-like growthfactor-I (IGF-I) to regulate synaptic plasticity during the estrous cycle in the rat mediobasal hypothalamus. It has been proposed that tanycytes, specialized glial cells lining the ventral region of the third ventricle, may regulate the availability of IGF-I to hypothalamic neurons. IGF-I levels in tanycytes fluctuate during the estrous cycle. Furthermore, estrogen administration to ovariectomized rats increases IGF-I levels in tanycytes, while progesterone, injected simultaneously with estrogen, blocks the estrogen-induced increase of IGF-I levels in tanycytes. To test whether hormonal regulation of IGF-I receptor (IGF-IR) and IGF binding protein-2 (IGFBP-2) may be involved in the accumulation of IGF-I in tanycytes, we assessed the effect of ovarian hormones on the levels of these molecules in the mediobasal hypothalamus of adult female rats. Ovariectomized animals were treated with either oil, estrogen, progesterone, or estrogen and progesterone simultaneously and then killed 6 or 24 h later. Some neurons, some astrocytes, and many tanycytes in the mediobasal hypothalamus were found by confocal microscopy to be immunoreactive for IGF-IR. IGFBP-2 immunoreactivity was restricted almost exclusively to tanycytes and ependymal cells and was colocalized with IGF-IR immunoreactivity in tanycytes. By electron microscope immunocytochemistry using colloidal gold labeling, IGF-IR and IGFBP-2 immunoreactivities were observed in the microvilli of tanycytes in the lumen of the third ventricle. IGF-IR and IGFBP-2 immunoreactive levels on the apical surface of tanycytes were significantly decreased by the administration of progesterone, either alone or in the presence of estradiol. IGF-IR levels in the mediobasal hypothalamus, measured by Western blotting, were not significantly affected by the separate administration of estradiol or progesterone to ovariectomized rats. However, the simultaneous administration of both hormones

  14. Radioiodsodestannylation. Convenient synthesis of a high affinity thromboxane A2/prostaglandin H2 receptor antagonist

    International Nuclear Information System (INIS)

    Mais, D.E.; Hamanaka, Nobuyuki

    1991-01-01

    Radioiodination of methyl-7-[(2R, 2S, 5R)-6,6-dimethyl-3-(4-trimethylstannylbenzenesulfononylamino3S) bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoate with [ 125 I] Na using a modification of the chloramine-T method in organic solvent is simple with high yields and site specific. The product, following hydrolysis of the ester, 7-[(2R, 2S, 3S, 5R)-6,6-dimethyl-3-(4[ 125 I]-iodobenzenesulfonylamino) bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid [( 125 I]-ISAP), was purified by HPLC. The high specific activity and specific binding will make the ligand a useful tool for the characterization of thromboxane A 2 /prostaglandin H 2 receptors. (author)

  15. [Regulatory effect and mechanism of RNA binding motif protein 38 on the expression of progesterone receptor in human breast cancer ZR-75-1 cells].

    Science.gov (United States)

    Lou, P P; Li, C L; Xia, T S; Shi, L; Wu, J; Zhou, X J; Wang, Y; Ding, Q

    2016-06-23

    To investigate the regulatory mechanism of RNA binding motif protein 38 (RNPC1) on the expression of progesterone receptor (PR) in breast cancer cell line ZR-75-1. Lentiviral vector was used to induce overexpression of RNPC1 in ZR-75-1 cells. qRT-PCR and Western blot were used to assess the regulatory effect of RNPC1 on PR expression. Actinomycin was used to detect the regulatory mechanism involved. Immunohistochemical (IHC) staining was used to determine the protein expression of RNPC1 and PR in 80 breast cancer tissues. IHC staining showed that the expression of RNPC1 was significantly higher in the PR positive breast cancer tissues than that in the PR negative breast cancer tissues (P<0.05). The qRT-PCR results showed that overexpression of RNPC1 in ZR-75-1 cells significantly upregulated the mRNA level of PR (1.764±0.028 vs. 1.001±0.037, P<0.01), whereas knockdown of RNPC1 did the opposite (0.579± 0.007 vs. 1.000±0.002, P<0.01). The Western blot results also showed that overexpression of RNPC1 up-regulated PR levels, while knockdown of RNPC1 resulted in down-regulation of PR levels in the ZR-75-1 cells.The actinomycin assay showed that overexpression of RNPC1 increased the mRNA stability of PR. The half-life of PR mRNA was increased from 4.0 h to 6.5 h. Knockdown of RNPC1 decreased the mRNA stability of PR and the half-life of PR transcript was decreased from 4.1 h to 3.0 h. RNPC1 plays a crucial role in regulating the expression of PR in breast cancer ZR-75-1 cells.

  16. A comparison of survival outcomes and side effects of toremifene or tamoxifen therapy in premenopausal estrogen and progesterone receptor positive breast cancer patients: a retrospective cohort study

    International Nuclear Information System (INIS)

    Gu, Ran; Long, Meijun; Chen, Kai; Chen, Lili; Xiao, Qiaozhen; Wu, Mei; Song, Erwei; Su, Fengxi; Jia, Weijuan; Zeng, Yunjie; Rao, Nanyan; Hu, Yue; Li, Shunrong; Wu, Jiannan; Jin, Liang; Chen, Lijuan

    2012-01-01

    In premenopausal women, endocrine adjuvant therapy for breast cancer primarily consists of tamoxifen alone or with ovarian suppressive strategies. Toremifene is a chlorinated derivative of tamoxifen, but with a superior risk-benefit profile. In this retrospective study, we sought to establish the role of toremifene as an endocrine therapy for premenopausal patients with estrogen and/or progesterone receptor positive breast cancer besides tamoxifen. Patients with early invasive breast cancer were selected from the breast tumor registries at the Sun Yat-Sen Memorial Hospital (China). Premenopausal patients with endocrine responsive breast cancer who underwent standard therapy and adjuvant therapy with toremifene or tamoxifen were considered eligible. Patients with breast sarcoma, carcinosarcoma, concurrent contralateral primary breast cancer, or with distant metastases at diagnosis, or those who had not undergone surgery and endocrine therapy were ineligible. Overall survival and recurrence-free survival were the primary outcomes measured. Toxicity data was also collected and compared between the two groups. Of the 810 patients reviewed, 452 patients were analyzed in the study: 240 received tamoxifen and 212 received toremifene. The median and mean follow up times were 50.8 and 57.3 months, respectively. Toremifene and tamoxifen yielded similar overall survival values, with 5-year overall survival rates of 100% and 98.4%, respectively (p = 0.087). However, recurrence-free survival was significantly better in the toremifene group than in the tamoxifen group (p = 0.022). Multivariate analysis showed that recurrence-free survival improved independently with toremifene (HR = 0.385, 95% CI = 0.154-0.961; p = 0.041). Toxicity was similar in the two treatment groups with no women experiencing severe complications, other than hot flashes, which was more frequent in the toremifene patients (p = 0.049). No patients developed endometrial cancer. Toremifene may be a valid and

  17. High-affinity α4β2 nicotinic receptors mediate the impairing effects of acute nicotine on contextual fear extinction.

    Science.gov (United States)

    Kutlu, Munir Gunes; Holliday, Erica; Gould, Thomas J

    2016-02-01

    Previously, studies from our lab have shown that while acute nicotine administered prior to training and testing enhances contextual fear conditioning, acute nicotine injections prior to extinction sessions impair extinction of contextual fear. Although there is also strong evidence showing that the acute nicotine's enhancing effects on contextual fear conditioning require high-affinity α4β2 nicotinic acetylcholine receptors (nAChRs), it is unknown which nAChR subtypes are involved in the acute nicotine-induced impairment of contextual fear extinction. In this study, we investigated the effects of acute nicotine administration on contextual fear extinction in knock-out (KO) mice lacking α4, β2 or α7 subtypes of nAChRs and their wild-type (WT) littermates. Both KO and WT mice were first trained and tested for contextual fear conditioning and received a daily contextual extinction session for 4 days. Subjects received intraperitoneal injections of nicotine (0.18 mg/kg) or saline 2-4 min prior to each extinction session. Our results showed that the mice that lack α4 and β2 subtypes of nAChRs showed normal contextual fear extinction but not the acute nicotine-induced impairment while the mice that lack the α7 subtype showed both normal contextual extinction and nicotine-induced impairment of contextual extinction. In addition, control experiments showed that acute nicotine-induced impairment of contextual fear extinction persisted when nicotine administration was ceased and repeated acute nicotine administrations alone did not induce freezing behavior in the absence of context-shock learning. These results clearly demonstrate that high-affinity α4β2 nAChRs are necessary for the effects of acute nicotine on contextual fear extinction. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Confirmation of 5p12 As a Susceptibility Locus for Progesterone-Receptor-Positive, Lower Grade Breast Cancer

    DEFF Research Database (Denmark)

    Milne, Roger L; Goode, Ellen L; García-Closas, Montserrat

    2011-01-01

    -negative disease; P(heterogeneity) = 2 × 10(-7)); heterogeneity by ER status was not observed (P = 0.2) once PR status was accounted for. The association was also stronger for lower grade tumors [per-allele OR (95% CI) = 1.20 (1.14-1.25), 1.13 (1.09-1.16), and 1.04 (0.99-1.08) for grade 1, 2, and 3/4, respectively......BACKGROUND: The single-nucleotide polymorphism (SNP) 5p12-rs10941679 has been found to be associated with risk of breast cancer, particularly estrogen receptor (ER)-positive disease. We aimed to further explore this association overall, and by tumor histopathology, in the Breast Cancer Association...... and histopathology were assessed using logistic regression. RESULTS: For white Europeans, the per-allele OR associated with 5p12-rs10941679 was 1.11 (95% CI = 1.08-1.14, P = 7 × 10(-18)) for invasive breast cancer and 1.10 (95% CI = 1.01-1.21, P = 0.03) for DCIS. For Asian women, the estimated OR for invasive...

  19. Crystal structure of an affinity-matured prolactin complexed to its dimerized receptor reveals the topology of hormone binding site 2

    DEFF Research Database (Denmark)

    Broutin, Isabelle; Jomain, Jean-Baptiste; Tallet, Estelle

    2010-01-01

    We report the first crystal structure of a 1:2 hormone.receptor complex that involves prolactin (PRL) as the ligand, at 3.8-A resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely relate...... and prostate cancer.......We report the first crystal structure of a 1:2 hormone.receptor complex that involves prolactin (PRL) as the ligand, at 3.8-A resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely related...... PRL receptor (PRLR) ligand. This structure allows one to draw up an exhaustive inventory of the residues involved at the PRL.PRLR site 2 interface, consistent with all previously reported site-directed mutagenesis data. We propose, with this description, an interaction model involving three structural...

  20. Estrogen receptor, progesterone receptor, HER-2, and response to postmastectomy radiotherapy in high-risk breast cancer: The Danish Breast Cancer Cooperative Group

    DEFF Research Database (Denmark)

    Kyndi, M.; Sorensen, F.B.; Overgaard, M.

    2008-01-01

    . End points were locoregional recurrence as isolated first event, distant metastases, and overall survival. For statistical analyses four subgroups were constructed from hormonal receptors (Rec). Rec+ was defined as ER+ and/or PgR+. Rec- as both ER- and PgR-. The four subgroups were Rec+/HER-2-, Rec......+/HER-2+, Rec-/HER-2- (triple negative), and Rec-/HER-2+. Results A significantly improved overall survival after PMRT was seen only among patients characterized by good prognostic markers such as hormonal receptor-positive and HER-2- patients (including the two Rec+ subtypes). No significant overall...... after PMRT were found for ER- and PgR- tumors compared with the ER+ and PgR+ tumors (P = .003 and 04, respectively), and for the triple-negative (P = .02), and the Rec-/HER-2+ subtypes (P = .003) compared with the Rec-/HER-2- subtype. Conclusion Hormonal receptor status, HER-2, and the constructed...

  1. Glucocorticoid up-regulation of high-affinity interleukin 6 receptors on human epithelial cells

    International Nuclear Information System (INIS)

    Snyers, L.; De Wit, L.; Content, J.

    1990-01-01

    Interleukin 6 (IL-6) is a potent pleiotropic cytokine, known, among others, to stimulate immunoglobulin production by B cells and to trigger acute-phase protein synthesis by hepatocytes. Similar to IL-1, it is produced by monocytes and macrophages following an inflammatory challenge. Analysis of IL-6 receptor (IL-6R) expression on different human cell lines indicates that dexamethasone could up-regulate the number of IL-6R on one epithelial cell line (UAC) and on two hepatoma cell lines (HepG2 and Hep3B). This effect was confirmed by Scatchard analysis of binding experiments, using [ 35 S]methionine and [ 35 S]cysteine metabolically labeled IL-6. It was confirmed at the level of mRNA expression by Northern blot analysis. These results provide evidence for a link between IL-6 and glucocorticoids. They could represent an example of a system in which one role of glucocorticoids is to define more accurately the target of cytokines, and they could explain, at least partly, the frequently observed synergy between IL-6 and glucocorticoids, notably in the case of hepatocytes

  2. Chiral dimethylamine flutamide derivatives-modeling, synthesis, androgen receptor affinities and carbon-11 labeling

    International Nuclear Information System (INIS)

    Jacobson, Orit; Laky, Desideriu; Carlson, Kathryn E.; Elgavish, Sharona; Gozin, Michael; Even-Sapir, Einat; Leibovitc, Ilan; Gutman, Mordechai; Chisin, Roland; Katzenellenbogen, John A.; Mishani, Eyal

    2006-01-01

    Most prostate cancers are androgen dependent upon initial diagnosis. On the other hand, some very aggressive forms of prostate cancer were shown to have lost the expression of the androgen receptor (AR). Although the AR is routinely targeted in endocrine treatment, the clinical outcome remains suboptimal. Therefore, it is crucial to demonstrate the presence and activity of the AR in each case of prostate cancer, before and after treatment. While noninvasive positron emission tomography (PET) has the potential to determine AR expression of tumor cells in vivo, fully optimized PET imaging agents are not yet available. Based on molecular modeling, three novel derivatives of hydroxyflutamide (Compounds 1-3) were designed and synthesized. They contain an electron-rich group (dimethylamine) located on the methyl moiety, which may confer a better stability to the molecule in vivo. Compounds 1-3 have AR binding that is similar or higher than that of the currently used commercial drugs. An automated carbon-11 radiolabeling route was developed, and the compounds were successfully labeled with a 10-15% decay-corrected radiochemical yield, 99% radiochemical purity and a specific activity of 4Ci/μmol end of bombardment (n=15). These labeled biomarkers may facilitate the future quantitative molecular imaging of AR-positive prostate cancer using PET and may also allow for image-guided treatment of prostate cancer

  3. Nuclear detection of Y-box protein-1 (YB-1) closely associates with progesterone receptor negativity and is a strong adverse survival factor in human breast cancer

    International Nuclear Information System (INIS)

    Dahl, Edgar; Dunn, Sandra E; Mertens, Peter R; En-Nia, Abdelaziz; Wiesmann, Frank; Krings, Renate; Djudjaj, Sonja; Breuer, Elisabeth; Fuchs, Thomas; Wild, Peter J; Hartmann, Arndt

    2009-01-01

    Y-box binding protein-1 (YB-1) is the prototypic member of the cold shock protein family that fulfills numerous cellular functions. In the nucleus YB-1 protein orchestrates transcription of proliferation-related genes, whereas in the cytoplasm it associates with mRNA and directs translation. In human tumor entities, such as breast, lung and prostate cancer, cellular YB-1 expression indicates poor clinical outcome, suggesting that YB-1 is an attractive marker to predict patients' prognosis and, potentially, is suitable to individualize treatment protocols. Given these predictive qualities of YB-1 detection we sought to establish a highly specific monoclonal antibody (Mab) for diagnostic testing and its characterization towards outcome prediction (relapse-free and overall survival). Hybridoma cell generation was carried out with recombinant YB-1 protein as immunogen and Mab characterization was performed using immunoblotting and ELISA with recombinant and tagged YB-1 proteins, as well as immunohistochemistry of healthy and breast cancer specimens. Breast tumor tissue array staining results were analyzed for correlations with receptor expression and outcome parameters. YB-1-specific Mab F-E2G5 associates with conformational binding epitopes mapping to two domains within the N-terminal half of the protein and detects nuclear YB-1 protein by immunohistochemistry in paraffin-embedded breast cancer tissues. Prognostic evaluation of Mab F-E2G5 was performed by immunohistochemistry of a human breast cancer tissue microarray comprising 179 invasive breast cancers, 8 ductal carcinoma in situ and 37 normal breast tissue samples. Nuclear YB-1 detection in human breast cancer cells was associated with poor overall survival (p = 0.0046). We observed a close correlation between nuclear YB-1 detection and absence of progesterone receptor expression (p = 0.002), indicating that nuclear YB-1 detection marks a specific subgroup of breast cancer. Likely due to limitation of sample

  4. Mid-aged and aged wild-type and progestin receptor knockout (PRKO) mice demonstrate rapid progesterone and 3alpha,5alpha-THP-facilitated lordosis.

    Science.gov (United States)

    Frye, C A; Sumida, K; Lydon, J P; O'Malley, B W; Pfaff, D W

    2006-05-01

    Progesterone (P) and its 5alpha-reduced metabolite, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP), facilitate sexual behavior of rodents via agonist-like actions at intracellular progestin receptors (PRs) and membrane GABA(A)/benzodiazepine receptor complexes (GBRs), respectively. Given that ovarian secretion of progestins declines with aging, whether or not senescent mice are responsive to progestins was of interest. Homozygous PR knockout (PRKO) or wild-type mice that were between 10-12 (mid-aged) or 20-24 (aged) months of age were administered P or 3alpha,5alpha-THP, and the effect on lordosis were examined. Effects of a progestin-priming regimen that enhances PR-mediated (experiment 1) or more rapid, PR-independent effects of progestins (experiments 2 and 3) on sexual behavior were examined. Levels of P, 3alpha,5alpha-THP, and muscimol binding were examined in tissues from aged mice (experiment 4). Wild-type, but not PRKO, mice were responsive when primed with 17beta-estradiol (E(2); 0.5 microg) and administered P (500 microg, subcutaneously). Mid-aged wild-type mice demonstrated greater increases in lordosis 6 h later compared to their pre-P, baseline test than did aged wild-type mice (experiment 1). Lordosis of younger and older wild-type, but not PRKO, mice was significantly increased within 5 min of intravenous (IV) administration of P (100 ng), compared with E(2)-priming alone (experiment 2). However, wild-type and PRKO mice demonstrated significant increases in lordosis 5 min after IV administration of 3alpha,5alpha-THP, an effect which was more pronounced in mid-aged than in aged animals (100 ng-experiment 3). In tissues from aged wild-type and PRKO mice, levels of P, 3alpha,5alpha-THP, and muscimol binding were increased by P administration (experiment 4). PR binding was lower in the cortex of PRKO than that of wild-type mice. Mid-aged and aged PRKO and wild-type mice demonstrated rapid P or 3alpha,5alpha-THP-facilitated lordosis that may be

  5. Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors

    International Nuclear Information System (INIS)

    Amagai, Yosuke; Tanaka, Akane; Ohmori, Keitaro; Matsuda, Hiroshi

    2008-01-01

    Much is known regarding participations of mast cells with innate and acquired immunity by secreting various cytokines and chemical mediators. However, details of mast cell biology still remain unclear. In this study, we successfully established a novel growth factor-independent mast cell line (MPT-1) derived from canine mast cell tumor. MPT-1 cells manifested factor-independent proliferation as floating cells containing a large amount of histamine, as well as chymase-like dog mast cell protease 3, in cytosolic granules. Particularly, MPT-1 cells expressed high-affinity IgE receptors (FcεRI) and wild-type c-kit receptors. Degranulation of MPT-1 cells was induced not only by stimulation with calcium ionophore but also by cross-linkage of the surface IgE. Given that MPT-1 is the first mast cell line with FcεRI which has no c-kit mutations, MPT-1 cells may provide great contribution for investigation of IgE-mediated activation mechanisms of mast cells, leading to development of effective treatment for allergic disorders

  6. Evaluation of estrogen receptor alpha and beta and progesterone receptor expression and correlation with clinicopathologic factors and proliferative marker Ki-67 in breast cancers

    DEFF Research Database (Denmark)

    Rosa, Fabíola E; Caldeira, José R F; Felipes, Joice

    2008-01-01

    To elucidate the molecular profile of hormonal steroid receptor status, we analyzed ER-alpha, ER-beta, and PGR mRNA and protein expression in 80 breast carcinomas using reverse transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR, and immunohistochemical analysis. Qualitative ana...

  7. Progesterone resistance in endometriosis: origins, consequences and interventions.

    Science.gov (United States)

    Patel, Bansari G; Rudnicki, Martin; Yu, Jie; Shu, Yimin; Taylor, Robert N

    2017-06-01

    Endometriosis is a common cause of pelvic pain and affects up to 10% of women of reproductive age. Aberrant progesterone signaling in the endometrium plays a significant role in impaired decidualization and establishment of ectopic endometrial implants. Eutopic endometrial cells from women with endometriosis fail to downregulate genes needed for decidualization, such as those involved in cell cycle regulation, leading to unbridled proliferation. Several causes of progesterone resistance in the endometrium have been postulated, including congenital "preconditioning", whereby the in utero environment renders infants susceptible to neonatal uterine bleeding and endometriosis. Progesterone action is crucial to decreasing inflammation in the endometrium, and deviant progesterone signaling results in a proinflammatory phenotype. Conversely, chronic inflammation can induce a progesterone-resistant state. Repetitive retrograde endometrial shedding begets chronic peritoneal inflammation, which further exacerbates progesterone resistance. Genetic causes of progesterone resistance include progesterone receptor gene polymorphisms, altered microRNA expression, and epigenetic modifications to progesterone receptors and their targets. Environmental toxins such as dioxin play a possible role in the genesis of endometriosis by permitting an inflammatory milieu. A consequence of impaired progesterone action is that hormonal therapy is rendered ineffective for a subset of women with endometriosis. Synthetic progestins, such as dienogest, may overcome this phenomenon by increasing progesterone receptor expression and decreasing proinflammatory cytokines. Other modalities include high dose depot formulations of progestins, medicated intrauterine devices and the likely advent of oral GnRH antagonists. Unearthing root causes of progesterone inaction in endometriosis will aid in the development of novel therapeutics geared toward prevention and treatment. © 2017 Nordic Federation of

  8. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    International Nuclear Information System (INIS)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N.; Moran, Jeffery H.; Prather, Paul L.

    2013-01-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB 1 Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB 2 Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB 2 Rs (hCB 2 Rs). The affinity of cannabinoids for hCB 2 Rs was determined by competition binding studies employing CHO-hCB 2 membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB 2 cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB 2 Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB 2 Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ 9 -tetrahydrocannabinol (Δ 9 -THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB 2 R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB 2 Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB 2 Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB 1 and CB 2 Rs. - Highlights: • JWH-018 and JWH-073 are synthetic cannabinoids present in abused K2

  9. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Moran, Jeffery H. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Arkansas Department of Public Health, Public Health Laboratory, Little Rock, AR 72205 (United States); Prather, Paul L., E-mail: pratherpaull@uams.edu [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States)

    2013-06-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018

  10. Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain.

    Science.gov (United States)

    Runge, Steffen; Schimmer, Susann; Oschmann, Jan; Schiødt, Christine Bruun; Knudsen, Sanne Möller; Jeppesen, Claus Bekker; Madsen, Kjeld; Lau, Jesper; Thøgersen, Henning; Rudolph, Rainer

    2007-05-15

    Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.

  11. The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity.

    Science.gov (United States)

    Abdiche, Yasmina Noubia; Yeung, Yik Andy; Chaparro-Riggers, Javier; Barman, Ishita; Strop, Pavel; Chin, Sherman Michael; Pham, Amber; Bolton, Gary; McDonough, Dan; Lindquist, Kevin; Pons, Jaume; Rajpal, Arvind

    2015-01-01

    The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.

  12. Estrogen receptor, progesterone receptor, HER-2, and response to postmastectomy radiotherapy in high-risk breast cancer: the Danish Breast Cancer Cooperative Group

    DEFF Research Database (Denmark)

    Kyndi, Marianne; Sørensen, Flemming Brandt; Knudsen, Helle

    2008-01-01

    . End points were locoregional recurrence as isolated first event, distant metastases, and overall survival. For statistical analyses four subgroups were constructed from hormonal receptors (Rec). Rec+ was defined as ER+ and/or PgR+. Rec-as both ER-and PgR-. The four subgroups were Rec+/HER-2-, Rec......+/HER-2+, Rec-/HER-2-(triple negative), and Rec-/HER-2+. RESULTS: A significantly improved overall survival after PMRT was seen only among patients characterized by good prognostic markers such as hormonal receptor-positive and HER-2- patients (including the two Rec+ subtypes). No significant overall...... after PMRT were found for ER-and PgR-tumors compared with the ER+ and PgR+ tumors (P = .003 and .04, respectively), and for the triple-negative (P = .02), and the Rec-/HER-2+ subtypes (P = .003) compared with the Rec+/HER-2-subtype. CONCLUSION: Hormonal receptor status, HER-2, and the constructed...

  13. Septide and neurokinin A are high-affinity ligands on the NK-1 receptor: evidence from homologous versus heterologous binding analysis.

    Science.gov (United States)

    Hastrup, H; Schwartz, T W

    1996-12-16

    The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK-1, NK-2 and NK-3 receptors. However, NKA also has actions which cannot be mediated through its normal NK-2 receptor and the synthetic peptide [pGlu6,Pro9]-Substance P9-11--called septide--is known to have tachykinin-like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK-1 receptor expressed in COS-7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK-1 receptor with high affinity: Kd values of 0.51 +/- 0.15 nM (NKA) and 0.55 +/- 0.03 nM (septide). It is concluded that NKA and septide are high-affinity ligands for the NK-1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.

  14. Technical note on the validation of a semi-automated image analysis software application for estrogen and progesterone receptor detection in breast cancer

    Science.gov (United States)

    2011-01-01

    Background The immunohistochemical detection of estrogen (ER) and progesterone (PR) receptors in breast cancer is routinely used for prognostic and predictive testing. Whole slide digitalization supported by dedicated software tools allows quantization of the image objects (e.g. cell membrane, nuclei) and an unbiased analysis of immunostaining results. Validation studies of image analysis applications for the detection of ER and PR in breast cancer specimens provided strong concordance between the pathologist's manual assessment of slides and scoring performed using different software applications. Methods The effectiveness of two connected semi-automated image analysis software (NuclearQuant v. 1.13 application for Pannoramic™ Viewer v. 1.14) for determination of ER and PR status in formalin-fixed paraffin embedded breast cancer specimens immunostained with the automated Leica Bond Max system was studied. First the detection algorithm was calibrated to the scores provided an independent assessors (pathologist), using selected areas from 38 small digital slides (created from 16 cases) containing a mean number of 195 cells. Each cell was manually marked and scored according to the Allred-system combining frequency and intensity scores. The performance of the calibrated algorithm was tested on 16 cases (14 invasive ductal carcinoma, 2 invasive lobular carcinoma) against the pathologist's manual scoring of digital slides. Results The detection was calibrated to 87 percent object detection agreement and almost perfect Total Score agreement (Cohen's kappa 0.859, quadratic weighted kappa 0.986) from slight or moderate agreement at the start of the study, using the un-calibrated algorithm. The performance of the application was tested against the pathologist's manual scoring of digital slides on 53 regions of interest of 16 ER and PR slides covering all positivity ranges, and the quadratic weighted kappa provided almost perfect agreement (κ = 0.981) among the two

  15. Critical biological parameters modulate affinity as a determinant of function in T-cell receptor gene-modified T-cells.

    Science.gov (United States)

    Spear, Timothy T; Wang, Yuan; Foley, Kendra C; Murray, David C; Scurti, Gina M; Simms, Patricia E; Garrett-Mayer, Elizabeth; Hellman, Lance M; Baker, Brian M; Nishimura, Michael I

    2017-11-01

    T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

  16. Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface

    DEFF Research Database (Denmark)

    Ahring, Philip K.; Olsen, Jeppe A.; Nielsen, Elsebet O.

    2015-01-01

    The nicotinic acetylcholine receptor alpha 4 beta 2 is important for normal mammalian brain function and is known to express in two different stoichiometries, (alpha 4)(2)(beta 2)(3) and (alpha 4)(3)(beta 2)(2). While these are similar in many aspects, the (alpha 4)(3)(beta 2)(2) stoichiometry...... differs by harboring a third orthosteric acetylcholine binding site located at the alpha 4-alpha 4 interface. Interestingly, the third binding site has, so far, only been documented using electrophysiological assays, actual binding affinities of nicotinic receptor ligands to this site are not known....... The present study was therefore aimed at determining binding affinities of nicotinic ligands to the alpha 4-alpha 4 interface. Given that epibatidine shows large functional potency differences at alpha 4-beta 2 vs. alpha 4-alpha 4 interfaces, biphasic binding properties would be expected at (alpha 4)(3)(beta...

  17. Radioiodinated ligands for the estrogen receptor: Effect of different 7-cyanoalkyl chains on the binding affinity of novel iodovinyl-6-dehydroestradiols

    International Nuclear Information System (INIS)

    Neto, Carina; Oliveira, Maria Cristina; Gano, Lurdes; Marques, Fernanda; Santos, Isabel; Morais, Goreti Ribeiro; Yasuda, Takumi; Thiemann, Thies; Botelho, Filomena; Oliveira, Carlos F.

    2009-01-01

    Three novel 17α-ethynyl-Δ 6,7 -estra-3,17β-diols and their 17α-[ 125 I]-iodovinyl derivatives, containing different C7-cyanoalkyl chains, were studied as potential radioligands for the estrogen receptor. The influence of the chain length on the biological behaviour of the compounds was assessed through in vitro ER binding assays of the ethynyl derivatives and breast cancer cell uptake studies of the 17α-[ 125 I]-iodovinyl-Δ 6,7 -estra-3,17β-diols. A difference in alkyl chain induced a decrease in ER binding affinities of substances, however, the receptor-binding affinities (RBA) of all compounds were lower than that of estradiol itself. In addition, a non-specific cell binding was observed which is in accordance with the encountered ethynyl RBA values suggesting that the uptake is not ER mediated

  18. Progesterone Exerts a Neuromodulatory Effect on Turning Behavior of Hemiparkinsonian Male Rats: Expression of 3α-Hydroxysteroid Oxidoreductase and Allopregnanolone as Suggestive of GABAA Receptors Involvement

    Directory of Open Access Journals (Sweden)

    Roberto Yunes

    2015-01-01

    Full Text Available There is a growing amount of evidence for a neuroprotective role of progesterone and its neuroactive metabolite, allopregnanolone, in animal models of neurodegenerative diseases. By using a model of hemiparkinsonism in male rats, injection of the neurotoxic 6-OHDA in left striatum, we studied progesterone’s effects on rotational behavior induced by amphetamine or apomorphine. Also, in order to find potential explanatory mechanisms, we studied expression and activity of nigrostriatal 3α-hydroxysteroid oxidoreductase, the enzyme that catalyzes progesterone to its active metabolite allopregnanolone. Coherently, we tested allopregnanolone for a possible neuromodulatory effect on rotational behavior. Also, since allopregnanolone is known as a GABAA modulator, we finally examined the action of GABAA antagonist bicuculline. We found that progesterone, in addition to an apparent neuroprotective effect, also increased ipsilateral expression and activity of 3α-hydroxysteroid oxidoreductase. It was interesting to note that ipsilateral administration of allopregnanolone reversed a clear sign of motor neurodegeneration, that is, contralateral rotational behavior. A possible GABAA involvement modulated by allopregnanolone was shown by the blocking effect of bicuculline. Our results suggest that early administration of progesterone possibly activates genomic mechanisms that promote neuroprotection subchronically. This, in turn, could be partially mediated by fast, nongenomic, actions of allopregnanolone acting as an acute modulator of GABAergic transmission.

  19. Progesterone for premenstrual syndrome

    NARCIS (Netherlands)

    Ford, Olive; Lethaby, Anne; Roberts, Helen; Mol, Ben Willem J.

    2009-01-01

    BACKGROUND: About 5% of women experience severe symptoms called premenstrual syndrome (PMS), only in the two weeks before their menstrual periods. Treatment with progesterone may restore a deficiency, balance menstrual hormone levels or reduce effects of falling progesterone levels on the brain or

  20. Progesterone for premenstrual syndrome

    NARCIS (Netherlands)

    Ford, Olive; Lethaby, Anne; Roberts, Helen; Mol, Ben Willem J.

    2012-01-01

    Background About 5% of women experience severe symptoms called premenstrual syndrome (PMS), only in the two weeks before their menstrual periods. Treatment with progesterone may restore a deficiency, balance menstrual hormone levels or reduce effects of falling progesterone levels on the brain or on

  1. Re-Appraisal of Estrogen Receptor Negative/Progesterone Receptor Positive (ER-/PR+) Breast Cancer Phenotype: True Subtype or Technical Artefact?

    Science.gov (United States)

    Foley, Niamh M; Coll, J M; Lowery, A J; Hynes, S O; Kerin, M J; Sheehan, M; Brodie, C; Sweeney, K J

    2017-09-11

    Expression of the ER and PR receptors is routinely quantified in breast cancer as a predictive marker of response to hormonal therapy. Accurate determination of ER and PR status is critical to the optimal selection of patients for targeted therapy. The existence of an ER-/PR+ subtype is controversial, with debate centred on whether this represents a true phenotype or a technical artefact on immunohistochemistry (IHC). The aim of this study was to investigate the true incidence and clinico-pathological features of ER-/PR+ breast cancers in a tertiary referral symptomatic breast unit. Clinico-pathological data were collected on invasive breast cancers diagnosed between 1995 and 2005. IHC for ER and PR receptors was repeated on all cases which were ER-/PR+, with the same paraffin block used for the initial diagnostic testing. Concordance between the diagnostic and repeat IHC was determined using validated testing. Complete data, including ER and PR status were available for 697 patients diagnosed during the study period. On diagnostic IHC, the immunophenotype of the breast tumours was: ER+/PR+ in 396 (57%), ER-/PR- in 157 (23%), ER+/PR- in 88 (12%) and ER-/PR+ in 56 (8.6%) patients. On repeat IHC of 48/56 ER-/PR+ tumours 45.8% were ER+/PR+, 6% were ER+/PR- and 43.7% were ER-/PR- None of the cases were confirmed to be ER-/PR+. The ER-/PR+ phenotypic breast cancer is likely to be the result of technical artefact. Prompt reassessment of patients originally assigned to this subtype who re-present with symptoms should be considered to ensure appropriate clinical management.

  2. A DFT and semiempirical model-based study of opioid receptor affinity and selectivity in a group of molecules with a morphine structural core.

    Science.gov (United States)

    Bruna-Larenas, Tamara; Gómez-Jeria, Juan S

    2012-01-01

    We report the results of a search for model-based relationships between mu, delta, and kappa opioid receptor binding affinity and molecular structure for a group of molecules having in common a morphine structural core. The wave functions and local reactivity indices were obtained at the ZINDO/1 and B3LYP/6-31G(∗∗) levels of theory for comparison. New developments in the expression for the drug-receptor interaction energy expression allowed several local atomic reactivity indices to be included, such as local electronic chemical potential, local hardness, and local electrophilicity. These indices, together with a new proposal for the ordering of the independent variables, were incorporated in the statistical study. We found and discussed several statistically significant relationships for mu, delta, and kappa opioid receptor binding affinity at both levels of theory. Some of the new local reactivity indices incorporated in the theory appear in several equations for the first time in the history of model-based equations. Interaction pharmacophores were generated for mu, delta, and kappa receptors. We discuss possible differences regulating binding and selectivity in opioid receptor subtypes. This study, contrarily to the statistically backed ones, is able to provide a microscopic insight of the mechanisms involved in the binding process.

  3. A DFT and Semiempirical Model-Based Study of Opioid Receptor Affinity and Selectivity in a Group of Molecules with a Morphine Structural Core

    Directory of Open Access Journals (Sweden)

    Tamara Bruna-Larenas

    2012-01-01

    Full Text Available We report the results of a search for model-based relationships between mu, delta, and kappa opioid receptor binding affinity and molecular structure for a group of molecules having in common a morphine structural core. The wave functions and local reactivity indices were obtained at the ZINDO/1 and B3LYP/6-31 levels of theory for comparison. New developments in the expression for the drug-receptor interaction energy expression allowed several local atomic reactivity indices to be included, such as local electronic chemical potential, local hardness, and local electrophilicity. These indices, together with a new proposal for the ordering of the independent variables, were incorporated in the statistical study. We found and discussed several statistically significant relationships for mu, delta, and kappa opioid receptor binding affinity at both levels of theory. Some of the new local reactivity indices incorporated in the theory appear in several equations for the first time in the history of model-based equations. Interaction pharmacophores were generated for mu, delta, and kappa receptors. We discuss possible differences regulating binding and selectivity in opioid receptor subtypes. This study, contrarily to the statistically backed ones, is able to provide a microscopic insight of the mechanisms involved in the binding process.

  4. Progesterone resistance in endometriosis

    DEFF Research Database (Denmark)

    Patel, Bansari G; Rudnicki, Martin; Yu, Jie

    2017-01-01

    Endometriosis is a common cause of pelvic pain and affects up to 10% of women of reproductive age. Aberrant progesterone signaling in the endometrium plays a significant role in impaired decidualization and establishment of ectopic endometrial implants. Eutopic endometrial cells from women...... with endometriosis fail to downregulate genes needed for decidualization, such as those involved in cell cycle regulation, leading to unbridled proliferation. Several causes of progesterone resistance in the endometrium have been postulated, including congenital "preconditioning", whereby the in utero environment...... renders infants susceptible to neonatal uterine bleeding and endometriosis. Progesterone action is crucial to decreasing inflammation in the endometrium, and deviant progesterone signaling results in a proinflammatory phenotype. Conversely, chronic inflammation can induce a progesterone resistant state...

  5. Clobazam and its active metabolite N-desmethylclobazam display significantly greater affinities for α₂- versus α₁-GABA(A-receptor complexes.

    Directory of Open Access Journals (Sweden)

    Henrik Sindal Jensen

    Full Text Available Clobazam (CLB, a 1,5-benzodiazepine (BZD, was FDA-approved in October 2011 for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS in patients 2 years and older. BZDs exert various CNS effects through allosteric modulation of GABAA receptors. The structurally distinct, 1,4-BZD clonazepam (CLN is also approved to treat LGS. The precise mechanisms of action and clinical efficacy of both are unknown. Data show that the GABAA α₁-subunit-selective compound zolpidem [ZOL] exhibits hypnotic/sedative effects. Conversely, data from knock-in mice carrying BZD binding site mutations suggest that the α₂ subunit mediates anticonvulsant effects, without sedative actions. Hence, the specific pattern of interactions across the GABAA receptor complexes of BZDs might be reflected in their clinical efficacies and adverse effect profiles. In this study, GABAA-receptor binding affinities of CLB, N-desmethylclobazam (N-CLB, the major metabolite of CLB, CLN, and ZOL were characterized with native receptors from rat-brain homogenates and on cloned receptors from HEK293 cells transfected with combinations of α (α₁, α₂, α₃, or α₅, β₂, and γ₂ subtypes. Our results demonstrate that CLB and N-CLB have significantly greater binding affinities for α₂- vs. α₁-receptor complexes, a difference not observed for CLN, for which no distinction between α₂ and α₁ receptors was observed. Our experiments with ZOL confirmed the high preference for α₁ receptors. These results provide potential clues to a new understanding of the pharmacologic modes of action of CLB and N-CLB.

  6. Synthetic Polymer Affinity Ligand for Bacillus thuringiensis ( Bt) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the Bt Protein-Insect Receptor Binding Mechanism.

    Science.gov (United States)

    Liu, Mingming; Huang, Rong; Weisman, Adam; Yu, Xiaoyang; Lee, Shih-Hui; Chen, Yalu; Huang, Chao; Hu, Senhua; Chen, Xiuhua; Tan, Wenfeng; Liu, Fan; Chen, Hao; Shea, Kenneth J

    2018-05-24

    We report a novel strategy for creating abiotic Bacillus thuringiensis ( Bt) protein affinity ligands by biomimicry of the recognition process that takes place between Bt Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R 1 proteins. Guided by this strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and screened for binding to three epitopes 280 FRGSAQGIEGS 290 , 368 RRPFNIGINNQQ 379 and 436 FRSGFSNSSVSIIR 449 located in loop α8, loop 2 and loop 3 of domain II of Bt Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was found to have high affinity to one of the epitopes, 368 RRPFNIGINNQQ 379 . This same NP also had specific binding ability to both Bt Cry1Ab and Bt Cry1Ac, proteins that share the same epitope, but very low affinity to Bt Cry2A, Bt Cry1C and Bt Cry1F closely related proteins that lack epitope homology. To locate possible NP- Bt Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the binding of 865 NITIHITDTNNK 876 , a specific recognition site in insect receptor Bt-R 1 , to 368 RRPFNIGINNQQ 379 . The inhibition by NP12 reached as high as 84%, indicating that NP12 binds to Bt Cry1Ab/Ac proteins mainly via 368 RRPFNIGINNQQ 379 . This epitope region was then utilized as a "target" or "bait" for the separation and concentration of Bt Cry1Ac protein from the extract of transgenic Bt cotton leaves by NP12. This strategy, based on the antigen-receptor recognition mechanism, can be extended to other biotoxins and pathogen proteins when designing biomimic alternatives to natural protein affinity ligands.

  7. beta-Arrestin 1 and 2 stabilize the angiotensin II type I receptor in distinct high-affinity conformations

    DEFF Research Database (Denmark)

    Sanni, S J; Hansen, J T; Bonde, M M

    2010-01-01

    The angiotensin II type 1 (AT(1)) receptor belongs to family A of 7 transmembrane (7TM) receptors. The receptor has important roles in the cardiovascular system and is commonly used as a drug target in cardiovascular diseases. Interaction of 7TM receptors with G proteins or beta-arrestins often...

  8. Radioimmunoassay of plasma progesterone

    Energy Technology Data Exchange (ETDEWEB)

    Langer, L; Veleminsky, J [Institute of Clinical Endocrinology, Lubochna (Czechoslovakia); Hampl, R; Starka, L [Vyzkumny Ustav Endokrinologicky, Prague (Czechoslovakia); Holan, J [Comenius Univ., School of Medicine, Martin (Czechoslovakia). Dept. of Physics and Nuclear Medicine

    1978-06-30

    A simple modification of plasma progesterone radioimmunoassay is described. 11..cap alpha..-Hydroxyprogesterone hemisuccinate - BSA conjugate was used as an immunogen. (1,2,6,7-H-3) Progesterone specific radioactivity 82 Ci.mmol/sup -1/ was purchased from Radiochemical Centre Amersham (England). The method has been applied for the analysis of more than 2000 plasma samples. The typical fluctuation of progesterone in plasma during the menstrual cycle, using data obtained with this method is illustrated. The reliability criteria of the method are given.

  9. Fundamental considerations in the design of fluorine-18 labeled progestins and androgens as imaging agents for receptor-positive tumors of the breast and prostate

    International Nuclear Information System (INIS)

    Brandes, S.J.; Katzenellenbogen, J.A.

    1988-01-01

    A review is given of the structural and functional features which are important in the design and development of imaging agents for the progesterone receptor (PR) and the androgen receptor (AR) directed towards imaging receptor-positive tumors in the breast and prostate respectively. In particular the effects of various substituents on the biological activities and homologous receptor binding of progesterone, testosterone, nortestosterone and dihydrotestosterone are discussed. The effect of fluorine substitution on the affinities of progestins and androgens for their respective receptors is described. Other ligand systems that have high affinity for AR and PR and which may provide good bases for the design of fluorine-substituted imaging agents are also discussed. Finally, previous studies with radiolabelled progestins and androgens are described. (U.K.)

  10. Mutation-induced quisqualic acid and ibotenic acid affinity at the metabotropic glutamate receptor subtype 4: ligand selectivity results from a synergy of several amino acid residues

    DEFF Research Database (Denmark)

    Hermit, Mette B; Greenwood, Jeremy R; Bräuner-Osborne, Hans

    2004-01-01

    The metabotropic glutamate receptors (mGluRs) are key modulators of excitatory neurotransmission in the central nervous system. The eight mGluR subtypes are seven trans-membrane-spanning proteins that possess a large extracellular amino-terminal domain in which the endogenous ligand binding pocket...... resides. In this study, we have identified four non-conserved amino acid residues that are essential for differentiating mGluR1 from mGluR4. Our approach has been to increase the affinity of the classic mGluR1 agonists, quisqualic acid and ibotenic acid, at mGluR4 by making various point mutations......, the mutations K74Y and K317R induced dramatic triple-order-of-magnitude increases in the affinity of ibotenic acid at mGluR4, making the affinity equivalent to that of mGluR1. Furthermore, the affinity of quisqualic acid at mGluR4 was increased to the same level as mGluR1 by the two double mutations, K74Y/K317R...

  11. Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

    Science.gov (United States)

    Dunn, Steven M.; Rizkallah, Pierre J.; Baston, Emma; Mahon, Tara; Cameron, Brian; Moysey, Ruth; Gao, Feng; Sami, Malkit; Boulter, Jonathan; Li, Yi; Jakobsen, Bent K.

    2006-01-01

    The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential. PMID:16600963

  12. Fluoxetine, a selective inhibitor of serotonin uptake, potentiates morphine analgesia without altering its discriminative stimulus properties or affinity for opioid receptors

    International Nuclear Information System (INIS)

    Hynes, M.D.; Lochner, M.A.; Bemis, K.G.; Hymson, D.L.

    1985-01-01

    The analgesic effect of morphine in the rat tail jerk assay was enhanced by the serotonin uptake inhibitor, fluoxetine. Tail jerk latency was not affected by fluoxetine alone. Morphine's affinity for opioid receptors labeled in vitro with 3 H-naloxone or 3 H-D-Ala 2 -D-Leu 5 -enkephalin was not altered by fluoxetine, which has no affinity for these sites at concentrations as high as 1000 nM. In rats trained to discriminate morphine from saline, fluoxetine at doses of 5 or 10 mg/kg were recognized as saline. Increasing the fluoxetine dose to 20 mg/kg did not result in generalization to either saline or morphine. The dose response curve for morphine generalization was not significantly altered by fluoxetine doses of 5 or 10 mg/kg. Those rats treated with the combination of morphine and 20 mg/kg of fluoxetine did not exhibit saline or morphine appropriate responding. Fluoxetine potentiates the analgesic properties of morphine without enhancing its affinity for opioid receptors or its discriminative stimulus properties. 30 references, 2 figures, 2 tables

  13. Synthesis, modelling, and mu-opioid receptor affinity of N-3(9)-arylpropenyl-N-9(3)-propionyl-3,9-diazabicycl.

    Science.gov (United States)

    Pinna, G A; Murineddu, G; Curzu, M M; Villa, S; Vianello, P; Borea, P A; Gessi, S; Toma, L; Colombo, D; Cignarella, G

    2000-08-01

    A series of N-3-arylpropenyl-N-9-propionyl-3,9-diazabicyclo[3.3.1]nonanes (1a-g) and of reverted N-3-propionyl-N-9-arylpropenyl isomers (2a-g), as homologues of the previously reported analgesic 3,8-diazabicyclo[3.2.1]octanes (I-II), were synthesized and evaluated for the binding affinity towards opioid receptor subtypes mu, delta and kappa. Compounds 1a-g and 2a-g exhibited a strong selective mu-affinity with Ki values in the nanomolar range, which favourably compared with those of I and II. In addition, contrary to the trend observed for DBO-I, II, the mu-affinity of series 2 is markedly higher than that of the isomeric series 1. This aspect was discussed on the basis of the conformational studies performed on DBN which allowed hypotheses on the mode of interaction of these compounds with the mu receptor.

  14. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains

    DEFF Research Database (Denmark)

    Behrendt, N; Ronne, E; Dano, K

    1996-01-01

    by chemical cross-linking, but quantitative binding/competition studies showed that the apparent ligand affinity was 100- to 1000-fold lower than that of the intact suPAR. This loss of affinity was comparable with the loss found after cleavage between the first domain (D1) and D(2 + 3), using chymotrypsin...

  15. Involvement of direct inhibition of NMDA receptors in the effects of sigma-receptor ligands on glutamate neurotoxicity in vitro.

    Science.gov (United States)

    Nishikawa, H; Hashino, A; Kume, T; Katsuki, H; Kaneko, S; Akaike, A

    2000-09-15

    This study was performed to examine the roles of the N-methyl-D-aspartate (NMDA) receptor/phencyclidine (PCP) channel complex in the protective effects of sigma-receptor ligands against glutamate neurotoxicity in cultured cortical neurons derived from fetal rats. A 1-h exposure of cultures to glutamate caused a marked loss of viability, as determined by Trypan blue exclusion. This acute neurotoxicity of glutamate was prevented by NMDA receptor antagonists. Expression of sigma(1) receptor mRNA in cortical cultures was confirmed by reverse transcription polymerase chain reaction (RT-PCR). sigma Receptor ligands with affinity for NMDA receptor channels including the PCP site, such as (+)-N-allylnormetazocine ((+)-SKF10,047), haloperidol, and R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane ((-)-PPAP), prevented glutamate neurotoxicity in a concentration-dependent manner. In contrast, other sigma-receptor ligands without affinity for NMDA receptors, such as carbetapentane and R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine ((+)-3-PPP), did not show neuroprotective effects. Putative endogenous sigma receptor ligands such as pregnenolone, progesterone, and dehydroepiandrosterone did not affect glutamate neurotoxicity. The protective effects of (+)-SKF10,047, haloperidol, and (-)-PPAP were not affected by the sigma(1) receptor antagonist rimcazole. These results suggested that a direct interaction with NMDA receptors but not with sigma receptors plays a crucial role in the neuroprotective effects of sigma receptor ligands with affinity for NMDA receptors.

  16. Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a highly immunogenic phenotype.

    Science.gov (United States)

    Decker, T; Schneller, F; Kronschnabl, M; Dechow, T; Lipford, G B; Wagner, H; Peschel, C

    2000-05-01

    CpG-oligodeoxynucleotides (CpG-ODN) have been shown to induce proliferation, cytokine production, and surface molecule regulation in normal and malignant human B cells. In the present study, we investigated the potential of CpG-ODN to induce functional high-affinity receptors in leukemic and normal B cells and the effects of costimulation with IL-2 on proliferation, cytokine secretion, and surface molecule regulation. Highly purified B cells from B-CLL patients and normal controls were stimulated with CpG-ODN with or without IL-2. Expression of CD25 was determined using FACS, and the presence of high-affinity IL-2 receptors was determined by scatchard analysis. Costimulatory effects of IL-2 and CpG-ODN were investigated using proliferation assays, ELISA (IL-6, TNF-alpha), and FACS analysis (CD80, CD86 expression). Reactivity of autologous and allogeneic T cells toward activated B-CLL cells was determined in mixed lymphocyte reactions and Interferon-gamma Elispot assays. The CpG-ODN DSP30 caused a significantly stronger induction of the IL-2 receptor alpha chain in malignant as compared with normal B cells (p = 0.03). This resulted in the expression of functional high-affinity IL-2 receptors in B-CLL cells, but fewer numbers of receptors with less affinity were expressed in normal B cells. Although addition of IL-2 to CpG-ODN-stimulated cells augmented proliferation in both normal B cells and B-CLL cells, no costimulatory effect on cytokine production or surface molecule expression could be observed in normal B cells. In contrast, TNF-alpha and IL-6 production was increased in B-CLL cells, and the expression of CD80 and CD86 was further enhanced when IL-2 was used as a costimulus. Autologous and allogeneic immune recognition of B-CLL cells stimulated with CpG-ODN and IL-2 was increased compared with B-CLL cells stimulated with CpG-ODN alone. Stimulation of B-CLL cells with CpG-ODN and IL-2 might be an attractive strategy for potential immunotherapies for B

  17. Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells

    Directory of Open Access Journals (Sweden)

    Chad M. Williams

    2017-07-01

    Full Text Available The discovery of naturally occurring T cell receptors (TCRs that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds rather than synergy (total CD8 cooperation alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our

  18. Study of progesterone mechanisms in radio-induced apoptosis prevention

    International Nuclear Information System (INIS)

    Vares, G.

    2004-10-01

    The purpose of this work was to study the modulation of radiation-induced cell death of human mammary tumoral cells by progesterone. On the one hand, we observed that progesterone protects cells against radiation-induced apoptosis and increases the proportion of surviving and proliferating damaged cells. On the other hand, a transcriptome analysis was undertaken in irradiated cells treated by progesterone, using DNA micro-arrays. This let us highlight several transcriptional dis-regulations that are likely to explain the protective effect of the hormone; in particular, we showed that progesterone regulates the expression of genes implicated in apoptosis signaling by death receptors. Knowing the crucial role of hormonal control and of apoptosis regulation in breast cancer initiation, our results may help to achieve a better understanding of the implication of progesterone in mammary carcinogenesis. (author)

  19. Affinity and selectivity of PD156707, a novel nonpeptide endothelin antagonist, for human ET(A) and ET(B) receptors.

    Science.gov (United States)

    Maguire, J J; Kuc, R E; Davenport, A P

    1997-02-01

    We have determined the affinity and selectivity of a new nonpeptide antagonist PD156707 (sodium 2-benzo(1,3ioxol-5-yl-4-(4-methoxy-pheny l)-4-oxo-3-(3,4,5-trime tho xybenzyl)-but-2-enoate) for human endothelin (ET)(A) and ET(B) receptors. In human coronary artery and saphenous vein the affinity of the ET(A) receptor for PD156707 was 0.15 +/- 0.06 nM and 0.5 +/- 0.13 nM, respectively. Competition experiments in human left ventricle and kidney revealed that PD156707 had 1,000- to 15,000-fold selectivity for the ET(A) receptor over the ET(B) receptor. This selectivity was confirmed autoradiographically. In human coronary artery, mammary artery and saphenous vein PD156707 (3-300 nM) potently antagonized the vasoconstrictor responses to ET-1. The pA2 values estimated from the Gaddum-Schild equation were 8.07 +/- 0.09, 8.45 +/- 0.11 and 8.70 +/- 0.13, respectively. The concentration-response curves to ET-1 were shifted to the right in parallel fashion, without reduction of the maximum response. However, the regression lines fitted to the resulting Schild data deviated significantly from one. PD156707 appeared to be a more effective antagonist at lower concentrations than at the higher ones. It is possible that PD156707, a sodium salt, was reverting to a less soluble form which results in underestimation of its potency. These data show that PD156707 is a potent and selective antagonist at human ET(A) receptors and will be useful in clarifying the role of the endothelin peptides in human cardiovascular disease.

  20. MiRNA-513a-5p inhibits progesterone receptor expression and constitutes a risk factor for breast cancer: the hOrmone and Diet in the ETiology of breast cancer prospective study.

    Science.gov (United States)

    Muti, Paola; Donzelli, Sara; Sacconi, Andrea; Hossain, Ahmed; Ganci, Federica; Frixa, Tania; Sieri, Sabina; Krogh, Vittorio; Berrino, Franco; Biagioni, Francesca; Strano, Sabrina; Beyene, Joseph; Yarden, Yosef; Blandino, Giovanni

    2018-02-09

    MicroRNAs (miRNAs) might be considered both predictors and players of cancer development. The aim of the present report was to investigate whether many years before the diagnosis of breast cancer miRNA expression is already disregulated. In order to test this hypothesis, we compared miRNAs extracted from leukocytes in healthy women who later developed breast cancer and in women who remain healthy during the whole 15-year follow-up time. Accordantly, we used a case-control study design nested in the hOrmone and Diet in the ETiology of breast cancer (ORDET) prospective cohort study addressing the possibility that miRNAs can serve as both early biomarkers and components of the hormonal etiological pathways leading to breast cancer development in premenopausal women. We compared leukocyte miRNA profiles of 191 incident premenopausal breast cancer cases and profiles of 191 women who remained healthy over a follow-up period of 20 years. The analysis identified 20 differentially expressed miRNAs in women candidate to develop breast cancer versus control women. The upregulated miRNAs, miR-513-a-5p, miR-513b-5p and miR-513c-5p were among the most significantly deregulated miRNAs. In multivariate analysis, miR-513a-5p upregulation was directly and statistically significant associated with breast cancer risk (OR = 1.69; 95% CI 1.08-2.64; P = 0.0293). In addition, the upregulation of miR-513-a-5p displayed the strongest direct association with serum progesterone and testosterone levels. The experimental data corroborated the inhibitory function of miR-513a-5p on progesterone receptor expression confirming that progesterone receptor is a target of miR-513a-5p. The identification of upregulated miR-513a-5p with its oncogenic potential further validates the use of miRNAs as long-term biomarker of breast cancer risk. © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Effects of the 3β-hydroxysteroid dehydrogenase inhibitor trilostane on luteal progesterone production in the dog

    NARCIS (Netherlands)

    de Gier, J.; Wolthers, C.H.; Galac, S.; Schaefers-Okkens, A.C.; Kooistra, H.S.

    2011-01-01

    Abstract Interference with the pregnancy-maintaining influence of progesterone is the basis of most methods for termination of unwanted pregnancy in dogs. The currently available methods are based on induction of luteolysis or blocking of the progesterone receptor. Inhibition of progesterone

  2. In vivo measurement of haloperidol affinity to dopamine D2/D3 receptors by [123I]IBZM and single photon emission computed tomography

    DEFF Research Database (Denmark)

    Videbaek, C; Toska, K; Friberg, L

    2001-01-01

    This study examines the feasibility of a steady-state bolus-integration method with the dopamine D2/D3 receptor single photon emission computer tomography (SPECT) tracer, [123I]IBZM, for determination of in vivo affinity of haloperidol. The nonspecific binding of [123I]IBZM was examined in the rat...... brain by infusion of haloperidol to plasma levels approximately 100 times the Kd level in man. In humans, Kd for haloperidol binding was measured in four healthy volunteers that were examined twice: once with partial dopamine D2/D3 receptor blockade obtained by a scheduled infusion of unlabeled...... haloperidol (0.7 mg total dosage), and once in an unblocked state. Blood sampling and SPECT were performed intermittently during 6 hours after intravenous [123I]IBZM bolus injection. Plasma [123I]IBZM was determined by octane extraction. Plasma haloperidol was determined by a radioimmunoassay, and plasma...

  3. Two classes of astrocytes in the adult human and pig retina in terms of their expression of high affinity NGF receptor (TrkA).

    Science.gov (United States)

    Ruiz-Ederra, Javier; Hitchcock, Peter F; Vecino, Elena

    2003-02-13

    Astrocytes have been implicated in axon guidance and synaptic regeneration in the retina and these processes involve activation of the high affinity nerve growth factor receptor, known as the tyrosine kinase A (TrkA) receptor. The purpose of the present study was to characterize the expression of TrkA in astrocytes of the adult pig and human retina. To this end, sections of human and pig retinas were immunolabeled with a combination of antibodies to glial fibrillary acidic protein (GFAP) and TrkA. Our study revealed that most of the GFAP-positive cells express TrkA, whereas a rare, novel subpopulation of astrocytes was found to be devoid of TrkA. Our results support the idea that astrocytes play an important neurotrophic role in the retina.

  4. Pharmacodynamics and safety of the novel selective progesterone receptor modulator vilaprisan: a double-blind, randomized, placebo-controlled phase 1 trial in healthy women.

    Science.gov (United States)

    Schütt, Barbara; Kaiser, Andreas; Schultze-Mosgau, Marcus-Hillert; Seitz, Christian; Bell, David; Koch, Manuela; Rohde, Beate

    2016-08-01

    Does administration of vilaprisan (VPR) to healthy women for 12 weeks reduce menstrual bleeding? In this 12-week proof-of-concept phase 1 trial, most women (30/33, 90%) who received VPR at daily doses of 1-5 mg reported the absence of menstrual bleeding. Vilaprisan (BAY 1002670) is a novel, highly potent selective progesterone receptor modulator that markedly reduces the growth of human leiomyoma tissue in a preclinical model of uterine fibroids (UFs). In this double-blind, parallel-group study, of the 163 healthy women enrolled 73 were randomized to daily VPR 0.1 mg (n = 12), 0.5 mg (n = 12), 1 mg (n = 13), 2 mg (n = 12), 5 mg (n = 12) or placebo tablets (n = 12) for 12 weeks. Participants were followed up until the start of the second menstrual bleeding after the end of treatment. Trial simulations were used to determine the minimum sample size required to estimate the non-bleeding rate (i.e. self-assessed bleeding intensity of 'none' or 'spotting') using Bayesian dose-response estimation with incorporated prior information. It was estimated that 48 participants in the per-protocol analysis population would be sufficient. Women aged 18-45 years who had been sterilized by tubal ligation were enrolled between November 2011 and May 2012. Participants kept a daily diary of bleeding intensity. Blood and urine samples were taken, and transvaginal ultrasound was performed before treatment, during treatment and follow-up. Endometrial biopsies were obtained during the pretreatment cycle, at the end of the treatment period and during the follow-up phase. The primary outcome was the estimated dose-response curve of the observed non-bleeding rate during Days 10-84 of treatment, excluding the endometrial biopsy day and 2 days after biopsy. Secondary outcomes included return of bleeding during follow-up, size of follicle-like structures and serum hormone levels. Safety assessments included adverse events (AEs), endometrial thickness and histology, laboratory parameters, vital

  5. N- and C-terminally truncated forms of glucose-dependent insulinotropic polypeptide are high-affinity competitive antagonists of the human GIP receptor

    DEFF Research Database (Denmark)

    Hansen, L S; Sparre-Ulrich, A H; Christensen, M.

    2016-01-01

    functions and pharmacological potential. GIP(1-30)NH2 is a naturally occurring truncation of GIP(1-42). Here we characterize eight N-terminal trrncations of human GIP(1-30)NH2 : GIP(2- to 9-30)NH2 . EXPERIMENTAL APPROACH: COS-7 cells were transiently transfected with the human GIP receptor and assessed...... displayed lower affinities (Ki 2.3-347 nM) with highest affinities of GIP(3-30)NH2 and (5-30)NH2 . Agonism was only observed for GIP(1-30)NH2 with an Emax on 100% of GIP(1-42) and GIP(2-30)NH2 (Emax 20%). GIP(2- to 9-30)NH2 displayed antagonism (IC50 12-450 nM) and right-shifts of the GIP(1-42)-response......, but superior antagonist GIP(3-30)NH2 , that together with GIP(5-30)NH2 were high-affinity competitive antagonist and thus may be suitable tool compounds for basic GIP research and future pharmacological interventions....

  6. Rodent Models of Non-classical Progesterone Action Regulating Ovulation

    Directory of Open Access Journals (Sweden)

    Melinda A. Mittelman-Smith

    2017-07-01

    Full Text Available It is becoming clear that steroid hormones act not only by binding to nuclear receptors that associate with specific response elements in the nucleus but also by binding to receptors on the cell membrane. In this newly discovered manner, steroid hormones can initiate intracellular signaling cascades which elicit rapid effects such as release of internal calcium stores and activation of kinases. We have learned much about the translocation and signaling of steroid hormone receptors from investigations into estrogen receptor α, which can be trafficked to, and signal from, the cell membrane. It is now clear that progesterone (P4 can also elicit effects that cannot be exclusively explained by transcriptional changes. Similar to E2 and its receptors, P4 can initiate signaling at the cell membrane, both through progesterone receptor and via a host of newly discovered membrane receptors (e.g., membrane progesterone receptors, progesterone receptor membrane components. This review discusses the parallels between neurotransmitter-like E2 action and the more recently investigated non-classical P4 signaling, in the context of reproductive behaviors in the rodent.

  7. Correlation of breast cancer subtypes, based on estrogen receptor, progesterone receptor, and HER2, with functional imaging parameters from {sup 68}Ga-RGD PET/CT and {sup 18}F-FDG PET/CT

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Hai-Jeon [Seoul National University College of Medicine, Department of Nuclear Medicine, Seoul (Korea, Republic of); Seoul National University College of Medicine, The Institute of Radiation Medicine, Seoul (Korea, Republic of); Ewha Womans University School of Medicine, Department of Nuclear Medicine, Seoul (Korea, Republic of); Kang, Keon Wook; Jeong, Jae Min; Chung, June-Key [Seoul National University College of Medicine, Department of Nuclear Medicine, Seoul (Korea, Republic of); Seoul National University College of Medicine, Department of Biomedical Sciences, Seoul (Korea, Republic of); Seoul National University College of Medicine, The Institute of Radiation Medicine, Seoul (Korea, Republic of); Seoul National University, Cancer Research Institute, Seoul (Korea, Republic of); Chun, In Kook [Seoul National University College of Medicine, Department of Nuclear Medicine, Seoul (Korea, Republic of); Kangwon National University Hospital, Department of Nuclear Medicine, Chuncheon, Kangwon-Do (Korea, Republic of); Cho, Nariya [Seoul National University College of Medicine, Department of Radiology, Jongno-gu, Seoul (Korea, Republic of); Im, Seock-Ah [Seoul National University College of Medicine, Department of Internal Medicine, Seoul (Korea, Republic of); Jeong, Sunjoo [Dankook University, Department of Molecular Biology, Yongin (Korea, Republic of); Lee, Song [Seoul National University College of Medicine, Department of Nuclear Medicine, Seoul (Korea, Republic of); Seoul National University College of Medicine, The Institute of Radiation Medicine, Seoul (Korea, Republic of); Jung, Kyeong Cheon [Seoul National University College of Medicine, Department of Pathology, Seoul (Korea, Republic of); Lee, Yun-Sang [Seoul National University College of Medicine, Department of Nuclear Medicine, Seoul (Korea, Republic of); Seoul National University College of Medicine, Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul (Korea, Republic of); Lee, Dong Soo [Seoul National University College of Medicine, Department of Nuclear Medicine, Seoul (Korea, Republic of); Seoul National University College of Medicine, The Institute of Radiation Medicine, Seoul (Korea, Republic of); Seoul National University, Department of Molecular Medicine and Biopharmaceutical Sciences, Seoul (Korea, Republic of); Moon, Woo Kyung [Seoul National University College of Medicine, Department of Radiology, Jongno-gu, Seoul (Korea, Republic of); Seoul National University College of Medicine, Department of Biomedical Sciences, Seoul (Korea, Republic of); Seoul National University College of Medicine, The Institute of Radiation Medicine, Seoul (Korea, Republic of)

    2014-08-15

    Imaging biomarkers from functional imaging modalities were assessed as potential surrogate markers of disease status. Specifically, in this prospective study, we investigated the relationships between functional imaging parameters and histological prognostic factors and breast cancer subtypes. In total, 43 patients with large or locally advanced invasive ductal carcinoma (IDC) were analyzed (47.6 ± 7.5 years old). {sup 68}Ga-Labeled arginine-glycine-aspartic acid (RGD) and {sup 18}F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) were performed. The maximum and average standardized uptake values (SUV{sub max} and SUV{sub avg}) from RGD PET/CT and SUV{sub max} and SUV{sub avg} from FDG PET/CT were the imaging parameters used. For histological prognostic factors, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression was identified using immunohistochemistry (IHC) or fluorescent in situ hybridization (FISH). Four breast cancer subtypes, based on ER/PR and HER2 expression (ER/PR+,Her2-, ER/PR+,Her2+, ER/PR-,Her2+, and ER/PR-,Her2-), were considered. Quantitative FDG PET parameters were significantly higher in the ER-negative group (15.88 ± 8.73 vs 10.48 ± 6.01, p = 0.02 for SUV{sub max}; 9.40 ± 5.19 vs 5.92 ± 4.09, p = 0.02 for SUV{sub avg}) and the PR-negative group (8.37 ± 4.94 vs 4.79 ± 3.93, p = 0.03 for SUV{sub avg}). Quantitative RGD PET parameters were significantly higher in the HER2-positive group (2.42 ± 0.59 vs 2.90 ± 0.75, p = 0.04 for SUV{sub max}; 1.60 ± 0.38 vs 1.95 ± 0.53, p = 0.04 for SUV{sub avg}) and showed a significant positive correlation with the HER2/CEP17 ratio (r = 0.38, p = 0.03 for SUV{sub max} and r = 0.46, p < 0.01 for SUV{sub avg}). FDG PET parameters showed significantly higher values in the ER/PR-,Her2- subgroup versus the ER/PR+,Her2- or ER/PR+,Her2+ subgroups, while RGD PET parameters showed significantly lower values in the ER

  8. Decreased endogenous progesterone and ratio of progesterone to ...

    African Journals Online (AJOL)

    Progesterone and estrogen are two steroid hormones whose exposure may decrease the risk and delay the onset of ischemic stroke. The main objective of this study was to determine the plasma level of progesterone, estrogen and ratio of progesterone/estrogen in ischemic stroke patients. The plasma levels of ...

  9. Large-scale overproduction, functional purification and ligand affinities of the His-tagged human histamine H1 receptor.

    NARCIS (Netherlands)

    Ratnala, V.R.; Swarts, H.G.P.; Oostrum, J. van; Leurs, R.; Groot, H.J.M. de; Bakker, R.; Grip, W.J. de

    2004-01-01

    This report describes an efficient strategy for amplified functional purification of the human H1 receptor after heterologous expression in Sf9 cells. The cDNA encoding a C-terminally histidine-tagged (10xHis) human histamine H1 receptor was used to generate recombinant baculovirus in a Spodoptera

  10. Synthesis, biodistribution and in vitro evaluation of brain permeable high affinity type 2 cannabinoid receptor agonists [11C]MA2 and [18F]MA3

    Directory of Open Access Journals (Sweden)

    Muneer Ahamed

    2016-09-01

    Full Text Available Abstract The type 2 cannabinoid receptor (CB2 is a member of the endocannabinoid system and is known for its important role in (neuroinflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([11C]MA2 and a fluorine-18 ([18F]MA3 labeled analogue of a highly potent N-arylamide oxadiazole CB2 agonist (EC50 = 0.015 nM. MA2 and MA3 behaved as potent CB2 agonist (EC50: 3 nM and 0.1 nM, respectively and their in vitro binding affinity for hCB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group was found to have higher binding affinity and EC50 values when compared to the originally reported trifluoromethyl analogue 12. [11C]MA2 and [18F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [11C]MA2 and [18F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted.

  11. Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3.

    Science.gov (United States)

    Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M

    2016-01-01

    The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([ 11 C]MA2) and a fluorine-18 ([ 18 F]MA3) labeled analog of a highly potent N -arylamide oxadiazole CB2 agonist (EC 50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC 50 : 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for h CB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC 50 values when compared to the originally reported trifluoromethyl analog 12 . [ 11 C]MA2 and [ 18 F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [ 11 C]MA2 and [ 18 F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted.

  12. Concomitant alteration in number and affinity of P2X and muscarinic receptors are associated with bladder dysfunction in early stage of diabetic rats.

    Science.gov (United States)

    Yoshizawa, Tsuyoshi; Hayashi, Yukio; Yoshida, Akira; Yoshida, Shohei; Ito, Yoshihiko; Yamaguchi, Kenya; Yamada, Shizuo; Takahashi, Satoru

    2018-03-01

    To investigate time course of bladder dysfunction and concurrent changes in number and affinity of the muscarinic and P 2 X receptor in the early stage of streptozotocin (STZ)-induced diabetic rats. Diabetic rats were prepared by the intraperitoneal injection of 50 mg/kg of STZ to 7-week-old female Wistar rats. We performed recording of 24-h voiding behavior and cystometry at 1, 4, 8, and 12 weeks after the induction of diabetes. A muscle strip experiments with electrical field stimulation (EFS), carbachol, and α,β-methylene adenosine 5'-triphosphate (α,β-MeATP) were also performed at the same time-points. Additionally, concurrent changes in number and affinity of bladder muscarinic and P 2 X receptor were measured by a radioreceptor assay using [N-methyl- 3 H] scopolamine methyl chloride ([ 3 H]NMS) and α,β-methylene-ATP (2,8- 3 H) tetrasodium salt ([ 3 H]α,β-MeATP). In STZ-induced diabetic rats, polydipsic polyuric pollakiuria were noted on recording of 24-h voiding behavior from early stage. Also, the residual urine volume markedly increased in diabetic rats on cystometry. In the muscle strip experiment, the detrusor contractions induced by EFS, carbachol, and α,β-MeATP were enhanced in STZ-induced diabetic rats. Based on the radioreceptor assay, the maximum number of sites (Bmax) for the specific binding of [ 3 H]NMS and [ 3 H]α,β-MeATP was concurrently increased in the bladder from diabetic rats. Increased bladder contractility is found in early stage of diabetic rats. Then, bladder dysfunction is associated with increased number of muscarinic and P 2 X receptors in STZ-induced diabetic rats.

  13. Quantitative analysis of multiple kappa-opioid receptors by selective and nonselective ligand binding in guinea pig spinal cord: Resolution of high and low affinity states of the kappa 2 receptors by a computerized model-fitting technique

    International Nuclear Information System (INIS)

    Tiberi, M.; Magnan, J.

    1990-01-01

    The binding characteristics of selective and nonselective opioids have been studied in whole guinea pig spinal cord, using a computer fitting method to analyze the data obtained from saturation and competition studies. The delineation of specific binding sites labeled by the mu-selective opioid [3H]D-Ala2,MePhe4,Gly-ol5-enkephalin (Kd = 2.58 nM, R = 4.52 pmol/g of tissue) and by the delta-selective opioid [3H]D-Pen2, D-Pen5-enkephalin (Kd = 2.02 nM, R = 1.47 pmol/g of tissue) suggests the presence of mu and delta-receptors in the spinal cord tissue. The presence of kappa receptors was probed by the kappa-selective opioid [3H]U69593 (Kd = 3.31 nM, R = 2.00 pmol/g of tissue). The pharmacological characterization of the sites labeled by [3H]U69593 confirms the assumption that this ligand discriminates kappa receptors in guinea pig spinal cord. The benzomorphan [3H]ethylketazocine labels a population of receptors with one homogeneous affinity state (Kd = 0.65 nM, R = 7.39 pmol/g of tissue). The total binding capacity of this ligand was not different from the sum of the binding capacities of mu, delta-, and kappa-selective ligands. Under mu- and delta-suppressed conditions, [3H]ethylketazocine still binds to receptors with one homogeneous affinity state (Kd = 0.45 nM, R = 1.69 pmol/g of tissue). Competition studies performed against the binding of [3H]ethylketazocine under these experimental conditions reveal that the pharmacological profile of the radiolabeled receptors is similar to the profile of the kappa receptors labeled with [3H]U69593. Saturation studies using the nonselective opioid [3H]bremazocine demonstrate that this ligand binds to spinal cord membranes with heterogeneous affinities (Kd1 = 0.28 nM, R1 = 7.91 pmol/g of tissue; Kd2 = 3.24 nM, R2 = 11.2 pmol/g of tissue)

  14. Quantitative analysis of multiple kappa-opioid receptors by selective and nonselective ligand binding in guinea pig spinal cord: Resolution of high and low affinity states of the kappa 2 receptors by a computerized model-fitting technique

    Energy Technology Data Exchange (ETDEWEB)

    Tiberi, M.; Magnan, J. (Universite de Montreal, Quebec (Canada))

    1990-05-01

    The binding characteristics of selective and nonselective opioids have been studied in whole guinea pig spinal cord, using a computer fitting method to analyze the data obtained from saturation and competition studies. The delineation of specific binding sites labeled by the mu-selective opioid (3H)D-Ala2,MePhe4,Gly-ol5-enkephalin (Kd = 2.58 nM, R = 4.52 pmol/g of tissue) and by the delta-selective opioid (3H)D-Pen2, D-Pen5-enkephalin (Kd = 2.02 nM, R = 1.47 pmol/g of tissue) suggests the presence of mu and delta-receptors in the spinal cord tissue. The presence of kappa receptors was probed by the kappa-selective opioid (3H)U69593 (Kd = 3.31 nM, R = 2.00 pmol/g of tissue). The pharmacological characterization of the sites labeled by (3H)U69593 confirms the assumption that this ligand discriminates kappa receptors in guinea pig spinal cord. The benzomorphan (3H)ethylketazocine labels a population of receptors with one homogeneous affinity state (Kd = 0.65 nM, R = 7.39 pmol/g of tissue). The total binding capacity of this ligand was not different from the sum of the binding capacities of mu, delta-, and kappa-selective ligands. Under mu- and delta-suppressed conditions, (3H)ethylketazocine still binds to receptors with one homogeneous affinity state (Kd = 0.45 nM, R = 1.69 pmol/g of tissue). Competition studies performed against the binding of (3H)ethylketazocine under these experimental conditions reveal that the pharmacological profile of the radiolabeled receptors is similar to the profile of the kappa receptors labeled with (3H)U69593. Saturation studies using the nonselective opioid (3H)bremazocine demonstrate that this ligand binds to spinal cord membranes with heterogeneous affinities (Kd1 = 0.28 nM, R1 = 7.91 pmol/g of tissue; Kd2 = 3.24 nM, R2 = 11.2 pmol/g of tissue).

  15. Androgen and Progesterone Receptors Are Targets for Bisphenol A (BPA, 4-Methyl-2,4-bis-(P-HydroxyphenylPent-1-Ene--A Potent Metabolite of BPA, and 4-Tert-Octylphenol: A Computational Insight.

    Directory of Open Access Journals (Sweden)

    Mohd Rehan

    Full Text Available Exposure to toxic industrial chemicals that have capacity to disrupt the endocrine system, also known as endocrine disrupting chemicals (EDCs, has been increasingly associated with reproductive problems in human population. Bisphenol A (BPA; 4,4'-(propane-2,2-diyldiphenol and 4-tert-octylphenol (OP; 4-(1,1,3,3-tetramethylbutylphenol are among the most common environmental contaminants possessing endocrine disruption properties and are present in plastics, epoxy resins, detergents and other commercial products of common personal and industrial use. A metabolite of BPA, 4-Methyl-2,4-bis(4-hydroxyphenylpent-1-ene (MBP is about 1000 times more biologically active compared to BPA. Epidemiological, clinical, and experimental studies have shown association of BPA and OP with adverse effects on male and female reproductive system in human and animals. The endocrine disruption activity can occur through multiple pathways including binding to steroid receptors. Androgen receptor (AR and progesterone receptor (PR are critical for reproductive tract growth and function. Structural binding characterization of BPA, MBP, and OP with AR and PR using molecular docking simulation approaches revealed novel interactions of BPA with PR, and MBP and OP with AR and PR. For BPA, MBP, and OP, five AR interacting residues Leu-701, Leu-704, Asn-705, Met-742, and Phe-764 overlapped with those of native AR ligand testosterone, and four PR interacting residues Leu-715, Leu-718, Met-756, and Met-759 overlapped with those of PR co-complex ligand, norethindrone. For both the receptors the binding strength of MBP was maximum among the three compounds. Thus, these compounds have the potential to block or interfere in the binding of the endogenous native AR and PR ligands and, hence, resulting in dysfunction. The knowledge of the key interactions and the important amino-acid residues also allows better prediction of potential of xenobiotic molecules for disrupting AR- and PR

  16. Mitogen-Activated Protein Kinase 8 (MAP3K8) Mediates the Signaling Pathway of Estradiol Stimulating Progesterone Production Through G Protein-Coupled Receptor 30 (GPR30) in Mouse Corpus Luteum.

    Science.gov (United States)

    Liu, Ying; Li, Yueqin; Zhang, Di; Liu, Jiali; Gou, Kemian; Cui, Sheng

    2015-05-01

    The corpus luteum (CL) is a transient endocrine gland developed from the ovulated follicles, and the most important function is to synthesize and secrete progesterone (P(4)), a key hormone to maintain normal pregnancy and estrous cycle in most mammals. It is known that estrogen has a vital role in stimulating P(4) synthesis in CL, but it still remains unclear about the mechanism of estradiol (E(2)) regulating P(4) production in CL. Our results here first show that all of the CL cells express MAPK 8 (MAP3K8), and the MAP3K8 level is much higher at the midstage than at the early and late stages during CL development. The further functional studies show that the forced inhibition of endogenous MAP3K8 by using MAP3K8 small interfering RNA and MAP3K8 signaling inhibitor (MAP3K8i) in the luteal cells significantly block the P(4) synthesis and neutralize the enhancing effect of E(2) on P(4) production in the CL. In addition, our results here demonstrate that the stimulating effect of E(2) on P(4) synthesis relies on the estrogen no-classical protein-coupled receptor 30, and MAP3K8 is involved in mediating the protein-coupled receptor 30signaling of E(2) affecting P(4) synthesis via stimulating ERK phosphorylation. These novel findings are critical for our understanding the ovary physiology and pathological mechanism.

  17. Effect of hyperthyroidism on circulating prolactin and hypothalamic expression of tyrosine hydroxylase, prolactin signaling cascade members and estrogen and progesterone receptors during late pregnancy and lactation in the rat.

    Science.gov (United States)

    Pennacchio, Gisela E; Neira, Flavia J; Soaje, Marta; Jahn, Graciela A; Valdez, Susana R

    2017-02-15

    Hyperthyroidism (HyperT) compromises pregnancy and lactation, hindering suckling-induced PRL release. We studied the effect of HyperT on hypothalamic mRNA (RT-qPCR) and protein (Western blot) expression of tyrosine hydroxylase (TH), PRL receptor (PRLR) and signaling pathway members, estrogen-α (ERα) and progesterone (PR) receptors on late pregnancy (days G19, 20 and 21) and early lactation (L2) in rats. HyperT advanced pre-partum PRL release, reduced circulating PRL on L2 and increased TH mRNA (G21 and L2), p-TH, PRLR mRNA, STAT5 protein (G19 and L2), PRLR protein (G21) and CIS protein (G19). PRs mRNAs and protein decreased on G19 but afterwards PRA mRNA (G20), PRB mRNA (G21) and PRA mRNA and protein (L2) increased. ERα protein increased on G19 and decreased on G20. Thus, the altered hypothalamic PRLR, STAT5, PR and ERα expression in hyperthyroid rats may induce elevated TH expression and activation, that consequently, elevate dopaminergic tone during lactation, blunting suckling-induced PRL release and litter growth. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

    Directory of Open Access Journals (Sweden)

    Yuichi Ikeda

    Full Text Available Identification of cognate ligands for G protein-coupled receptors (GPCRs provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS. In a reporter cell, complementary fragments of β-lactamase (α and ω were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω, and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3. We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR and neuromedin B receptor (NMBR. Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

  19. Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently cross-link to galactosyl receptors on isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Herzig, M.C.S.; Weigel, P.H.

    1989-01-01

    The authors have developed chemical affinity reagents for the hepatic galactosyl receptor. Asialoorosomucoid (ASOR) was derivatized with five homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers. NHS/ASOR derivatives were synthesized, purified, and applied within 10 min to isolated rat hepatocytes at 4 degree C. Specific binding of these 125 I-labeled derivatives was ∼90% in the presence of either EGTA or excess ASOR. Specific cross-linking assessed by the resistance of specifically bound NHS/ 125 I-ASOR to release by EGTA, was 50-75% of the specifically bound ligand. The extent of specific cross-linking correlated with the average number of NHS groups per ASOR and was controlled by varying the molar ratio of cross-linker to ASOR during the synthesis. After being cross-linked with any of the NHS/ 125 I-ASOR derivatives, cells were washed with EGTA, solubilized in Triton X-100, and analyzed by SDA-PAGE and autoradiography. They conclude that all three receptor subunits can cross-link to ligand. They propose a model in which the native receptor is a heterohexamer composed of four subunits of RHL 1 and two subunits of RHL 2 and/or RHL 3

  20. Glioblastoma chemotherapy adjunct via potent serotonin receptor-7 inhibition using currently marketed high-affinity antipsychotic medicines

    Science.gov (United States)

    Kast, RE

    2010-01-01

    Glioblastoma treatment as now constituted offers increased survival measured in months over untreated patients. Because glioblastomas are active in synthesizing a bewildering variety of growth factors, a systematic approach to inhibiting these is being undertaken as treatment adjunct. The serotonin 7 receptor is commonly overexpressed in glioblastoma. Research documentation showing agonists at serotonin receptor 7 cause increased extracellular regulated kinase 1/2 activation, increased interleukin-6 synthesis, increased signal transducer and activator of transcription-3 activation, increased resistance to apoptosis and other growth enhancing changes in glioblastoma is reviewed in this paper. Because three drugs in wide use to treat thought disorders – paliperidone, pimozide and risperidone – are also potent and well-tolerated inhibitors at serotonin receptor 7, these drugs should be studied for growth factor deprivation in an adjunctive role in glioblastoma treatment. PMID:20880389

  1. Synthesis, binding affinity at glutamic acid receptors, neuroprotective effects, and molecular modeling investigation of novel dihydroisoxazole amino acids

    DEFF Research Database (Denmark)

    Conti, Paola; De Amici, Marco; Grazioso, Giovanni

    2005-01-01

    stereoisomers of the bicyclic analogue 5-amino-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-3,5-dicarboxylic acid (+)-2, (-)-2, (+)-3, and (-)-3 were tested at ionotropic and metabotropic glutamate receptor subtypes. The most potent NMDA receptor antagonists [(+)-2, (-)-4, and (+)-5] showed a significant......The four stereoisomers of 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acid(+)-4, (-)-4, (+)-5, and (-)-5 were prepared by stereoselective synthesis of two pairs of enantiomers, which were subsequently resolved by enzymatic procedures. These four stereoisomers and the four...

  2. New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics.

    Science.gov (United States)

    Zeilinger, Markus; Pichler, Florian; Nics, Lukas; Wadsak, Wolfgang; Spreitzer, Helmut; Hacker, Marcus; Mitterhauser, Markus

    2017-12-01

    Resolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A 3 R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the K i of eight different A 3 R antagonists, using CHO-K1 cells stably expressing the hA 3 R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants k on and k off , as well as the dedicated K d of the A 3 R agonist [ 125 I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds. Two novel real-time cell-binding approaches were successfully developed and established. Both experimental procedures were found to visualize the kinetic binding characteristics with high spatial and temporal resolution, resulting in reliable affinity values, which are in good agreement with values previously reported with traditional methods. Taking into account the lipophilicity of the A 3 R antagonists, no influences on the experimental performance and the resulting affinity were investigated. Both kinetic binding approaches comprise tracer administration and subsequent binding to living cells, expressing the dedicated target protein. Therefore, the experiments resemble better the true in vivo physiological conditions and provide important markers of cellular feedback and biological response.

  3. An efficient analytical platform for on line microfluidic profiling of neurotoxic snake venoms towards nicotinic receptor like affinity.

    NARCIS (Netherlands)

    Heus, F.A.H.; Vonk, F.; Otvos, R.A.; Bruyneel, B.; Smit, A.B.; Lingeman, H.; Richardson, M.; Niessen, W.M.A.; Kool, J.

    2013-01-01

    Venomous snakes have evolved their efficient venomous arsenals mainly to immobilize prey. The highly variable toxic peptides in these venoms target a myriad of neurotoxic and haemotoxic receptors and enzymes and comprise highly interesting candidates for drug discovery. Discovery of bioactive

  4. Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs.

    Science.gov (United States)

    Soheilypour, M; Mofrad, M R K

    2016-11-02

    Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus.

  5. Activation of high and low affinity dopamine receptors generates a closed loop that maintains a conductance ratio and its activity correlate

    Directory of Open Access Journals (Sweden)

    Wulf-Dieter Christian Krenz

    2013-10-01

    Full Text Available Neuromodulators alter network output and have the potential to destabilize a circuit. The mechanisms maintaining stability in the face of neuromodulation are not well described. Using the pyloric network in the crustacean stomatogastric nervous system, we show that dopamine (DA does not simply alter circuit output, but activates a closed loop in which DA-induced alterations in circuit output consequently drive a change in an ionic conductance to preserve a conductance ratio and its activity correlate. DA acted at low affinity type 1 receptors (D1Rs to induce an immediate modulatory decrease in the transient potassium current (IA of a pyloric neuron. This, in turn, advanced the activity phase of that component neuron, which disrupted its network function and thereby destabilized the circuit. DA simultaneously acted at high affinity D1Rs on the same neuron to confer activity-dependence upon the hyperpolarization activated current (Ih such that the DA-induced changes in activity subsequently reduced Ih. This DA-enabled, activity-dependent, intrinsic plasticity exactly compensated for the modulatory decrease in IA to restore the IA:Ih ratio and neuronal activity phase, thereby closing an open loop created by the modulator. Activation of closed loops to preserve conductance ratios may represent a fundamental operating principle neuromodulatory systems use to ensure stability in their target networks.

  6. N(G)-Acyl-argininamides as NPY Y(1) receptor antagonists: Influence of structurally diverse acyl substituents on stability and affinity.

    Science.gov (United States)

    Weiss, Stefan; Keller, Max; Bernhardt, Günther; Buschauer, Armin; König, Burkhard

    2010-09-01

    N(G)-Acylated argininamides, covering a broad range of lipophilicity (calculated logD values: -1.8-12.5), were synthesized and investigated for NPY Y(1) receptor (Y(1)R) antagonism, Y(1)R affinity and stability in buffer (N(G)-deacylation, yielding BIBP 3226). Broad structural variation of substituents was tolerated. The K(i) (binding) and K(b) values (Y(1)R antagonism) varied from low nM to one-digit muM. Most of the compounds proved to be sufficiently stable at pH 7.4 over 90min to determine reliable pharmacological data in vitro. Exceptionally high instability was detected when a succinyl moiety was attached to the guanidine, probably, due to an intramolecular cleavage mechanism. Copyright 2010 Elsevier Ltd. All rights reserved.

  7. Preliminary assessment of extrastriatal dopamine d-2 receptor binding in the rodent and nonhuman primate brains using the high affinity radioligand, {sup 18}F-fallypride

    Energy Technology Data Exchange (ETDEWEB)

    Mukherjee, Jogeshwar E-mail: jogeshwar-mukherjee@ketthealth.com; Yang, Z.-Y.; Brown, Terry; Lew, Robert; Wernick, Miles; Ouyang Xiaohu; Yasillo, Nicholas; Chen, C.-T.; Mintzer, Robert; Cooper, Malcolm

    1999-07-01

    We have identified the value of {sup 18}F-fallypride {l_brace}(S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-[{sup 18}F]fluoropropyl)-2,3-dim= ethoxybenzamide{r_brace}, as a dopamine D-2 receptor radiotracer for the study of striatal and extrastriatal receptors. Fallypride exhibits high affinities for D-2 and D-3 subtypes and low affinity for D-4 ({sup 3}H-spiperone IC{sub 50}s: D-2=0.05 nM [rat striata], D-3=0.30 nM [SF9 cell lines, rat recombinant], and D-4=240 nM [CHO cell lines, human recombinant]). Biodistribution in the rat brain showed localization of {sup 18}F-fallypride in striata and extrastriatal regions such as the frontal cortex, parietal cortex, amygdala, hippocampus, thalamus, and hypothalamus. In vitro autoradiographic studies in sagittal slices of the rat brain showed localization of {sup 18}F-fallypride in striatal and several extrastriatal regions, including the medulla. Positron emission tomography (PET) experiments with {sup 18}F-fallypride in male rhesus monkeys were carried out in a PET VI scanner. In several PET experiments, apart from the specific binding seen in the striatum, specific binding of {sup 18}F-fallypride was also identified in extracellular regions (in a lower brain slice, possibly the thalamus). Specific binding in the extrastriata was, however, significantly lower compared with that observed in the striata of the monkeys (extrastriata/cerebellum = 2, striata/cerebellum = 10). Postmortem analysis of the monkey brain revealed significant {sup 18}F-fallypride binding in the striata, whereas binding was also observed in extrastriatal regions such as the thalamus, cortical areas, and brain stem.

  8. Response to platelet-activating factor in human platelets stored and aged in plasma. Decrease in aggregation, phosphoinositide turnover, and receptor affinity

    International Nuclear Information System (INIS)

    Shukla, S.D.; Morrison, W.J.; Klachko, D.M.

    1989-01-01

    Human platelet concentrates were stored in polyolefin bags at 22 to 24 degrees C on a horizontal shaker for up to 8 days. At different intervals, aliquots of platelet-rich plasma (PRP) were removed aseptically and five variables, i.e., platelet counts, morphology, platelet-activating factor (PAF)-stimulated aggregation, phosphoinositide turnover, and [3H]PAF binding to platelet receptors, were studied. The number of platelets did not change during the 8 days of storage. Scanning electron microscopy of the platelets revealed a gradual morphologic change from biconcave flat discs to irregular, crenated forms. The PAF-induced aggregation of platelets declined with time of storage. A decrease to 50 percent of the Day 1 aggregatory response to PAF was evident on Day 2, and there was a further decline to about 20 percent by Day 6. Similarly, PAF receptor-coupled phosphoinositide turnover, as monitored by 32P incorporation into individual phosphoinositides, decreased dramatically with storage. After 2 to 3 days of storage, the phosphoinositide turnover was reduced to 50 percent of the original response, and it continued to decline to about 25 percent of original response by Day 5 or 6. The binding of [3H]PAF to washed human platelets indicated subtle changes between Days 2 and 4, which became more noticeable by Day 6. These results have raised the possibility of changes in the number of the receptors and/or their affinity for the ligand during storage. We conclude that although the number of platelets was maintained during storage for 8 days, a general deterioration of their responses to PAF occurred at the levels of cell surface receptor, transmembrane signaling (phosphoinositide turnover), and response (aggregation)

  9. Phase I hydroxylated metabolites of the K2 synthetic cannabinoid JWH-018 retain in vitro and in vivo cannabinoid 1 receptor affinity and activity.

    Directory of Open Access Journals (Sweden)

    Lisa K Brents

    Full Text Available K2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ(9-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2's high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R.JWH-018, five potential monohydroxylated metabolites (M1-M5, and one carboxy metabolite (M6 were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [(3H]CP-55,940, and then for CB1R intrinsic efficacy using an [(35S]GTPγS binding assay. JWH-018 and M1-M5 bound CB1Rs with high affinity, exhibiting K(i values that were lower than or equivalent to Δ(9-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ(9-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ(9-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251.Unlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher CB1R affinity and activity relative to Δ(9

  10. The role of progesterone signaling in the pathogenesis of uterine leiomyoma

    Science.gov (United States)

    Kim, J. Julie; Sefton, Elizabeth C.

    2011-01-01

    Uterine leiomyomas are benign tumors that originate from the myometrium. Evidence points to ovarian steroid hormones, in particular, progesterone as major promoters of leiomyoma development and growth. While progesterone action in leiomyomas involves the classical nuclear receptor effects on gene regulation, there is growing evidence that signaling pathways are directly activated by the progesterone receptor (PR) and that PR can interact with growth factor signaling systems to promote proliferation and survival of leiomyomas. Studies investigating the genomic and non-genomic actions of PR and its role in leiomyoma growth are summarized here. Studies testing various selective progesterone receptor modulators for the treatment of leiomyomas are also highlighted. An increased understanding of the mechanisms associated with progesterone-driven growth of leiomyomas is critical in order to develop more efficient and targeted therapies for this prevalent disease. PMID:21672608

  11. Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that [3H]dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

    International Nuclear Information System (INIS)

    Leff, S.E.; Creese, I.

    1985-01-01

    The interactions of dopaminergic agonists and antagonists with 3 H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of [ 3 H]dopamine and [ 3 H]apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/[ 3 H]dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific [ 3 H]dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and [ 3 H]flupentixol-binding activities. The affinities of agonists to inhibit D3 specific [ 3 H]dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/[ 3 H]flupentixol competition curves. Both D3 specific [ 3 H] dopamine binding and the high affinity agonist-binding component of dopamine/[ 3 H]flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor

  12. The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

    Science.gov (United States)

    Klippel, A; Escobedo, J A; Fantl, W J; Williams, L T

    1992-01-01

    Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor. Images PMID:1312663

  13. Additional lesions detected in therapeutic scans with 177Lu-DOTATATE reflect higher affinity of 177Lu-DOTATATE for somatostatin receptors.

    Science.gov (United States)

    Mirzaei, Siroos; Bastati, Brigitte; Lipp, Rainer W; Knoll, Peter; Zojer, Niklas; Ludwig, Heinz

    2011-01-01

    Peptide receptor-targeted radionuclide therapy (PRRT) of somatostatin receptor (SR)-expressing neuroendocrine tumors (NETs) has become an established therapeutic option in patients with advanced NETs. The aim of this study was to compare the lesion detection rate of (99m)Tc-EDDA/HYNIC-TOC, a newly developed tracer for NET imaging, with (177)Lu-DOTATATE used for PRRT. 8 patients (4 women, 4 men, age range 46-76 years) with histologically proven NETs, who showed high SR loads by (99m)Tc-EDDA/HYNIC-TOC scintigraphy, were treated with (177)Lu-DOTATATE. After treatment, all patients were subjected to whole-body scintigraphy with additional low-dose single-photon emission computed tomography (SPECT-CT) of the chest and abdomen. All patients demonstrated (177)Lu-DOTATATE accumulation in all lesions previously detected by (99m)Tc- EDDA/HYNIC-TOC scintigraphy. Three patients showed additional lesions in the liver and lungs. SPECT-CT after (177)Lu-DOTATATE therapy may be helpful in detecting additional lesions not seen using (99m)Tc-EDDA/HYNIC-TOC. This could reflect the broader affinity of (177)Lu-DOTATATE for SRs compared with (99m)Tc-EDDA/HYNIC-TOC. Copyright © 2011 S. Karger AG, Basel.

  14. Carbon-11 labelling of eticlopride in two different positions - a selective high-affinity ligand for the study of dopamine D-2 receptors using PET

    International Nuclear Information System (INIS)

    Halldin, Christer; Hall, Haakan

    1990-01-01

    A new highly selective high-affinity dopamine D-2 receptor antagonist, eticlopride ((-)-(S)-5-chloro-3-ethyl-N-(1-ethyl-2-pyrrolidinyl)methyl)-6-methoxysalicylamide), was labelled with 11 C in two different positions ([N-ethyl- 11 C]eticlopride (I) and ([methyl- 11 C]eticlopride (II)). Product I was prepared by N-alkylation of the N-desethyl compound with [ 11 C]ethyl iodide. II was prepared by O-alkylation of the diphenolic precursor with [ 11 C]methyl iodide followed by separation of the two methylated products. The radiochemical yields were 15-20% (EOB) with an overall synthesis time of 45-60 min. Both compounds were isolated by semi-preparative HPLC and the radiochemical purity was in both cases > 99%. I was injected i.v. in a Cynomolgus monkey and brain radioactivity was measured by positron emission tomography (PET). The specific activity was 70 Ci/mmol at time of injection. There was a marked accumulation of radioactivity in the basal ganglia, regions known to have a high density of dopamine D-2 receptors. (author)

  15. Humoral Immunity Provides Resident Intestinal Eosinophils Access to Luminal Antigen via Eosinophil-Expressed Low-Affinity Fcγ Receptors.

    Science.gov (United States)

    Smith, Kalmia M; Rahman, Raiann S; Spencer, Lisa A

    2016-11-01

    Eosinophils are native to the healthy gastrointestinal tract and are associated with inflammatory diseases likely triggered by exposure to food allergens (e.g., food allergies and eosinophilic gastrointestinal disorders). In models of allergic respiratory diseases and in vitro studies, direct Ag engagement elicits eosinophil effector functions, including degranulation and Ag presentation. However, it was not known whether intestinal tissue eosinophils that are separated from luminal food Ags by a columnar epithelium might similarly engage food Ags. Using an intestinal ligated loop model in mice, in this study we determined that resident intestinal eosinophils acquire Ag from the lumen of Ag-sensitized but not naive mice in vivo. Ag acquisition was Ig-dependent; intestinal eosinophils were unable to acquire Ag in sensitized Ig-deficient mice, and passive immunization with immune serum or Ag-specific IgG was sufficient to enable intestinal eosinophils in otherwise naive mice to acquire Ag in vivo. Intestinal eosinophils expressed low-affinity IgG receptors, and the activating receptor FcγRIII was necessary for Ig-mediated acquisition of Ags by isolated intestinal eosinophils in vitro. Our combined data suggest that intestinal eosinophils acquire lumen-derived food Ags in sensitized mice via FcγRIII Ag focusing and that they may therefore participate in Ag-driven secondary immune responses to oral Ags. Copyright © 2016 by The American Association of Immunologists, Inc.

  16. Ligand binding affinity at the insulin receptor isoform A (IR-A and subsequent IR-A tyrosine phosphorylation kinetics are important determinants of mitogenic biological outcomes.

    Directory of Open Access Journals (Sweden)

    Harinda eRajapaksha

    2015-07-01

    Full Text Available The insulin receptor (IR is a tyrosine kinase receptor that can mediate both metabolic and mitogenic biological actions. The IR isoform-A (IR-A arises from alternative splicing of exon 11 and has different ligand binding and signalling properties compared to the IR isoform-B. The IR-A not only binds insulin but also insulin-like growth factor-II (IGF-II with high affinity. IGF-II acting through the IR-A promotes cancer cell proliferation, survival and migration by activating some unique signalling molecules compared to those activated by insulin. This observation led us to investigate whether the different IR-A signalling outcomes in response to IGF-II and insulin could be attributed to phosphorylation of a different subset of IR-A tyrosine residues or to the phosphorylation kinetics. We correlated IR-A phosphorylation to activation of molecules involved in mitogenic and metabolic signalling (MAPK and Akt and receptor internalisation rates (related to mitogenic signalling. We also extended this study to incorporate two ligands that are known to promote predominantly mitogenic ([His4, Tyr15, Thr49, Ile51] IGF-I, qIGF-I or metabolic (S597 peptide biological actions, to see if common mechanisms can be used to define mitogenic or metabolic signalling through the IR-A. The 3-fold lower mitogenic action of IGF-II compared to insulin was associated with a decreased potency in activation of Y960, Y1146, Y1150, Y1151, Y1316 and Y1322, in MAPK phosphorylation and in IR-A internalization. With the poorly mitogenic S597 peptide it was a decreased rate of tyrosine phosphorylation rather than potency that was associated with a low mitogenic potential. We conclude that both decreased affinity of IR-A binding and the kinetics of IR-A phosphorylation can independently lead to a lower mitogenic activity. None of the studied parameters could account for the lower metabolic activity of qIGF-I.

  17. Expression and localization of ghrelin and its functional receptor in corpus luteum during different stages of estrous cycle and the modulatory role of ghrelin on progesterone production in cultured luteal cells in buffalo.

    Science.gov (United States)

    Gupta, M; Dangi, S S; Chouhan, V S; Hyder, I; Babitha, V; Yadav, V P; Khan, F A; Sonwane, A; Singh, G; Das, G K; Mitra, A; Bag, S; Sarkar, M

    2014-07-01

    Evidence obtained during recent years provided has insight into the regulation of corpus luteum (CL) development, function, and regression by locally produced ghrelin. The present study was carried out to evaluate the expression and localization of ghrelin and its receptor (GHS-R1a) in bubaline CL during different stages of the estrous cycle and investigate the role of ghrelin on progesterone (P4) production along with messenger RNA (mRNA) expression of P4 synthesis intermediates. The mRNA and protein expression of ghrelin and GHS-R1a was significantly greater in mid- and late luteal phases. Both factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of ghrelin and GHS-R1a was greater during mid- and late luteal phases. Luteal cells were cultured in vitro and treated with ghrelin each at 1, 10, and 100 ng/mL concentrations for 48 h after obtaining 75% to 80% confluence. At a dose of 1 ng/mL, there was no significant difference in P4 secretion between control and treatment group. At 10 and 100 ng/mL, there was a decrease (P production in buffalo. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. DOTA-NOC, a high-affinity ligand of somatostatin receptor subtypes 2, 3 and 5 for labelling with various radiometals

    International Nuclear Information System (INIS)

    Wild, Damian; Schmitt, Joerg S.; Ginj, Mihaela; Maecke, Helmut R.; Bernard, Bert F.; Krenning, Eric; Jong, Marion de; Wenger, Sandra; Reubi, Jean-Claude

    2003-01-01

    Earlier studies have shown that modification of the octapeptide octreotide in positions 3 and 8 may result in compounds with increased somatostatin receptor affinity that, if radiolabelled, display improved uptake in somatostatin receptor-positive tumours. The aim of a recent research study in our laboratory was to employ the parallel peptide synthesis approach by further exchanging the amino acid in position 3 of octreotide and coupling the macrocyclic chelator DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to these peptides for labelling with radiometals like gallium-67 or -68, indium-111, yttrium-90 and lutetium-177. The purpose was to find radiopeptides with an improved somatostatin receptor binding profile in order to extend the spectrum of targeted tumours. A first peptide, [ 111 In, 90 Y-DOTA]-1-Nal 3 -octreotide ( 111 In, 90 Y-DOTA-NOC), was isolated which showed an improved profile. In III -DOTA-NOC exhibited the following IC 50 values (nM) when studied in competition with [ 125 I][Leu 8 , d-Trp 22 , Tyr 25 ]somatostatin-28 (values for Y III -DOTA-NOC are shown in parentheses): sstr2, 2.9±0.1 (3.3±0.2); sstr3, 8±2 (26±1.9); sstr5, 11.2±3.5 (10.4±1.6). Affinity towards sstr1 and 4 was very low or absent. In III -DOTA-NOC is superior to all somatostatin-based radiopeptides having this particular type of binding profile, including DOTA-lanreotide, and has three to four times higher binding affinity to sstr2 than In III ,Y III -DOTA-Tyr 3 -octreotide (In III ,Y III -DOTA-TOC). In addition, [ 111 In]DOTA-NOC showed a specific and high rate of internalization into AR4-2J rat pancreatic tumour cells which, after 4 h, was about two times higher than that of [ 111 In]DOTA-TOC and three times higher than that of [ 111 In]DOTA-octreotide ([ 111 In]DOTA-OC). The internalized radiopeptides were externalized intact upon 2 h of internalization followed by an acid wash. After 2-3 h of externalization a plateau is reached, indicating a steady

  19. Exogenous progesterone exacerbates running response of adolescent female mice to repeated food restriction stress by changing α4-GABAA receptor activity of hippocampal pyramidal cells.

    Science.gov (United States)

    Wable, G S; Chen, Y-W; Rashid, S; Aoki, C

    2015-12-03

    Adolescent females are particularly vulnerable to mental illnesses with co-morbidity of anxiety, such as anorexia nervosa (AN). We used an animal model of AN, called activity-based anorexia (ABA), to investigate the neurobiological basis of vulnerability to repeated, food restriction (FR) stress-evoked anxiety. Twenty-one of 23 adolescent female mice responded to the 1st FR with increased wheel-running activity (WRA), even during the limited period of food access, thereby capturing AN's symptoms of voluntary FR and over-exercise. Baseline WRA was an excellent predictor of FR-elicited WRA (severity of ABA, SOA), with high baseline runners responding to FR with minimal SOA (i.e., negative correlation). Nine gained resistance to ABA following the 1st FR. Even though allopregnanolone (3α-OH-5α-pregnan-20-one, THP), the metabolite of progesterone (P4), is a well-recognized anxiolytic agent, subcutaneous P4 to these ABA-resistant animals during the 2nd FR was exacerbative, evoking greater WRA than the counterpart resistant group that received oil vehicle, only. Moreover, P4 had no WRA-reducing effect on animals that remained ABA-vulnerable. To explain the sensitizing effect of P4 upon the resistant mice, we examined the relationship between P4 treatment and levels of the α4 subunit of GABAARs at spines of pyramidal cells of the hippocampal CA1, a parameter previously shown to correlate with resistance to ABA. α4 levels at spine membrane correlated strongly and negatively with SOA during the 1st ABA (prior to P4 injection), confirming previous findings. α4 levels were greater among P4-treated animals that had gained resistance than of vehicle-treated resistant animals or of the vulnerable animals with or without P4. We propose that α4-GABAARs play a protective role by counterbalancing the ABA-induced increase in excitability of CA1 pyramidal neurons, and although exogenous P4's metabolite, THP, enhances α4 expression, especially among those that can gain resistance

  20. A role for progesterone and α4-containing GABAA receptors of hippocampal pyramidal cells in the exacerbated running response of adolescent female mice to repeated food restriction stress

    Science.gov (United States)

    Wable, Gauri; Chen, Yi-Wen; Rashid, Shannon; Aoki, Chiye

    2015-01-01

    Adolescent females are particularly vulnerable to mental illnesses with comorbidity of anxiety, such as anorexia nervosa (AN). We used an animal model of AN, called activity-based anorexia (ABA), to investigate the neurobiological basis of vulnerability to repeated, food restriction (FR) stress-evoked anxiety. Twenty-one of 23 adolescent female mice responded to the 1st FR with increased wheel running activity (WRA), even during the limited period of food access, thereby capturing AN's symptoms of voluntary FR and over-exercise. Baseline WRA was an excellent predictor of FR-elicited WRA (severity of ABA, SOA), with high baseline-runners responding to FR with minimal SOA (i.e., negative correlation). Nine gained resistance to ABA following the 1st FR. Even though allopregnanolone (3α-OH-5α-pregnan-20-one, THP), the metabolite of progesterone (P4), is a well-recognized anxiolytic agent, subcutaneous P4 to these ABA-resistant animals during the 2nd FR was exacerbative, evoking greater WRA than the counterpart resistant group that received oil vehicle, only. Moreover, P4 had no WRA-reducing effect on animals that remained ABA-vulnerable. To explain the sensitizing effect of P4 upon the resistant mice, we examined the relationship between P4 treatment and levels of the α4 subunit of GABAARs at spines of pyramidal cells of the hippocampal CA1, a parameter previously shown to correlate with resistance to ABA. α4 levels at spine membrane correlated strongly and negatively with SOA during the 1st ABA (prior to P4 injection), confirming previous findings. α4 expression levels were greater among P4-treated animals that had gained resistance than of vehicle-treated resistant animals or of the vulnerable animals with or without P4. We propose that α4-GABAARs play a protective role by counterbalancing the ABA-induced increase in excitability of CA1 pyramidal neurons, and although exogenous P4's metabolite, THP, enhances α4 expression, especially among those that can gain

  1. Quantification of human opiate receptor concentration and affinity using high and low specific activity ( sup 11 C)diprenorphine and positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Sadzot, B.; Price, J.C.; Mayberg, H.S.; Douglass, K.H.; Dannals, R.F.; Lever, J.R.; Ravert, H.T.; Wilson, A.A.; Wagner, H.N. Jr.; Feldman, M.A. (Johns Hopkins Medical Institutions, Baltimore, MD (USA))

    1991-03-01

    (11C)Diprenorphine, a weak partial opiate agonist, and positron emission tomography were used to obtain noninvasive regional estimates of opiate receptor concentration (Bmax) and affinity (Kd) in human brain. Different compartmental models and fitting strategies were compared statistically to establish the most reliable method of parameter estimation. Paired studies were performed in six normal subjects using high (769-5,920 Ci/mmol) and low (27-80 Ci/mmol) specific activity (SA) (11C)diprenorphine. Two subjects were studied a third time using high SA (11C)diprenorphine after a pretreatment with 1-1.5 mg/kg of the opiate antagonist naloxone. After the plasma radioactivity was corrected for metabolites, the brain data were analyzed using a three-compartment model and nonlinear least-squares curve fitting. Linear differential equations were used to describe the high SA (low receptor occupancy) kinetics. The k3/k4 ratio varied from 1.0 +/- 0.2 (occipital cortex) to 8.6 +/- 1.6 (thalamus). Nonlinear differential equations were used to describe the low SA (high receptor occupancy) kinetics and the curve fits provided the konf2 product. The measured free fraction of (11C)diprenorphine in plasma (f1) was 0.30 +/- 0.03, the average K1/k2 ratio from the two naloxone studies was 1.1 +/- 0.2, and the calculated free fraction of (11C)diprenorphine in the brain (f2) was 0.3. Using the paired SA studies, the estimated kinetic parameters, and f2, separate estimates of Bmax and Kd were obtained. Bmax varied from 2.3 +/- 0.5 (occipital cortex) to 20.6 +/- 7.3 (cingulate cortex) nM. The average Kd (eight brain regions) was 0.85 +/- 0.17 nM.

  2. Z-(-,-)-[76Br]BrQNP: a high affinity PET radiotracer for central and cardiac muscarinic receptors

    International Nuclear Information System (INIS)

    Strijckmans, V.; Coulon, C.; Loc'h, C.; Maziere, B.; Luo, H.; McPherson, D.W.; Knapp, F.F.

    1996-01-01

    Racemic E-1-azabicyclo[2.2.2]oct-3-yl α-(1-bromo-1-1-propen-3-yl)-α -hydroxy-α-phenylacetate (BrQNP) was prepared and evaluated in vivo as a potential candidate for imaging muscarinic acetylcholinergic receptors by Positron Emission Tomography. Initial in vivo blocking studies utilizing Z-(-,-)-[ 125 I]IQNP as a radiolabelled muscarinic probe demonstrated that a preinjection of cold E-BrQNP effectively blocks the uptake of the radiolabelled probe in the brain and heart, by 71% and 86% respectively. Z-(-,-)-[ 76 Br]BrQNP was prepared by electrophilic substitution from a tributylstannyl precursor. Peracetic acid and chloramine T was evaluated as oxidizing agents. After purification by SPE and RP-HPLC, radiolabelling yields of 85% and 95% were obtained with peracetic acid and chloramine T, respectively. The final radiochemical yield was 70% for both oxidizing agents. (author)

  3. Influence of Dexamethasone on Some Reproductive Hormones and Uterine Progesterone Receptor Localization in Pregnant Yankasa Sheep in Semiarid Zones of Nigeria

    OpenAIRE

    Yahi, Dauda; Ojo, Nicholas Adetayo; Mshelia, Gideon Dauda

    2017-01-01

    Dexamethasone is widely used in both veterinary and human medical practices. However, it seems to cause some deleterious effects on pregnancy probably by causing changes in the reproductive hormone levels and their corresponding receptor concentrations. This study investigated the effects of dexamethasone on these parameters. Twenty healthy adult Yankasa sheep comprising 18 ewes and 2 rams were used for this study. Pregnancies were achieved by natural mating after estrus synchronization. Dexa...

  4. Prediction of N-Methyl-D-Aspartate Receptor GluN1-Ligand Binding Affinity by a Novel SVM-Pose/SVM-Score Combinatorial Ensemble Docking Scheme.

    Science.gov (United States)

    Leong, Max K; Syu, Ren-Guei; Ding, Yi-Lung; Weng, Ching-Feng

    2017-01-06

    The glycine-binding site of the N-methyl-D-aspartate receptor (NMDAR) subunit GluN1 is a potential pharmacological target for neurodegenerative disorders. A novel combinatorial ensemble docking scheme using ligand and protein conformation ensembles and customized support vector machine (SVM)-based models to select the docked pose and to predict the docking score was generated for predicting the NMDAR GluN1-ligand binding affinity. The predicted root mean square deviation (RMSD) values in pose by SVM-Pose models were found to be in good agreement with the observed values (n = 30, r 2  = 0.928-0.988,  = 0.894-0.954, RMSE = 0.002-0.412, s = 0.001-0.214), and the predicted pK i values by SVM-Score were found to be in good agreement with the observed values for the training samples (n = 24, r 2  = 0.967,  = 0.899, RMSE = 0.295, s = 0.170) and test samples (n = 13, q 2  = 0.894, RMSE = 0.437, s = 0.202). When subjected to various statistical validations, the developed SVM-Pose and SVM-Score models consistently met the most stringent criteria. A mock test asserted the predictivity of this novel docking scheme. Collectively, this accurate novel combinatorial ensemble docking scheme can be used to predict the NMDAR GluN1-ligand binding affinity for facilitating drug discovery.

  5. The methoxychlor metabolite, HPTE, inhibits rat luteal cell progesterone production.

    Science.gov (United States)

    Akgul, Yucel; Derk, Raymond C; Meighan, Terence; Rao, K Murali Krishna; Murono, Eisuke P

    2011-07-01

    The methoxychlor metabolite, HPTE, was shown to inhibit P450-cholesterol side-chain cleavage (P450scc) activity resulting in decreased progesterone production by cultured ovarian follicular cells in previous studies. It is not known whether HPTE has any effect on progesterone formation by the corpus luteum. Exposure to 100 nM HPTE reduced progesterone production by luteal cells with progressive declines to progesterone formation and P450scc catalytic activity of hCG- or 8 Br-cAMP-stimulated luteal cells. However, HPTE did not alter mRNA and protein levels of P450scc. Compounds acting as estrogen (17 β-estradiol, bisphenol-A or octylphenol), antiestrogen (ICI) or antiandrogen (monobutyl phthalate, flutamide or M-2) added alone to luteal cells did not mimic the action of HPTE on progesterone and P450scc activity. These results suggest that HPTE directly inhibits P450scc catalytic activity resulting in reduced progesterone formation, and this action was not mediated through estrogen or androgen receptors. Published by Elsevier Inc.

  6. The role of progesterone in prevention of preterm birth

    Directory of Open Access Journals (Sweden)

    Jodie M Dodd

    2009-07-01

    Full Text Available Jodie M Dodd, Caroline A CrowtherDiscipline of Obstetrics and Gynaecology, The University of Adelaide, Adelaide, South Australia, AustraliaAbstract: Preterm birth continues to provide an enormous challenge in the delivery of perinatal health care, and is associated with considerable short and long-term health consequences for surviving infants. Progesterone has a role in maintaining pregnancy, by suppression of the calcium–calmodulin–myosin light chain kinase system. Additionally, progesterone has recognized anti-inflammatory properties, raising a possible link between inflammatory processes, alterations in progesterone receptor expression and the onset of preterm labor. Systematic reviews of randomized controlled trials evaluating the use of intramuscular and vaginal progesterone in women considered to be at increased risk of preterm birth have been published, with primary outcomes of perinatal death, preterm birth <34 weeks, and neurodevelopmental handicap in childhood. Eleven randomized controlled trials were included in the systematic review, involving 2714 women and 3452 infants, with results presented according to the reason women were considered to be at increased risk of preterm birth. While there is a potential beneficial effect in the use of progesterone for some women considered to be at increased risk of preterm birth, primarily in the reduction in the risk of preterm birth before 34 weeks gestation, it remains unclear if the observed prolongation of pregnancy translates into improved health outcomes for the infant.Keywords: progesterone, preterm birth, systematic review, randomized trial

  7. Progesterone Upregulates Gene Expression in Normal Human Thyroid Follicular Cells

    Directory of Open Access Journals (Sweden)

    Ana Paula Santin Bertoni

    2015-01-01

    Full Text Available Thyroid cancer and thyroid nodules are more prevalent in women than men, so female sex hormones may have an etiological role in these conditions. There are no data about direct effects of progesterone on thyroid cells, so the aim of the present study was to evaluate progesterone effects in the sodium-iodide symporter NIS, thyroglobulin TG, thyroperoxidase TPO, and KI-67 genes expression, in normal thyroid follicular cells, derived from human tissue. NIS, TG, TPO, and KI-67 mRNA expression increased significantly after TSH 20 μUI/mL, respectively: 2.08 times, P<0.0001; 2.39 times, P=0.01; 1.58 times, P=0.0003; and 1.87 times, P<0.0001. In thyroid cells treated with 20 μUI/mL TSH plus 10 nM progesterone, RNA expression of NIS, TG, and KI-67 genes increased, respectively: 1.78 times, P<0.0001; 1.75 times, P=0.037; and 1.95 times, P<0.0001, and TPO mRNA expression also increased, though not significantly (1.77 times, P=0.069. These effects were abolished by mifepristone, an antagonist of progesterone receptor, suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone. This work provides evidence that progesterone has a direct effect on thyroid cells, upregulating genes involved in thyroid function and growth.

  8. Production of Monoclonal Antibodies specific for Progesterone

    OpenAIRE

    YÜCEL, Fatıma

    2014-01-01

    Progesterone levels in milk and serum are indicators of pregnancy in cattle. The progesterone level reaches a peak on the 21 st and 22 nd days of pregnancy. Monoclonal antibodies specific to progesterone could be used for the immunodetection of milk and serum progesterone levels. We report here the development of hybrid cells prdoducing monoclonal antibodies specific for progesterone using hybridoma technology. Hybridoma cells secreting monoclonal antibodies against progesterone (MAM 2H1...

  9. Intestinal tumorigenesis is not affected by progesterone signaling in rodent models.

    Directory of Open Access Journals (Sweden)

    Jarom Heijmans

    Full Text Available Clinical data suggest that progestins have chemopreventive properties in the development of colorectal cancer. We set out to examine a potential protective effect of progestins and progesterone signaling on colon cancer development. In normal and neoplastic intestinal tissue, we found that the progesterone receptor (PR is not expressed. Expression was confined to sporadic mesenchymal cells. To analyze the influence of systemic progesterone receptor signaling, we crossed mice that lacked the progesterone receptor (PRKO to the Apc(Min/+ mouse, a model for spontaneous intestinal polyposis. PRKO-Apc(Min/+ mice exhibited no change in polyp number, size or localization compared to Apc(Min/+. To examine effects of progestins on the intestinal epithelium that are independent of the PR, we treated mice with MPA. We found no effects of either progesterone or MPA on gross intestinal morphology or epithelial proliferation. Also, in rats treated with MPA, injection with the carcinogen azoxymethane did not result in a difference in the number or size of aberrant crypt foci, a surrogate end-point for adenoma development. We conclude that expression of the progesterone receptor is limited to cells in the intestinal mesenchyme. We did not observe any effect of progesterone receptor signaling or of progestin treatment in rodent models of intestinal tumorigenesis.

  10. Two synthetic progestins and natural progesterone are responsible for most of the progestagenic activities in municipal wastewater treatment plant effluents in the Czech and Slovak republics.

    Science.gov (United States)

    Šauer, Pavel; Stará, Alžběta; Golovko, Oksana; Valentová, Olga; Bořík, Adam; Grabic, Roman; Kroupová, Hana Kocour

    2018-06-15

    Vast numbers of xenobiotics are known still to be present in treated municipal wastewater treatment plant (WWTP) effluents. Some of these possess endocrine-disrupting potency and pose risks for exposed aquatic animals. We searched for 17 potential environmental contaminants having affinity to the progesterone receptor. Relative potency values of these progesterone receptor-active chemicals were obtained. On the basis of relative potencies and measured environmental concentrations, the contribution of progestins to measured progestagenic activities was evaluated. Wastewaters (influent and effluent) and surrounding surface waters (upstream and downstream) at six municipal WWTPs were screened using instrumental chemical analysis and in vitro reporter gene bioassay. We showed the presence of target compounds and (anti-)progestagenic activities in municipal wastewater and surface water. Nine and seven progestins were identified in influent and effluent wastewaters, respectively. Only two compounds, progesterone and medroxyprogesterone were found in surface waters. Progestagenic agonistic activities in influents were partially masked by strong anti-progestagenic activities that were detected in all influents and ranged from 2.63 to 83 ng/L of mifepristone equivalents (EQs). Progestagenic activities were detected in all effluents and ranged from 0.06 to 0.47 ng/L of reference compound ORG 2058 EQs (a synthetic progestin equivalents), thus indicating incomplete removal of progestins during wastewater treatment processing. This activity poses a continuing risk for the aquatic environment. By contrast, anti-progestagenic activities showed better removal efficiency in WWTPs compared to progestagenic agonistic activities. Anti-progestagenic activities were found in only three of six effluents and ranged from 0.26 to 2.1 ng/L mifepristone EQs. We explained most of the progestagenic activity in municipal WWTP effluents by the presence of synthetic progestins and

  11. Progesterone for preterm birth prevention

    Directory of Open Access Journals (Sweden)

    Miha Lucovnik

    2015-10-01

    Full Text Available Background: Progesterone is important in maintaining pregnancy. Progesterone supplementation may reduce risk of preterm birth in certain populations of pregnant women. The objective of this review was to develop evidence-based clinical recommendation for progesterone treatment in the prevention of preterm birth.Methods: A search in the Medline database was performed using keywords: progesterone, pregnancy, preterm birth, preterm labour, preterm delivery, randomized trial, and randomized controlled trial. We only included studies of vaginal progesterone treatments for the prevention of preterm birth and excluded studies on 17-α-hydroksiprogesterone caproate.Results: We report findings from twelve randomized trials conducted since 2003. These trials differ regarding inclusion criteria, progesterone dose, vehicle used, and duration of treatment. Inclusion criteria were: short uterine cervix (two trials, history of previous preterm birth (two trials, signs and symptoms of preterm labour (three trials, twin pregnancies (three trials, and multiple risk factors (among these history of previous preterm birth was the most common (two trials. Six of these twelve trials showed a significant reduction in preterm birth in the progesterone groups.Conclusions: Based on current evidence we recommend treatment with 200 mg of micronized progesterone daily, administered vaginally, in pregnant women found to have a short cervix (≤ 25 mm at 19-24 weeks. The treatment should be continued until 37 weeks.

  12. Dietary fiber intake and risk of breast cancer defined by estrogen and progesterone receptor status: the Japan Public Health Center-based Prospective Study.

    Science.gov (United States)

    Narita, Saki; Inoue, Manami; Saito, Eiko; Abe, Sarah K; Sawada, Norie; Ishihara, Junko; Iwasaki, Motoki; Yamaji, Taiki; Shimazu, Taichi; Sasazuki, Shizuka; Shibuya, Kenji; Tsugane, Shoichiro

    2017-06-01

    Epidemiological studies have suggested a protective effect of dietary fiber intake on breast cancer risk while the results have been inconsistent. Our study aimed to investigate the association between dietary fiber intake and breast cancer risk and to explore whether this association is modified by reproductive factors and hormone receptor status of the tumor. A total of 44,444 women aged 45 to 74 years from the Japan Public Health Center-based Prospective Study were included in analyses. Dietary intake assessment was performed using a validated 138-item food frequency questionnaire (FFQ). Hazard ratios (HRs) and 95% confidence intervals (CIs) for breast cancer incidence were calculated by multivariate Cox proportional hazards regression models. During 624,423 person-years of follow-up period, 681 breast cancer cases were identified. After adjusting for major confounders for breast cancer risk, inverse trends were observed but statistically non-significant. Extremely high intake of fiber was associated with decreased risk of breast cancer but this should be interpreted with caution due to limited statistical power. In stratified analyses by menopausal and hormone receptor status, null associations were observed except for ER-PR- status. Our findings suggest that extreme high fiber intake may be associated with decreased risk of breast cancer but the level of dietary fiber intake among Japanese population might not be sufficient to examine the association between dietary fiber intake and breast cancer risk.

  13. (3H)leukotriene B4 binding to the guinea pig spleen membranes: a rich tissue source for a high affinity leukotriene B4 receptor site

    International Nuclear Information System (INIS)

    Cheng, J.B.; Kohi, F.; Townley, R.G.

    1986-01-01

    To select a tissue rich for the high affinity leukotriene (LT)B 4 receptor site, they compared binding of 1 nM ( 3 H)LTB 4 (180 Ci/mmol) to the crude membrane preparations of guinea pig spleen, thymus, lung, uterus, bladder, brain, adrenal gland, small intestine, liver, kidney and heart. They found that the membrane preparations from spleen contained the highest binding activity per mg protein. They characterized the LTB 4 binding to the spleen preparation in detail. LTB 4 binding was rapid, reversible, stereoselective and saturable. The data from equilibrium experiments showed a linear Scatchard plot with a K/sub d/ of 1.6 nM and a binding site density of 259 fmol/mg prot. The rank order of agents competing for spleen ( 3 H)LTB 4 binding at 25 0 C was: LTB 4 (K/sub i/ = 2.8 nM) > 20-OH-LTB 4 (23 nM) > LTA 4 (48 nM) > LTA 4 methyl ester (0.13 μM) > 20-COOH-LTB 4 (> 6.6 μM) ≥ arachidonic acid (0.15 mM) similarly ordered FPL-55,712 (0.11 mM). At 4 0 C, LTB 4 (2.3 nM) competed at least 10x more effectively than 20-OH-LTB 4 (29 nM) and 20-COOH-LTB 4 (> 6.6 μM). HPLC analysis indicated that incubation of 84 ng LTB 4 with the spleen membrane at 25 0 C did not result in the formation of 20-OH-LTB 4 ( 3 H)LTB 4 receptor binding sites

  14. Study of progesterone mechanisms in radio-induced apoptosis prevention; Etude des mecanismes de prevention de l'apoptose radioinduite par la progesterone

    Energy Technology Data Exchange (ETDEWEB)

    Vares, G.

    2004-10-15

    The purpose of this work was to study the modulation of radiation-induced cell death of human mammary tumoral cells by progesterone. On the one hand, we observed that progesterone protects cells against radiation-induced apoptosis and increases the proportion of surviving and proliferating damaged cells. On the other hand, a transcriptome analysis was undertaken in irradiated cells treated by progesterone, using DNA micro-arrays. This let us highlight several transcriptional dis-regulations that are likely to explain the protective effect of the hormone; in particular, we showed that progesterone regulates the expression of genes implicated in apoptosis signaling by death receptors. Knowing the crucial role of hormonal control and of apoptosis regulation in breast cancer initiation, our results may help to achieve a better understanding of the implication of progesterone in mammary carcinogenesis. (author)

  15. Predicting the affinity of Farnesoid X Receptor ligands through a hierarchical ranking protocol: a D3R Grand Challenge 2 case study

    Science.gov (United States)

    Réau, Manon; Langenfeld, Florent; Zagury, Jean-François; Montes, Matthieu

    2018-01-01

    The Drug Design Data Resource (D3R) Grand Challenges are blind contests organized to assess the state-of-the-art methods accuracy in predicting binding modes and relative binding free energies of experimentally validated ligands for a given target. The second stage of the D3R Grand Challenge 2 (GC2) was focused on ranking 102 compounds according to their predicted affinity for Farnesoid X Receptor. In this task, our workflow was ranked 5th out of the 77 submissions in the structure-based category. Our strategy consisted in (1) a combination of molecular docking using AutoDock 4.2 and manual edition of available structures for binding poses generation using SeeSAR, (2) the use of HYDE scoring for pose selection, and (3) a hierarchical ranking using HYDE and MM/GBSA. In this report, we detail our pose generation and ligands ranking protocols and provide guidelines to be used in a prospective computer aided drug design program.

  16. Premature Aging Phenotype in Mice Lacking High-Affinity Nicotinic Receptors: Region-Specific Changes in Layer V Pyramidal Cell Morphology.

    Science.gov (United States)

    Konsolaki, Eleni; Skaliora, Irini

    2015-08-01

    The mechanisms by which aging leads to alterations in brain structure and cognitive deficits are unclear. Α deficient cholinergic system has been implicated as one of the main factors that could confer a heightened vulnerability to the aging process, and mice lacking high-affinity nicotinic receptors (β2(-/-)) have been proposed as an animal model of accelerated cognitive aging. To date, however, age-related changes in neuronal microanatomy have not been studied in these mice. In the present study, we examine the neuronal structure of yellow fluorescent protein (YFP(+)) layer V neurons in 2 cytoarchitectonically distinct cortical regions in wild-type (WT) and β2(-/-) animals. We find that (1) substantial morphological differences exist between YFP(+) cells of the anterior cingulate cortex (ACC) and primary visual cortex (V1), in both genotypes; (2) in WT animals, ACC cells are more susceptible to aging compared with cells in V1; and (3) β2 deletion is associated with a regionally and temporally specific increase in vulnerability to aging. ACC cells exhibit a prematurely aged phenotype already at 4-6 months, whereas V1 cells are spared in adulthood but strongly affected in old animals. Collectively, our data reveal region-specific synergistic effects of aging and genotype and suggest distinct vulnerabilities in V1 and ACC neurons. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Antinociceptive action of DBO 17 and DBO 11 in mice: two 3,8 diazabicyclo (3.2.1.) octane derivates with selective mu opioid receptor affinity.

    Science.gov (United States)

    Fadda, P; Barlocco, D; Tronci, S; Cignarella, G; Fratta, W

    1997-11-01

    Two 3,8 diazabicyclo (3.2.1.) octane derivates, namely DBO 17 and DBO 11, were studied for the opioid-like activity. In the rat brain membrane preparation binding studies, DBO 17 and DBO 11 showed a high affinity and selectivity for the mu opioid receptor (Ki's: 5.1 and 25 nM, respectively). DBO 17 and DBO 11 inhibited the nociceptive response in the hot-plate test of mice with ED50 values of 0.16 mg/kg and 0.44 mg/kg, respectively. The antinociceptive action of both DBO 17 and DBO 11 was blocked by naloxone. Tolerance to the antinociceptive action of DBO 17 and DBO 11 was present after 13 and 7 days of repeated treatment, respectively. Both DBO 17 and DBO 11 were ineffective in morphine-tolerant mice and vice versa. Chronic treatments (three times daily for seven consecutive days) of DBO 17 and DBO 11 induced a naloxone-precipitated withdrawal syndrome in DBO 17 treated mice similar to that in morphine treated mice, whereas in DBO 11 treated mice abstinence signs were virtually absent. These results indicate an interesting pharmacological profile that suggests these compounds as possible new candidates for the clinical treatment of pain.

  18. Cost-effectiveness of a 21-gene recurrence score assay versus Canadian clinical practice in women with early-stage estrogen- or progesterone-receptor-positive, axillary lymph-node negative breast cancer

    International Nuclear Information System (INIS)

    Hannouf, Malek B; Xie, Bin; Brackstone, Muriel; Zaric, Gregory S

    2012-01-01

    A 21-gene recurrence score (RS) assay may inform adjuvant systematic treatment decisions in women with early stage breast cancer. We sought to investigate the cost effectiveness of using the RS-assay versus current clinical practice (CCP) in women with early-stage estrogen- or progesterone-receptor-positive, axilliary lymph-node negative breast cancer (ER+/ PR + LN- ESBC) from the perspective of the Canadian public healthcare system. We developed a Markov model to project the lifetime clinical and economic consequences of ESBC. We evaluated adjuvant therapy separately in post- and pre-menopausal women with ER+/ PR + LN- ESBC. We assumed that the RS-assay would reclassify pre- and post-menopausal women among risk levels (low, intermediate and high) and guide adjuvant systematic treatment decisions. The model was parameterized using 7 year follow up data from the Manitoba Cancer Registry, cost data from Manitoba administrative databases, and secondary sources. Costs are presented in 2010 CAD. Future costs and benefits were discounted at 5%. The RS-assay compared to CCP generated cost-savings in pre-menopausal women and had an ICER of $60,000 per QALY gained in post-menopausal women. The cost effectiveness was most sensitive to the proportion of women classified as intermediate risk by the RS-assay who receive adjuvant chemotherapy and the risk of relapse in the RS-assay model. The RS-assay is likely to be cost effective in the Canadian healthcare system and should be considered for adoption in women with ER+/ PR + LN- ESBC. However, ongoing assessment and validation of the assay in real-world clinical practice is warranted

  19. cis- and trans-2,3,3a,4,5,9b-Hexahydro-1H-benz[e]indoles: synthesis and evaluation of dopamine D2, and D3 receptor binding affinity

    DEFF Research Database (Denmark)

    Song, Xiaodong; Crider, Michael A.; Cruse, Sharon F.

    1999-01-01

    cis- and trans-2,3,3a,4,5,9b-hexahydro-1H-benz [e]indoles were synthesized as conformationally rigid analogues of 3-phenylpyrrolidine and evaluated for dopamine (DA) D2S and D3 receptor binding affinity. The tricyclic benz[e]indole nucleus was constructed by a previously reported reductive...... configuration. These novel ligands may be useful tools for gaining additional information about the DA D3 receptor. Copyright Elsevier, Paris.dopamine / D2S receptor / D3 receptor / cis- and trans-2,3,3a,4,5,9b-hexahydro-1H-benz[e]indoles / receptor binding affinity....... receptors was shown by compounds substituted with N-n-propyl or N-allyl groups. The cis-(+-)-N-allyl derivative 21e demonstrated a D2S/D3 selectivity of 290. Resolution of cis-(+-)-5 and trans-(+-)- 21c into individual enantiomers showed that in both series the more active isomer had 3aR absolute...

  20. Decreased endogenous progesterone and ratio of progesterone to ...

    African Journals Online (AJOL)

    PRECIOUS

    2010-02-01

    Feb 1, 2010 ... 30 control subjects (15 men and 15 women) of comparable age. There were ... estrogens and progestins makes the brain more vulnerable to acute insults ... estrogen or ratio of progesterone to estrogen is different in ischemia ...

  1. Synthesis and characterization of [{sup 76}Br]-labeled high-affinity A{sub 3} adenosine receptor ligands for positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Kiesewetter, Dale O. [Positron Emission Tomography Radiochemistry Group, NIBIB, Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States)], E-mail: dk7k@nih.gov; Lang Lixin; Ma Ying; Bhattacharjee, Abesh Kumar [Positron Emission Tomography Radiochemistry Group, NIBIB, Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Gao, Zhan-Guo; Joshi, Bhalchandra V.; Melman, Artem; Castro, Sonia de; Jacobson, Kenneth A. [Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)

    2009-01-15

    Introduction: Bromine-76-radiolabeled analogues of previously reported high-affinity A{sub 3} adenosine receptor (A{sub 3}AR) nucleoside ligands have been prepared as potential radiotracers for positron emission tomography. Methods: The radiosyntheses were accomplished by oxidative radiobromination on the N{sup 6}-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics of uptake were conducted in awake rats. Results: We prepared an agonist ligand {l_brace}[{sup 76}Br](1'S,2'R,3'S,4'R,5'S)-4'-{l_brace}2-chloro-6-[(3-bromophenylmethyl)amino] purin-9-yl{r_brace}-1'-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2',3'-diol (MRS3581){r_brace} in 59% radiochemical yield with a specific activity of 19.5 GBq/{mu}mol and an antagonist ligand {l_brace}[{sup 76}Br](1'R,2'R,3'S,4'R,5'S)-4'-(6-(3-bromobenzylamino) -2-chloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2',3'-diol (MRS5147){r_brace} in 65% radiochemical yield with a specific activity of 22 GBq/{mu}mol. The resultant products exhibited the expected high affinity (K{sub i}{approx}0.6 nM) and specific binding at the human A{sub 3}AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A{sub 3}AR, over a 2-h time course, which suggests the presence of a specific A{sub 3}AR retention mechanism. Conclusion: We were able to compare uptake of the [{sup 76}Br]-labeled antagonist MRS5147 to [{sup 76}Br]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A{sub 3}AR-rich tissue, suggesting that this ligand may have promise as a molecular imaging agent.

  2. In vitro and in vivo binding of neuroactive steroids to the sigma-1 receptor as measured with the positron emission tomography radioligand [18F]FPS.

    Science.gov (United States)

    Waterhouse, Rikki N; Chang, Raymond C; Atuehene, Nana; Collier, Thomas Lee

    2007-07-01

    Sigma-1 receptors are widely expressed in the mammalian brain and also in organs of the immune, endocrine and reproductive systems. Based on behavioral and pharmacological assessments, sigma-1 receptors are important in memory and cognitive processes, and are thought to be involved in specific psychiatric illnesses, including schizophrenia, depression, and drug addiction. It is thought that specific neuroactive steroids are endogenous ligands for these sites. In addition, several sigma-1 receptor binding steroids including progesterone, dihydroepiandrosterone (DHEA), and testosterone are being examined clinically for specific therapeutic purposes; however, their mechanisms of action have not been clearly defined. We previously described the high affinity sigma-1 receptor selective PET tracer [(18)F]FPS. This study examines the effect of neuroactive steroids on [(18)F]FPS binding in vitro and in vivo. Inhibition constants were determined in vitro for progesterone, testosterone, DHEA, estradiol, and estriol binding to the [(18)F]FPS labeled receptor. The affinity order (K(i) values) for these steroids ranged from 36 nM for progesterone to >10,000 nM for estrodiol and estriol. Biodistribution studies revealed that i.v. coadministration of progesterone (10 mg/kg), testosterone (20 mg/kg), or DHEA (20 mg/kg) significantly decreased [(18)F]FPS uptake (%ID/g) by up to 50% in nearly all of eight brain regions examined. [(18)F]FPS uptake in several peripheral organs that express sigma-1 receptors (heart, spleen, muscle, lung) was also reduced (54-85%). These studies clearly demonstrate that exogenously administered steroids can occupy sigma-1 receptors in vivo, and that [(18)F]FPS may provide an effective tool for monitoring sigma-1 receptor occupancy of specific therapeutic steroids during clinical trials.

  3. Immunohistochemical expression of oestrogen and progesterone receptors during experimental acute and chronic murine Schistosomiasis mansoni Expressão imunohistoquímica de receptores para estrogênio e progesterona nas fases aguda e crônica da esquistossomose mansônica experimental em camundongos

    Directory of Open Access Journals (Sweden)

    Fawzia Ahmed Habib

    2010-10-01

    Full Text Available INTRODUCTION: The responsibility of Schistosoma mansoni in female infertility is still controversial. This study was conducted to evaluate the effect of acute and chronic schistosomiasis mansoni infection on the endometrium using immunohistochemical analysis of uterine hormone receptor expression. METHODS: Twenty-four nonpregnant swiss albino mice were divided into three groups: control, noninfected; acute; and chronic Schistosoma mansoni infection. Histological sections of uterine specimens were examined by light microscope with an image analyzing system to detect structural histological, estrogen receptor (ER and progesterone receptor (PR expression in the endometrium. RESULTS: No secretory phase was detected in the endometrium in acute and chronic Schistosoma infection. Hormone receptor expression (ER and PR showed statistically significant differences among the groups (pINTRODUÇÃO: A responsabilidade do Schistosoma mansoni em esterilidade feminina é ainda controversa. Este estudo é conduzido para avaliar o efeito da esquistossomose mansoni aguda e crônica no endométrio usando análise de imuno-histoquímíca da expressão de receptor hormonal uterina. MÉTODOS: Vinte e quatro camundongos fêmeas albinas suíças não grávidas foram divididas em 3 grupos (controle não-infectado, grupos agudos e crônicos infeccionados com Schistosoma mansoni. As seções histológicas de espécimes uterinos foram examinadas por microscópio leve com imagem, analisando sistema para detectar no endométrio expressões histológicas estruturais, receptor de estrogênio (ER e receptor de progesterona (PR. RESULTADOS: Nenhuma fase secretora foi detectada no endométrio com infecção aguda e crônica de Schistosoma. A expressão hormonal de receptor (ER e PR mostrou diferenças estatisticamente significantes entre grupos diferentes (p<0,05 com baixa significativa hormonal de ER com infecção crônica (comparado com controle proliferativo, controle secret

  4. Progesterone in Breast Cancer Angiogenesis

    OpenAIRE

    Botelho, Monica C.; Soares, Raquel; Alves, Helena

    2015-01-01

    The involvement of steroid hormones in breast carcinogenesis is well established. Recent evidence suggests that angiogenesis can be regulated by hormones. Both oestrogen and progesterone have been implicated in the angiogenic process of hormone-dependent cancers, such as breast cancer. Vascular Endothelial Growth Factor (VEGF) is a growth factor involved in angiogenesis in breast cancer that is up-regulated by estrogens. In our study we evaluated the role of progesterone in the expression of ...

  5. Pulmonary Administration of GW0742, a High-Affinity Peroxisome Proliferator-Activated Receptor Agonist, Repairs Collapsed Alveoli in an Elastase-Induced Mouse Model of Emphysema.

    Science.gov (United States)

    Ozawa, Chihiro; Horiguchi, Michiko; Akita, Tomomi; Oiso, Yuki; Abe, Kaori; Motomura, Tomoki; Yamashita, Chikamasa

    2016-01-01

    Pulmonary emphysema is a disease in which lung alveoli are irreversibly damaged, thus compromising lung function. Our previous study revealed that all-trans-retinoic acid (ATRA) induces the differentiation of human lung alveolar epithelial type 2 progenitor cells and repairs the alveoli of emphysema model mice. ATRA also reportedly has the ability to activate peroxisome proliferator-activated receptor (PPAR) β/δ. A selective PPARβ/δ ligand has been reported to induce the differentiation of human keratinocytes during wound repair. Here, we demonstrate that treatment using a high-affinity PPARβ/δ agonist, GW0742, reverses the lung tissue damage induced by elastase in emphysema-model mice and improves respiratory function. Mice treated with elastase, which collapsed their alveoli, were then treated with either 10% dimethyl sulfoxide (DMSO) in saline (control group) or GW0742 (1.0 mg/kg twice a week) by pulmonary administration. Treatment with GW0742 for 2 weeks increased the in vivo expression of surfactant proteins A and D, which are known alveolar type II epithelial cell markers. GW0742 treatment also shortened the average distance between alveolar walls in the lungs of emphysema model mice, compared with a control group treated with 10% DMSO in saline. Treatment with GW0742 for 3 weeks also improved tissue elastance (cm H2O/mL), as well as the ratio of the forced expiratory volume in the first 0.05 s to the forced vital capacity (FEV 0.05/FVC). In each of these experiments, GW0742 treatment reversed the damage caused by elastase. In conclusion, PPARβ/δ agonists are potential therapeutic agents for pulmonary emphysema.

  6. Effects of perinatal hypo- and hyperthyroidism on the levels of nerve growth factor and its low-affinity receptor in cerebellum.

    Science.gov (United States)

    Figueiredo, B C; Otten, U; Strauss, S; Volk, B; Maysinger, D

    1993-04-16

    Deficits or excesses of thyroid hormones during critical periods of mammalian cerebellar development can lead to profound biochemical and morphological abnormalities in this system. The goal of this study was to investigate the effects of perinatal hypo- and hyperthyroidism on the ontogeny of nerve growth factor (NGF) and its low-affinity receptor (p75NGFR) in the rat cerebellum. The concentration of NGF and of p75NGFR immunoreactivity (IR) were measured, several days after birth, in cerebella of rats which had received propylthiouracil (PTU) or thyroxine. NGF concentration was markedly enhanced only on postnatal day 2 (P2) in hyperthyroid rats, whereas in hypothyroid (PTU-treated) rats NGF values were similar to age-matched controls. These observations suggest that thyroid hormone affects NGF synthesis during early periods of cerebellar development. In Purkinje cells of control animals, p75NGFR IR peaked at P10. In hypothyroid rats, the expression of p75NGFR was retarded, peaking at P15, whereas in hyperthyroid rats it was advanced, peaking at P8. The increased p75NGFR IR found in Purkinje cell bodies and the delayed disappearance of p75NGFR IR from the external granular layer of hypothyroid rats suggest different roles for thyroid hormone in the developing cerebellum. We conclude that p75NGFR and NGF are independently regulated by thyroid hormone during critical periods of cerebellar development. The effect of thyroid hormone deficiency on p75NGFR content in Purkinje cells may involve complex mechanisms such as impaired efficiency of axonal transport.

  7. 5-(sulfonyl)oxy-tryptamines and ethylamino side chain restricted derivatives. Structure-affinity relationships for h5-HT1B and h5-HT1D receptors

    NARCIS (Netherlands)

    Barf, T; Wikstrom, H; Pauwels, PJ; Palmier, C; Tardif, S; Lundmark, M; Sundell, S

    A number of sulfonic acid ester derivatives of serotonin (5-hydroxytryptamine; 5-HT; 1) were prepared and their affinities are compared to that of the reference compound 5-[[(trifluoromethyl)sulfonyl]oxy]-tryptamine (8b). The structure-affinity relationship (SAFIR) is discussed in terms of in vitro

  8. Providing affinity

    DEFF Research Database (Denmark)

    Guglielmi, Michel; Johannesen, Hl

    2004-01-01

    , Essex, Hertfordshire, Norfolk and Suffolk. Research found that there was a lack of identity or sense of belonging and nothing anchoring people to the region as a whole. Common affinity is somehow forced to the people of East England and thereby we came to the conclusion that a single landmark...... and potential situations but also virtual events that calls for an undeterminated process of resolution. This process is activated by the user who co-produces the actualisation as an answer to a virtual reality that we defined at the first place. The potential situations or the possible it is a fantomatic real....... The possible is like the real. It is determinated and it only lakes existence. While the possible is already made, the virtual is like a problematic which needs to be resolved and actualized. Our installations are based on high tech interactivity where we use sensors and remote communication to offer a sense...

  9. Progesterone is essential for protecting against LPS-induced pregnancy loss. LIF as a potential mediator of the anti-inflammatory effect of progesterone.

    Directory of Open Access Journals (Sweden)

    Julieta Aisemberg

    Full Text Available Lipopolysaccharide (LPS administration to mice on day 7 of gestation led to 100% embryonic resorption after 24 h. In this model, nitric oxide is fundamental for the resorption process. Progesterone may be responsible, at least in part, for a Th2 switch in the feto-maternal interface, inducing active immune tolerance against fetal antigens. Th2 cells promote the development of T cells, producing leukemia inhibitory factor (LIF, which seems to be important due to its immunomodulatory action during early pregnancy. Our aim was to evaluate the involvement of progesterone in the mechanism of LPS-induced embryonic resorption, and whether LIF can mediate hormonal action. Using in vivo and in vitro models, we provide evidence that circulating progesterone is an important component of the process by which infection causes embryonic resorption in mice. Also, LIF seems to be a mediator of the progesterone effect under inflammatory conditions. We found that serum progesterone fell to very low levels after 24 h of LPS exposure. Moreover, progesterone supplementation prevented embryonic resorption and LPS-induced increase of uterine nitric oxide levels in vivo. Results show that LPS diminished the expression of the nuclear progesterone receptor in the uterus after 6 and 12 h of treatment. We investigated the expression of LIF in uterine tissue from pregnant mice and found that progesterone up-regulates LIF mRNA expression in vitro. We observed that LIF was able to modulate the levels of nitric oxide induced by LPS in vitro, suggesting that it could be a potential mediator of the inflammatory action of progesterone. Our observations support the view that progesterone plays a critical role in a successful pregnancy as an anti-inflammatory agent, and that it could have possible therapeutic applications in the prevention of early reproductive failure associated with inflammatory disorders.

  10. Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

    Science.gov (United States)

    Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

    2017-01-01

    CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

  11. Estrogen and progesterone decrease let-7f microRNA expression and increase IL-23/IL-23 receptor signaling and IL-17A production in patients with severe asthma.

    Science.gov (United States)

    Newcomb, Dawn C; Cephus, Jacqueline Yvonne; Boswell, Madison G; Fahrenholz, John M; Langley, Emily W; Feldman, Amy S; Zhou, Weisong; Dulek, Daniel E; Goleniewska, Kasia; Woodward, Kimberly B; Sevin, Carla M; Hamilton, Robert G; Kolls, Jay K; Peebles, R Stokes

    2015-10-01

    Women have an increased prevalence of severe asthma compared with men. IL-17A is associated with severe asthma and requires IL-23 receptor (IL-23R) signaling, which is negatively regulated by let-7f microRNA. We sought to Determine the mechanism by which 17β-estradiol (E2) and progesterone (P4) increase IL-17A production. IL-17A production was determined by using flow cytometry in TH17 cells from women (n = 14) and men (n = 15) with severe asthma. Cytokine levels were measured by using ELISA, and IL-23R and let-7f expression was measured by using quantitative PCR in TH17-differentiated cells from healthy women (n = 13) and men (n = 14). In sham-operated or ovariectomized female mice, 17β-E2, P4, 17β-E2+P4, or vehicle pellets were administered for 3 weeks before ex vivo TH17 cell differentiation. Airway neutrophil infiltration and CXCL1 (KC) expression were also determined in ovalbumin (OVA)-challenged wild-type female recipient mice with an adoptive transfer of OVA-specific TH17 cells from female and male mice. In patients with severe asthma and healthy control subjects, IL-17A production was increased in TH17 cells from women compared with men. IL-23R expression was increased and let-7f expression was decreased in TH17-differentiated cells from women compared with men. In ovariectomized mice IL-17A and IL-23R expression was increased and Let-7f expression was decreased in TH17 cells from mice administered 17β-E2+P4 compared with those administered vehicle. Furthermore, transfer of female OVA-specific TH17 cells increased acute neutrophil infiltration in the lungs of OVA-challenged recipient mice compared with transfer of male OVA-specific TH17 cells. 17β-E2+P4 increased IL-17A production from TH17 cells, providing a potential mechanism for the increased prevalence of severe asthma in women compared with men. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  12. Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

    International Nuclear Information System (INIS)

    Raymond, J.R.; Fargin, A.; Lohse, M.J.; Regan, J.W.; Senogles, S.E.; Lefkowitz, R.J.; Caron, M.G.

    1989-01-01

    The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor

  13. N1-Substituted 2,3-Quinoxalinediones as Kainate Receptor Antagonists: X-ray Crystallography, Structure-Affinity Relationships and in vitro Pharmacology

    DEFF Research Database (Denmark)

    Pallesen, Jakob Staun; Møllerud, Stine; Frydenvang, Karla Andrea

    2018-01-01

    Among the ionotropic glutamate receptors, the physiological role of kainate receptors is less well understood than AMPA and NMDA receptors, partly due to a lack of selective pharmacological tool compounds. Although ligands with selectivity towards the kainate receptor subtype GluK1 are available,...

  14. Affinity biosensors: techniques and protocols

    National Research Council Canada - National Science Library

    Rogers, Kim R; Mulchandani, Ashok

    1998-01-01

    ..., and government to begin or expand their biosensors research. This volume, Methods in Biotechnology vol. 7: Affinity Biosensors: Techniques and Protocols, describes a variety of classical and emerging transduction technologies that have been interfaced to bioaffinity elements (e.g., antibodies and receptors). Some of the reas...

  15. Effects of Kaixin Powder on melatonin receptor expression and (125)I-Mel binding affinity in a rat model of depression.

    Science.gov (United States)

    Huang, Yan-li; Liang, Xue-bing; Qian, Li-qi; Cai, Chuan; Guo, Jun; Gao, Chao; Guan, Jian-hua; Zhao, Guo-ping

    2015-07-01

    To explore the effects of Kaixin Powder (, KXP) on melatonin receptor (MR) expression and (125)I-Mel binding affinity in a depression rat model. Seventy-two male Wistar rats were divided into six groups: a blank control group, model group, ramelteon group, KXP high-dosage group (HKXP), medium-dosage group (MKXP) and low-dosage group (LKXP). To establish the depression model, all groups except the blank control group were singly housed and exposed to chronic unpredictable mild stress. Weight gain, sucrose consumption and the open-field test were used to evaluate induction of depression. KXP at 260, 130 and 65 mg/(kg•d) was also respectively administered to the rats in the HKXP, MKXP and LKXP groups for 21 days. Ramelteon [0.83 mg/(kg•d)] was given to the positive drug control group. An equivalent volume of physiological saline was given to the blank and model groups. The liquid chip method was used to measure the concentration of plasma melatonin (MT). Mel1a (MT1) and Mel1b (MT2) expression levels were determined by Western blotting. In addition, a radioactive ligand-binding assay was used to analyze the specific binding properties and dynamic characteristics between MR and (125)I-Mel. The results of weight gain, sucrose consumption and the open-field test showed that our model successfully produced depressive symptoms and depressive-like behavior. The concentration of plasma MT in the model group decreased significantly at night but increased in the MKXP group (P0.05). The maximum binding capacity (B(max)) for specific binding between MR and 125I-Mel in the MKXP group was significantly higher than that in the model group (P0.05). KXP may have a similar effect as ramelteon. KXP improved depressive-like behavior by increasing the concentration of plasma MT and MT1 expression, thereby increasing three B(max) of MR to achieve the desired antidepressant effect.

  16. Synthesis, in vitro validation and in vivo pharmacokinetics of [{sup 125}I]N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl) ethylamine: A high-affinity ligand for imaging sigma receptor positive tumors

    Energy Technology Data Exchange (ETDEWEB)

    John, Christy S; Gulden, Mary E; Vilner, Bertold J; Bowen, Wayne D

    1996-08-01

    N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl)ethylamine, IPEMP, and the corresponding bromo derivative, BrPEMP, have been synthesized and characterized. Both BrPEMP and IPEMP were evaluated for sigma-1 and sigma-2 subtype receptor affinities and found to possess very high affinities for both receptor subtypes. The precursor for radioiodination n-tributylstannylphenylethylpiperidinylethylamine was prepared from its bromo derivative by palladium-catalyzed stannylation reaction. Radioiodinated 4-[{sup 125}I]PEMP was readily prepared in high yields and high specific activity by oxidative iododestannylation reaction using chloramine-T as oxidizing agent. Sites labeled by 4-[{sup 125}I]PEMP in guinea pig brain membranes showed high affinity for BD1008, haloperidol, and (+)-pentazocine (Ki = 5.06 {+-} 0.40, 32.6 {+-} 2.75, and 48.1 {+-} 8.60 nM, respectively), which is consistent with sigma receptor pharmacology. Competition binding studies of 4-[{sup 125}I]PEMP in melanoma (A375) and MCF-7 breast cancer cells showed a high affinity, dose-dependent inhibition of binding with known sigma ligand N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl) ethylamine, BD1008 (Ki = 5, 11 nM, respectively), supporting the labeling of sigma sites in these cells. Haloperidol, however showed a weaker (Ki 100-200 nM) affinity for the sites labeled by 4-[{sup 125}I]PEMP in these cells. Biodistribution studies of 4-[{sup 125}I]PEMP in rats showed a fast clearance of this radiopharmaceutical from blood, liver, lung, and other organs. A co-injection of 4-IPEMP with 4-[{sup 125}I]PEMP resulted in 37%, 69%, and 35% decrease in activity in liver, kidney, and brain (organs possessing sigma receptors), respectively at 1-h postinjection. These results suggest that 4-[{sup 125}I]PEMP is a promising radiopharmaceutical for pursuing further studies in animal models with tumors.

  17. Radiosynthesis and in vitro evaluation of 2-(N-alkyl-N-1'-11C-propyl)amino-5-hydroxytetralin analogs as high affinity agonists for dopamine D-2 receptors

    International Nuclear Information System (INIS)

    Shi Bingzhi; Narayanan, Tanjore K.; Yang, Z.-Y.; Christian, Bradley T.; Mukherjee, Jogeshwar

    1999-01-01

    We have developed radiotracers based on agonists that may potentially allow the in vivo assessment of the high affinity (HA) state of the dopamine D-2 receptors. The population of HA state, which is likely the functional state of the receptor, may be altered in certain diseases. We carried out radiosyntheses and evaluated the binding affinities, lipophilicity, and in vitro autoradiographic binding characteristics of three dopamine D-2 receptor agonists: (±)-2-(N,N-dipropyl)amino-5-hydroxytetralin (5-OH-DPAT), (±)-2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), and (±)-2-(N-cyclohexylethyl-N-propyl)amino-5-hydroxytetralin (ZYY-339). In 3 H-spiperone assays using rat striata, ZYY-339 exhibited subnanomolar affinity for D-2 receptor sites ( IC 50 =0.010 nM), PPHT was somewhat weaker ( IC 50 =0.65 nM), and 5-OH-DPAT exhibited the weakest affinity ( IC 50 =2.5 nM) of the three compounds. Radiosynthesis of these derivatives, 2-(N-propyl-N-1'- 11 C-propyl)amino-5-hydroxytetralin ( 11 C-5-OH-DPAT), 2-(N-phenethyl-N-1'- 11 C-propyl)amino-5-hydroxytetralin ( 11 C-PPHT), and 2-(N-cyclohexylethyl-N-1'- 11 C-propyl)amino-5-hydroxytetralin ( 11 C-ZYY-339) was achieved by first synthesizing 11 C-1-propionyl chloride and subsequent coupling with the appropriate secondary amine precursor to form the respective amide, which was then reduced to provide the desired tertiary amine products. The final products were obtained by reverse-phase high performance liquid chromatography (HPLC) purification in radiochemical yields of 5-10% after 60-75 min from the end of 11 CO 2 trapping and with specific activities in the range of 250-1,000 Ci/mmol. In vitro autoradiographs in rat brain slices with 11 C-5-OH-DPAT, 11 C-PPHT, and 11 C-ZYY-339 revealed selective binding of the three radiotracers to the dopamine D-2 receptors in the striata

  18. Transcriptional regulation of genes related to progesterone production.

    Science.gov (United States)

    Mizutani, Tetsuya; Ishikane, Shin; Kawabe, Shinya; Umezawa, Akihiro; Miyamoto, Kaoru

    2015-01-01

    Steroid hormones are synthesized from cholesterol in various tissues, mainly in the adrenal glands and gonads. Because these lipid-soluble steroid hormones immediately diffuse through the cells in which they are produced, their secretion directly reflects the activity of the genes related to their production. Progesterone is important not only for luteinization and maintenance of pregnancy, but also as a substrate for most other steroids. Steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-HSD) are well-known proteins essential for progesterone production. In addition to them, glutathione S-transferase A1-1 and A3-3 are shown to exert Δ(5)-Δ(4) isomerization activity to produce progesterone in a cooperative fashion with 3β-HSD. 5-Aminolevulinic acid synthase 1, ferredoxin 1, and ferredoxin reductase also play a role in steroidogenesis as accessory factors. Members of the nuclear receptor 5A (NR5A) family (steroidogenic factor 1 and liver receptor homolog 1) play a crucial role in the transcriptional regulation of these genes. The NR5A family activates these genes by binding to NR5A responsive elements present within their promoter regions, as well as to the elements far from their promoters. In addition, various NR5A-interacting proteins including peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear receptor subfamily 0, group B, member 1 (DAX-1), and CCAAT/enhancer-binding proteins (C/EBP) are involved in the transcription of NR5A target genes and regulate the transcription either positively or negatively under both basal and tropic hormone-stimulated conditions. In this review, we describe the transcriptional regulation of genes related to progesterone production.

  19. Intrahippocampal administration of Vitamin C and progesterone ...

    African Journals Online (AJOL)

    Vitamin C and progesterone alone improved spatial memory in comparison to lesion group. Effective doses of vitamin C + effective dose of progesterone had more improving effect on memory. Keywords: Neuroscience, Neurosteroid, Antioxidant, Demylination, Progesterone, Learning and memory impairments, Multiple ...

  20. Disturbances in production of progesterone and their implications in plant studies.

    Science.gov (United States)

    Janeczko, Anna; Oklestkova, Jana; Novak, Ondrej; Śniegowska-Świerk, Katarzyna; Snaczke, Zuzanna; Pociecha, Ewa

    2015-04-01

    Progesterone is a mammalian hormone that has also been discovered in plants but its physiological function in plants is not explained. Experiments using inhibitors of progesterone synthesis and binding would be useful in studies on the significance of this compound in plants. Until now, trilostane and mifepristone have been used in medical sciences as progesterone biosynthesis and binding inhibitors, respectively. We tested these synthetic steroids for the first time in plants and found that they reduced the content of progesterone in wheat. The aim of further experiments was to answer whether the potential disturbances in the production/binding of progesterone, influence resistance to environmental stress (drought) and the development of wheat. Inhibitors and progesterone were applied to plants via roots in a concentration of 0.25-0.5mg/l water. Both inhibitors lowered the activity of CO2 binding enzyme (Rubisco) in wheat exposed to drought stress and trilostane additionally lowered the chlorophyll content. However, trilostane-treated plants were rescued by treatment with exogenous progesterone. The inhibitors also modulated the development of winter wheat, which indicated the significance of steroid regulators and their receptors in this process. In this study, in addition to progesterone and its inhibitors, brassinosteroid (24-epibrassinolide) and an inhibitor of biosynthesis of brassinosteroids were also applied. Mifepristone inhibited the generative development of wheat (like 24-epibrassinolide), while trilostane (like progesterone and an inhibitor of biosynthesis of brassinosteroids) stimulated the development. We propose a model of steroid-induced regulation of the development of winter wheat, where brassinosteroids act as inhibitors of generative development, while progesterone or other pregnane derivatives act as stimulators. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Report: Affinity Chromatography.

    Science.gov (United States)

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  2. Binding of (/sup 3/H) progesterone to normal and neoplastic tissue samples from tumour bearing breasts

    Energy Technology Data Exchange (ETDEWEB)

    Pollow, K; Sinnecker, R; Schmidt-Gollwitzer, M; Boquoi, E; Pollow, B [Institut fuer Molekularbiologie und Biochemie, Frauenklinik Charlottenburg der Freien Universitat, Berlin (G.F.R.)

    1977-01-01

    Macromolecular components of normal human mammary cytosol (obtained from 'non-malignant tissue samples' from cancer bearing breasts) which bind (/sup 3/H)progesterone in vitro were characterized by sucrose gradient centrifugation, gel filtration on Agarose, ion exchange chromatography, isoelectric focusing, competition studies and kinetic parameters. The size of the cytoplasmic binding components vary with the concentration of KCl. In the absence of KCl, the major components are characterized by sedimentation coefficients of about 4 S and 8 S. In solutions containing 0.3M KCl, the cytoplasmic components sediment at 4 S in sucrose gradient. The corticosteroid-binding component of normal human mammary cytosol both sediment at about the same rate in the presence of 0.3M KCl and chromatograph as a single component on Agarose. The isoelectric point of the progesterone-binding component of normal human mammary cytosol was located around pH 5.0. The progesterone-binding component was more thermo-labile than serum CBG. CBG was inactivated at temperatures above 45 deg C but temperature above 20 deg C destroyed specific progesterone receptor binding. Progesterone receptor concentrations in normal mammary cytosol of premenopausal women depended on the menstrual cycle. The binding of progesterone was highest around the time of ovulation. In breast tumor tissue samples the progesterone receptor concentration was lower than in the normal mammary cytosol (obtained in each case from the same tumor-bearing breast). In 5 out of 37 breast tumor samples progesterone binding activity could not be detected.

  3. Estrogen receptor, Progesterone receptor, HER2 status and Ki67 index and responsiveness to adjuvant tamoxifen in postmenopausal high-risk breast cancer patients enrolled in the DBCG 77C trial

    DEFF Research Database (Denmark)

    Knoop, Ann; Lænkholm, Anne Vibeke; Jensen, M. B.

    2014-01-01

    BCRR and BCM in postmenopausal patients with ER positive breast cancers. The relative benefit from tamoxifen was not significantly different in luminal A and B subtypes. Funding: The Danish Breast Cancer Cooperative Group (DBCG) prepared the original protocol (DBCG 77C) and was the sponsor of the study......Background: The DBCG 77C trial compared one year of tamoxifen in postmenopausal, steroid-receptor unknown, high-risk breast cancer patients to no adjuvant systemic therapy. After a potential follow-up of 30 years we report overall efficacy of the study and results according to subtypes subsequently...... assessed by immunohistochemistry and fluorescent in situ hybridisation (FISH). Methods: Between 1977 and 1982, 1716 postmenopausal patients with tumours larger than 5 cm or positive axillary nodes were randomly assigned to no systemic therapy or tamoxifen 30 mg daily for one year. Archival tumour tissue...

  4. (D-Pen2,4 prime -125I-Phe4,D-Pen5)enkephalin: A selective high affinity radioligand for delta opioid receptors with exceptional specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Knapp, R.J.; Sharma, S.D.; Toth, G.; Duong, M.T.; Fang, L.; Bogert, C.L.; Weber, S.J.; Hunt, M.; Davis, T.P.; Wamsley, J.K. (Department of Pharmacology, University of Arizona, College of Medicine, Tucson (United States))

    1991-09-01

    (D-Pen2,4{prime}-125I-Phe4,D-Pen5)enkephalin ((125I)DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. (125I)DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 {plus minus} 67 pM and a receptor density (Bmax) value of 36.4 {plus minus} 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM (125I) DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 {plus minus} 0.88 {times} 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM (3H) DPDPE and 0.8 nM (3H) (D-Pen2,4{prime}-Cl-Phe4, D-Pen5)enkephalin, respectively. The dissociation rate of (125I)DPDPE (0.917 {plus minus} 0.117 {times} 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of (125I)DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes (125I)DPDPE a valuable new radioligand for studies of delta opioid receptors.

  5. Redesigning Protein Cavities as a Strategy for Increasing Affinity in Protein-Protein Interaction: Interferon-gamma Receptor 1 as a Model

    Czech Academy of Sciences Publication Activity Database

    Černý, Jiří; Biedermannová, Lada; Mikulecký, Pavel; Zahradník, Jiří; Charnavets, Tatsiana; Šebo, Peter; Schneider, Bohdan

    2015-01-01

    Roč. 716945, 28 April 2015 (2015) ISSN 2314-6133 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA ČR GAP305/10/2184 Institutional support: RVO:86652036 Keywords : CONFORMATIONAL ENTROPY * MOLECULAR RECOGNITION * BINDING-AFFINITY Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.134, year: 2015

  6. Efficacy of antipsychotic agents at human 5-HT(1A) receptors determined by [3H]WAY100,635 binding affinity ratios: relationship to efficacy for G-protein activation.

    Science.gov (United States)

    Newman-Tancredi, A; Verrièle, L; Touzard, M; Millan, M J

    2001-10-05

    5-HT(1A) receptors are implicated in the aetiology of schizophrenia. Herein, the influence of 15 antipsychotics on the binding of the selective 'neutral' antagonist, [3H]WAY100,635 ([3H]N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-cyclo-hexanecarboxamide), was examined at human 5-HT(1A) receptors expressed in Chinese Hamster Ovary cells. In competition binding experiments, 5-HT displayed biphasic isotherms which were shifted to the right in the presence of the G-protein uncoupling agent, GTPgammaS (100 microM). In analogy, the isotherms of ziprasidone, quetiapine and S16924 (((R-2-[1-[2-(2,3-dihydro-benzo[1,4]dioxin-5-yloxy)-ethyl]-pyrrolidin-3yl]-1-(4-fluoro-phenyl)-ethanone), were displaced to the right by GTPgammaS, consistent with agonist actions. Binding of several other antipsychotics, such as ocaperidone, olanzapine and risperidone, was little influenced by GTPgammaS. Isotherms of the neuroleptics, haloperidol, chlorpromazine and thioridazine were shifted to the left in the presence of GTPgammaS, suggesting inverse agonist properties. For most ligands, the magnitude of affinity changes induced by GTPgammaS (alteration in pK(i) values) correlated well with their previously determined efficacies in [35S]GTPgammaS binding studies [Eur. J. Pharmacol. 355 (1998) 245]. In contrast, the affinity of the 'atypical' antipsychotic agent, clozapine, which is a known partial agonist at 5-HT(1A) receptors, was less influenced by GTPgammaS. When the ratio of high-/low-affinity values was plotted against efficacy, hyperbolic isotherms were obtained, consistent with a modified ternary complex model which assumes that receptors can adopt active conformations in the absence of agonist. In conclusion, modulation of [3H]-WAY100,635 binding by GTPgammaS differentiated agonist vs. inverse agonist properties of antipsychotics at 5-HT(1A) receptors. These may contribute to differing profiles of antipsychotic activity.

  7. Survival analysis according to the receiver tumoral expression profile of the epidermal growth factor - 2 (HER2), the estrogen receptor (ER) and progesterone receptor (RP) in Uruguayan patients with breast cancer

    International Nuclear Information System (INIS)

    Camejo, N.; Gonzalez, V.; Ferrero, L.; Castillo, C.; Delgado, L.; Fresco, R.; Santander, G.; Aguiar, S.; Heinzen, S.; Martinez, A.; Maurizt, S.; Meyer, C.; Sena, G.; Spera, G.; Ubillos, L.; Xavier, F.; Deneo, H.; Aghazarian, M.; Rodriguez, R.; Sabini, G.

    2010-01-01

    Breast cancer (CM), the leading cause of death from cancer in Uruguayan women, is a heterogeneous disease. The study of the expression level of tumor hormone receptor (H R) and Her-2 neu can recognize subtypes with different characteristics. We have previously reported the relationship of these with the clinico pathological features. To analyze the PFS (SVLP) as the biological subtype (patients HR + / HER2 - triple negative (TN) and HER2 +) in Uruguayan patients with breast cancer EI-IIII Methodology: A retrospective study where SVLP 169 cancer patients carrying analyzed breast E I-III, diagnosed between March 2006 and March 2008 from the Clinical Hospital, Military Hospital, INCA and CASMU. SVLP analysis was performed according to different biological subtypes using the Kaplan method Meier and statistical significance of differences was assessed by the log-rank test. Results: The median follow-up was 43 months. At the time of analysis 160 patients (94.7%) are alive and 141 (83.4%) are relapse-free. One hundred twenty-three patients were HR + / HER2 - (72.7%), 32 patients were TN (18.9%) and 14 were HER2 + patients (8.2%). The SVLP to two years for the total of patients was 92.3%, 94% for HR + / HER2 - 91% for TN and 71.4% for HER2 +. Comparing the curves for different subtypes SVLP showed lower for SVLP He r2 + patients compared to patients HR + / HER2 - (p = 0.03) and TN (p 0.11). The median survival was not reached globally or in the subgroup analysis. Conclusions: He r2 + patients have a shorter time to relapse which coincides as reported in the literature. SVLP similar to 2 years and overlapping of curves SVLP Patients HR + / HER2 - and TN not be explained by differences in characteristics clinico pathological

  8. The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region

    DEFF Research Database (Denmark)

    Tapken, Daniel; Steffensen, Thomas Bielefeldt; Leth, Rasmus

    2017-01-01

    Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D...

  9. Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system.

    Science.gov (United States)

    Hur, Byung-ung; Yoon, Jae-bong; Liu, Li-Kun; Cha, Sang-hoon

    2010-11-30

    Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naïve immunoglobulin repertoires without the use of lengthy affinity optimization procedures. Here, we designed a hierarchical phage-displayed antibody library system to generate an enormous diversity of combinatorial Fab fragments (6×10(17)) and attempted to isolate high-affinity Fabs against the human epidermal growth factor receptor (EGFR). A primary antibody library, designated HuDVFab-8L, comprising 4.5×10(9) human naïve heavy chains and eight unspecified human naïve light chains was selected against the EGFR-Fc protein by biopanning, and four anti-EGFR Fab clones were isolated. Because one of the Fab clones, denoted EG-L2-11, recognized a native EGFR expressed on A431 cells, the heavy chain of the Fab was shuffled with a human naïve light chain repertoire with a diversity of 1.4×10(8) and selected a second time against the EGFR-Fc protein again. One EG-L2-11 variant, denoted EG-19-11, recognized an EGFR epitope that was almost the same as that bound by cetuximab and had a K(D) of approximately 540 pM for soluble EGFR, which is about 7-fold higher than that of the FabC225 derived from cetuximab. This variant was also internalized by A431 cells, likely via receptor-mediated endocytosis, and it efficiently inhibited EGF-mediated tyrosine phosphorylation of the EGFR. These results demonstrate that the use of our hierarchical antibody library system is advantageous in generating fully human antibodies especially with a therapeutic purpose. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors

    International Nuclear Information System (INIS)

    Garayoa, Elisa Garcia-; Allemann-Tannahill, Lesley; Blaeuenstein, Peter; Willmann, Martine; Carrel-Remy, Nathalie; Tourwe, Dirk; Iterbeke, Koen; Conrath, Peter; Schubiger, P. August

    2001-01-01

    The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [ 99m Tc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise

  11. Transition Metal Chelator Induces Progesterone Production in Mouse Cumulus-Oocyte Complexes and Corpora Lutea.

    Science.gov (United States)

    Tian, X; Anthony, K; Diaz, Francisco J

    2017-04-01

    Progesterone production is upregulated in granulosa cells (cumulus and mural) after the LH surge, but the intra-follicular mechanisms regulating this transition are not completely known. Recent findings show that the transition metal chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN), impairs ovarian function. In this study, we provide evidence that chelating transition metals, including zinc, enhances progesterone production. The findings show that TPEN (transition metal chelator) increases abundance of Cyp11a1 and Star messenger RNA (mRNA) between 8- and 20-fold and progesterone production more than 3-fold in cultured cumulus-oocyte complexes (COC). Feeding a zinc-deficient diet for 10 days, but not 3 days, increased Star, Hsd3b, and prostaglandin F2 alpha receptor (Ptgfr) mRNA ~2.5-fold, suggesting that the effect of TPEN is through modulation of zinc availability. Progesterone from cumulus cells promotes oocyte developmental potential. Blocking progesterone production with epostane during maturation reduced subsequent blastocyst formation from 89 % in control to 18 % in epostane-treated complexes, but supplementation with progesterone restored blastocyst developmental potential to 94 %. Feeding a zinc-deficient diet for 5 days before ovulation did not affect the number of CL, STAR protein, or serum progesterone. However, incubating luteal tissue with TPEN increased abundance of Star, Hsd3b, and Ptgfr mRNA 2-3-fold and increased progesterone production 3-fold. TPEN is known to abolish SMAD2/3 signaling in cumulus cells. However, treatment of COC with the SMAD2/3 phosphorylation inhibitor, SB421542, did not by itself induce steroidogenic transcripts but did potentiate EGF-induced Star mRNA expression. Collectively, the results show that depletion of transition metals with TPEN acutely enhances progesterone biosynthesis in COC and luteal tissue.

  12. The development of fluoroandrogens and fluoroprogestins as potential imaging agents for receptor-positive prostate and breast tumors

    International Nuclear Information System (INIS)

    Brandes, S.J.; Katzenellenbogen, J.A.

    1986-01-01

    The assay of progesterone receptor (PR) concentration in breast tumors and androgen receptor (AR) concentration in prostate tumors enables hormone responsive neoplasms to be distinguished from those that are non-responsive. In principle, a positron-emitting progestin or androgen with suitably high affinity and selectivity for PR and AR, respectively, and an adequately high specific activity might provide a means for imaging receptor-positive tumors and quantifying their receptor content in vivo. The use of fluorine-18 as a radiolabel, coupled with the use of positron emission transaxial tomography, appears to be a most favorable approach in the development of receptor binding radiopharmaceuticals for in vivo imaging. Therefore, we have begun a systematic investigation of the development of fluorine-substituted androgens and progestins that might be prepared in F-18 labeled form as probes for AR and PR. (author)

  13. Cloning of the human androgen receptor cDNA

    International Nuclear Information System (INIS)

    Govindan, M.V.; Burelle, M.; Cantin, C.; Kabrie, C.; Labrie, F.; Lachance, Y.; Leblanc, G.; Lefebvre, C.; Patel, P.; Simard, J.

    1988-01-01

    The authors discuss how in order to define the functional domains of the human androgen receptor, complementary DNA (cDNA) clones encoding the human androgen receptor (hAR) have been isolated from a human testis λgtll cDNA library using synthetic oligonnucleotide probes, homologous to segments of the human glucocorticoid, estradiol and progesterone receptors. The cDNA clones corresponding to the human glucocorticoid, estradiol and progesterone receptors were eliminated after cross-hybridization with their respective cDNA probes and/or after restriction mapping of the cDNA clones. The remaining cDNA clones were classified into different groups after analysis by restriction digestion and cross-hybridization. Two of the largest cDNA clones from each group were inserted into an expression vector in both orientations. The linearized plasmids were used as templates in in vitro transcription with T7 RNA polymerase. Subsequent in vitro translation of the purified transcripts in rabbit reticulocyte lysate followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) permitted the characterization of the encoded polyeptides. The expressed proteins larger than 30,000 Da were analyzed for their ability to bind tritium-labelled dihydrotestosterone ([ 3 H] DHT) with high affinity and specificity

  14. In Vitro and In Vivo Evaluation of Alexa Fluor 680-Bombesin[7–14]NH2 Peptide Conjugate, a High-Affinity Fluorescent Probe with High Selectivity for the Gastrin-Releasing Peptide Receptor

    Directory of Open Access Journals (Sweden)

    Lixin Ma

    2007-05-01

    Full Text Available Gastrin-releasing peptide (GRP receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN, a 14–amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H2N-glycylglycylglycine-BBN[7–14]NH2 peptide with the following general sequence: H2N-G-G-G-Q-W-A-V-G-H-L-M-(NH2. This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7–14]NH2 conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H2N-G-G-G-BBN[7–14]NH2 in dimethylformamide (DMF. In vitro competitive binding assays, using 125I-Tyr4-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 ± 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7–14]NH2 in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.

  15. Radioimmunoassay of progesterone in unextracted serum

    International Nuclear Information System (INIS)

    Haynes, S.P.; Corcoran, J.M.; Eastman, C.J.; Doy, F.A.

    1980-01-01

    A rapid, precise radioimmunoassay for progesterone in 25 μL of unextracted serum is described. Progesterone is released from its binding protein by adding an optimal amount of cortisol, which binds to the same protein (cortisol binding globulin) as progesterone. The amount of cortisol required does not cross react with the specific progesterone antibody used. This approach considerably shortens assay time and removes a tedious and imprecise stage in the conventional assay of serum progesterone. Results correlated well (r = 0.97) with a method involving organic solvent extraction of progesterone from serum. During the two years we have used this mehod in a busy diagnostic endocrine laboratory, the between-assay precision (CV) for low-, medium-, and high-concentration quality control sera was 12, 7, and 9%, respectively. Data from participation in an independent external quality-control program verified the adequacies of the method

  16. Cutting Edge: The murine high-affinity IgG receptor FcγRIV is sufficient for autoantibody-induced arthritis.

    Science.gov (United States)

    Mancardi, David A; Jönsson, Friederike; Iannascoli, Bruno; Khun, Huot; Van Rooijen, Nico; Huerre, Michel; Daëron, Marc; Bruhns, Pierre

    2011-02-15

    K/BxN serum-induced passive arthritis was reported to depend on the activation of mast cells, triggered by the activating IgG receptor FcγRIIIA, when engaged by IgG1 autoantibodies present in K/BxN serum. This view is challenged by the fact that FcγRIIIA-deficient mice still develop K/BxN arthritis and because FcγRIIIA is the only activating IgG receptor expressed by mast cells. We investigated the contribution of IgG receptors, IgG subclasses, and cells in K/BxN arthritis. We found that the activating IgG2 receptor FcγRIV, expressed only by monocytes/macrophages and neutrophils, was sufficient to induce disease. K/BxN arthritis occurred not only in mast cell-deficient W(sh) mice, but also in mice whose mast cells express no activating IgG receptors. We propose that at least two autoantibody isotypes, IgG1 and IgG2, and two activating IgG receptors, FcγRIIIA and FcγRIV, contribute to K/BxN arthritis, which requires at least two cell types other than mast cells, monocytes/macrophages, and neutrophils.

  17. Estradiol and Progesterone Strongly Inhibit the Innate Immune Response of Mononuclear Cells in Newborns ▿

    Science.gov (United States)

    Giannoni, Eric; Guignard, Laurence; Knaup Reymond, Marlies; Perreau, Matthieu; Roth-Kleiner, Matthias; Calandra, Thierry; Roger, Thierry

    2011-01-01

    Newborns are particularly susceptible to bacterial infections due to qualitative and quantitative deficiencies of the neonatal innate immune system. However, the mechanisms underlying these deficiencies are poorly understood. Given that fetuses are exposed to high concentrations of estradiol and progesterone during gestation and at time of delivery, we analyzed the effects of these hormones on the response of neonatal innate immune cells to endotoxin, bacterial lipopeptide, and Escherichia coli and group B Streptococcus, the two most common causes of early-onset neonatal sepsis. Here we show that at concentrations present in umbilical cord blood, estradiol and progesterone are as powerful as hydrocortisone for inhibition of cytokine production by cord blood mononuclear cells (CBMCs) and newborn monocytes. Interestingly, CBMCs and newborn monocytes are more sensitive to the effects of estradiol and progesterone than adult peripheral blood mononuclear cells and monocytes. This increased sensitivity is associated with higher expression levels of estrogen and membrane progesterone receptors but is independent of a downregulation of Toll-like receptor 2 (TLR2), TLR4, and myeloid differentiation primary response gene 88 in newborn cells. Estradiol and progesterone mediate their anti-inflammatory activity through inhibition of the NF-κB pathway but not the mitogen-activated protein kinase pathway in CBMCs. Altogether, these results suggest that elevated umbilical cord blood concentrations of estradiol and progesterone acting on mononuclear cells expressing high levels of steroid receptors contribute to impair innate immune responses in newborns. Therefore, intrauterine exposure to estradiol and progesterone may participate in increasing susceptibility to infection during the neonatal period. PMID:21518785

  18. Mechanisms and Regulation of Gene Expression by Androgen Receptor in Prostate Cancer

    National Research Council Canada - National Science Library

    Wang, Zhengxin

    2003-01-01

    ...) in the cytoplasm of LNCaP cells. Transient transfection assay revealed that p44 enhances AR-, glucocorticoid receptor-, and progesterone receptor- dependent transcription but not estrogen receptor- or thyroid hormone receptor-dependent transcription...

  19. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Garayoa, Elisa Garcia-; Allemann-Tannahill, Lesley; Blaeuenstein, Peter; Willmann, Martine; Carrel-Remy, Nathalie; Tourwe, Dirk; Iterbeke, Koen; Conrath, Peter; Schubiger, P. August E-mail: schubiger@psi.ch

    2001-01-01

    The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [{sup 99m}Tc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise.

  20. Affinity capillary electrophoresis and density functional theory employed for the characterization of hexaarylbenzene-based receptor complexation with alkali metal ions

    Czech Academy of Sciences Publication Activity Database

    Ehala, Sille; Toman, Petr; Rathore, R.; Makrlík, E.; Kašička, Václav

    2011-01-01

    Roč. 32, č. 9 (2011), s. 981-987 ISSN 0173-0835 R&D Projects: GA ČR(CZ) GA203/08/1428; GA ČR(CZ) GA203/09/0675; GA ČR(CZ) GAP205/10/2280; GA AV ČR 1ET400500402 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z40500505 Keywords : affinity capillary electrophoresis * alkali metal ions * binding constant Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.303, year: 2011

  1. in silico identification of cross affinity towards Cry1Ac pesticidal protein with receptor enzyme in Bos taurus and sequence, structure analysis of crystal proteins for stability.

    Science.gov (United States)

    Ebenezer, King Solomon; Nachimuthu, Ramesh; Thiagarajan, Prabha; Velu, Rajesh Kannan

    2013-01-01

    Any novel protein introduced into the GM crops need to be evaluated for cross affinity on living organisms. Many researchers are currently focusing on the impact of Bacillus thuringiensis cotton on soil and microbial diversity by field experiments. In spite of this, in silico approach might be helpful to elucidate the impact of cry genes. The crystal a protein which was produced by Bt at the time of sporulation has been used as a biological pesticide to target the insectivorous pests like Cry1Ac for Helicoverpa armigera and Cry2Ab for Spodoptera sp. and Heliothis sp. Here, we present the comprehensive in silico analysis of Cry1Ac and Cry2Ab proteins with available in silico tools, databases and docking servers. Molecular docking of Cry1Ac with procarboxypeptidase from Helicoverpa armigera and Cry1Ac with Leucine aminopeptidase from Bos taurus has showed the 125(th) amino acid position to be the preference site of Cry1Ac protein. The structures were compared with each other and it showed 5% of similarity. The cross affinity of this toxin that have confirmed the earlier reports of ill effects of Bt cotton consumed by cattle.

  2. Antihistamines suppress upregulation of histidine decarboxylase gene expression with potencies different from their binding affinities for histamine H1 receptor in toluene 2,4-diisocyanate-sensitized rats

    Directory of Open Access Journals (Sweden)

    Hiroyuki Mizuguchi

    2016-04-01

    Full Text Available Antihistamines inhibit histamine signaling by blocking histamine H1 receptor (H1R or suppressing H1R signaling as inverse agonists. The H1R gene is upregulated in patients with pollinosis, and its expression level is correlated with the severity of nasal symptoms. Here, we show that antihistamine suppressed upregulation of histidine decarboxylase (HDC mRNA expression in patients with pollinosis, and its expression level was correlated with that of H1R mRNA. Certain antihistamines, including mepyramine and diphenhydramine, suppress toluene-2,4-diisocyanate (TDI-induced upregulation of HDC gene expression and increase HDC activity in TDI-sensitized rats. However, d-chlorpheniramine did not demonstrate any effect. The potencies of antihistamine suppressive effects on HDC mRNA elevation were different from their H1R receptor binding affinities. In TDI-sensitized rats, the potencies of antihistamine inhibitory effects on sneezing in the early phase were related to H1R binding. In contrast, the potencies of their inhibitory effects on sneezing in the late phase were correlated with those of suppressive effects on HDC mRNA elevation. Data suggest that in addition to the antihistaminic and inverse agonistic activities, certain antihistamines possess additional properties unrelated to receptor binding and alleviate nasal symptoms in the late phase by inhibiting synthesis and release of histamine by suppressing HDC gene transcription.

  3. Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

    Science.gov (United States)

    Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin

    2016-03-10

    Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

  4. Radiosynthesis and in vitro validation of 3H-NS14492 as a novel high affinity alpha7 nicotinic receptor radioligand

    DEFF Research Database (Denmark)

    Magnussen, Janus H.; Ettrup, Anders; Donat, Cornelius K.

    2015-01-01

    The neuronal alpha 7 nicotinic acetylcholine receptor is a homo-pentameric ligand-gated ion channel that is a promising drug target for cognitive deficits in Alzheimer's disease and schizophrenia. We have previously described 11C-NS14492 as a suitable agonist radioligand for in vivo positron...... emission tomography (PET) occupancy studies of the alpha 7 nicotinic receptor in the pig brain. In order to investigate the utility of the same compound for in vitro studies, 3H-NS14492 was synthesized and its binding properties were characterized using in vitro autoradiography and homogenate binding...... assays in pig frontal cortex. 3H-NS14492 showed specific binding to alpha 7 nicotinic receptors in autoradiography, revealing a dissociation constant (Kd) of 2.1 ± 0.7 nM and a maximum number of binding sites (Bmax) of 15.7±2.0 fmol/mg tissue equivalent. Binding distribution was similar...

  5. Progesterone promotes maternal–fetal tolerance by reducing human maternal T‐cell polyfunctionality and inducing a specific cytokine profile

    Science.gov (United States)

    Eldershaw, Suzy A.; Inman, Charlotte F.; Coomarasamy, Aravinthan; Moss, Paul A. H.; Kilby, Mark D.

    2015-01-01

    Progesterone is a steroid hormone essential for the maintenance of human pregnancy, and its actions are thought to include promoting maternal immune tolerance of the semiallogenic fetus. We report that exposure of maternal T cells to progesterone at physiological doses induced a unique skewing of the cytokine production profile of CD4+ and CD8+ T cells, with reductions not only in potentially deleterious IFN‐γ and TNF‐α production but also in IL‐10 and IL‐5. Conversely, production of IL‐4 was increased. Maternal T cells also became less polyfunctional, focussing cytokine production toward profiles including IL‐4. This was accompanied by reduced T‐cell proliferation. Using fetal and viral antigen‐specific CD8+ T‐cell clones, we confirmed that this as a direct, nonantigen‐specific effect. Yet human T cells lacked conventional nuclear progesterone receptors, implicating a membrane progesterone receptor. CD4+ and CD8+ T cells responded to progesterone in a dose‐dependent manner, with subtle effects at concentrations comparable to those in maternal blood, but profound effects at concentrations similar to those at the maternal–fetal interface. This characterization of how progesterone modulates T‐cell function is important in understanding the normal biology of pregnancy and informing the rational use of progesterone therapy in pregnancies at risk of fetal loss. PMID:26249148

  6. Progesterone promotes maternal-fetal tolerance by reducing human maternal T-cell polyfunctionality and inducing a specific cytokine profile.

    Science.gov (United States)

    Lissauer, David; Eldershaw, Suzy A; Inman, Charlotte F; Coomarasamy, Aravinthan; Moss, Paul A H; Kilby, Mark D

    2015-10-01

    Progesterone is a steroid hormone essential for the maintenance of human pregnancy, and its actions are thought to include promoting maternal immune tolerance of the semiallogenic fetus. We report that exposure of maternal T cells to progesterone at physiological doses induced a unique skewing of the cytokine production profile of CD4(+) and CD8(+) T cells, with reductions not only in potentially deleterious IFN-γ and TNF-α production but also in IL-10 and IL-5. Conversely, production of IL-4 was increased. Maternal T cells also became less polyfunctional, focussing cytokine production toward profiles including IL-4. This was accompanied by reduced T-cell proliferation. Using fetal and viral antigen-specific CD8(+) T-cell clones, we confirmed that this as a direct, nonantigen-specific effect. Yet human T cells lacked conventional nuclear progesterone receptors, implicating a membrane progesterone receptor. CD4(+) and CD8(+) T cells responded to progesterone in a dose-dependent manner, with subtle effects at concentrations comparable to those in maternal blood, but profound effects at concentrations similar to those at the maternal-fetal interface. This characterization of how progesterone modulates T-cell function is important in understanding the normal biology of pregnancy and informing the rational use of progesterone therapy in pregnancies at risk of fetal loss. © 2015 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Solution and solid-state studies on the halide binding affinity of perfluorophenyl-armed uranyl-salophen receptors enhanced by anion-π interactions

    Energy Technology Data Exchange (ETDEWEB)

    Leoni, Luca; Mele, Andrea; Giannicchi, Ilaria; Mihan, Francesco Yafteh; Dalla Cort, Antonella [Dipartimento di Chimica and IMC-CNR, Universita di Roma La Sapienza (Italy); Puttreddy, Rakesh; Jurcek, Ondrej; Rissanen, Kari [University of Jyvaeskylae, Department of Chemistry, Nanoscience Center (Finland)

    2016-12-23

    The enhancement of the binding between halide anions and a Lewis acidic uranyl-salophen receptor has been achieved by the introduction of pendant electron-deficient arene units into the receptor skeleton. The association and the occurrence of the elusive anion-π interaction with halide anions (as tetrabutylammonium salts) have been demonstrated in solution and in the solid state, providing unambiguous evidence on the interplay of the concerted interactions responsible for the anion binding. (copyright 2016 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim)

  8. A Gata2-Dependent Transcription Network Regulates Uterine Progesterone Responsiveness and Endometrial Function

    Directory of Open Access Journals (Sweden)

    Cory A. Rubel

    2016-10-01

    Full Text Available Altered progesterone responsiveness leads to female infertility and cancer, but underlying mechanisms remain unclear. Mice with uterine-specific ablation of GATA binding protein 2 (Gata2 are infertile, showing failures in embryo implantation, endometrial decidualization, and uninhibited estrogen signaling. Gata2 deficiency results in reduced progesterone receptor (PGR expression and attenuated progesterone signaling, as evidenced by genome-wide expression profiling and chromatin immunoprecipitation. GATA2 not only occupies at and promotes expression of the Pgr gene but also regulates downstream progesterone responsive genes in conjunction with the PGR. Additionally, Gata2 knockout uteri exhibit abnormal luminal epithelia with ectopic TRP63 expressing squamous cells and a cancer-related molecular profile in a progesterone-independent manner. Lastly, we found a conserved GATA2-PGR regulatory network in both human and mice based on gene signature and path analyses using gene expression profiles of human endometrial tissues. In conclusion, uterine Gata2 regulates a key regulatory network of gene expression for progesterone signaling at the early pregnancy stage.

  9. Chronic potassium depletion increases adrenal progesterone production that is necessary for efficient renal retention of potassium.

    Science.gov (United States)

    Elabida, Boutaïna; Edwards, Aurélie; Salhi, Amel; Azroyan, Anie; Fodstad, Heidi; Meneton, Pierre; Doucet, Alain; Bloch-Faure, May; Crambert, Gilles

    2011-08-01

    Modern dietary habits are characterized by high-sodium and low-potassium intakes, each of which was correlated with a higher risk for hypertension. In this study, we examined whether long-term variations in the intake of sodium and potassium induce lasting changes in the plasma concentration of circulating steroids by developing a mathematical model of steroidogenesis in mice. One finding of this model was that mice increase their plasma progesterone levels specifically in response to potassium depletion. This prediction was confirmed by measurements in both male mice and men. Further investigation showed that progesterone regulates renal potassium handling both in males and females under potassium restriction, independent of its role in reproduction. The increase in progesterone production by male mice was time dependent and correlated with decreased urinary potassium content. The progesterone-dependent ability to efficiently retain potassium was because of an RU486 (a progesterone receptor antagonist)-sensitive stimulation of the colonic hydrogen, potassium-ATPase (known as the non-gastric or hydrogen, potassium-ATPase type 2) in the kidney. Thus, in males, a specific progesterone concentration profile induced by chronic potassium restriction regulates potassium balance.

  10. Crystal Structure of Human Interferon-[lamda]1 in Complex with Its High-Affinity Receptor Interferon-[lamda]R1

    Energy Technology Data Exchange (ETDEWEB)

    Miknis, Zachary; Magracheva, Eugenia; Li, Wei; Zdanov, Alexander; Kotenko, Sergei V.; Wlodawer, Alexander (NJMS); (NCI)

    2010-12-01

    Interferon (IFN)-{lambda}1 [also known as interleukin (IL)-29] belongs to the recently discovered group of type III IFNs. All type III IFNs initiate signaling processes through formation of specific heterodimeric receptor complexes consisting of IFN-{lambda}R1 and IL-10R2. We have determined the structure of human IFN-{lambda}1 complexed with human IFN-{lambda}R1, a receptor unique to type III IFNs. The overall structure of IFN-{lambda}1 is topologically similar to the structure of IL-10 and other members of the IL-10 family of cytokines. IFN-{lambda}R1 consists of two distinct domains having fibronectin type III topology. The ligand-receptor interface includes helix A, loop AB, and helix F on the IFN site, as well as loops primarily from the N-terminal domain and inter-domain hinge region of IFN-{lambda}R1. Composition and architecture of the interface that includes only a few direct hydrogen bonds support an idea that long-range ionic interactions between ligand and receptor govern the process of initial recognition of the molecules while hydrophobic interactions finalize it.

  11. Overview of progesterone profiles in dairy cows

    DEFF Research Database (Denmark)

    Blavy, P.; Derks, M.; Martin, O.

    2016-01-01

    The aim of this study was to gain a better understanding of the variability in shape and features of all progesterone profiles during oestrus cycles in cows, and to create templates for cycle shapes and features as a base for further research. Milk progesterone data from 1418 oestrus cycles, coming...

  12. Effects of phytoestrogens genistein and daidzein on progesterone and estrogen (estradiol) production of human term trophoblast cells in vitro.

    Science.gov (United States)

    Richter, Dagmar Ulrike; Mylonas, Ioannis; Toth, Bettina; Scholz, Christoph; Briese, Volker; Friese, Klaus; Jeschke, Udo

    2009-01-01

    Phytoestrogens are a diverse group of nonsteroidal plant compounds that occur naturally in many plants. Because they possess a ring system similar to estrogens they are able to bind on estrogen receptors alpha and beta in humans. The effects of the phytoestrogens genistein and daidzein on the production of progesterone and estrogen in isolated human term trophoblast cells in vitro were tested in this study. Cytotrophoblast cells were isolated from human term placentas. Phytoestrogens genistein and daidzein were incubated in different concentrations with trophoblast cells. Untreated cells were used as controls. After 24 h aliquots were removed and tested for progesterone and estrogen production. The production of the steroid hormones progesterone and estrogen are influenced by phytoestrogens genistein and daidzein in human term trophoblast cells. A strong inhibition effect of both phytoestrogens tested in the production of progesterone was demonstrated. In addition, a significant stimulating effect on estrogen production by genistein and daidzein was observed. Results obtained with this study show that phytoestrogens (genistein and daidzein) sufficiently reduce progesterone production in human term trophoblast cells. Because blockade of progesterone is a possible mechanism involved in initiation of labor, we may speculate that high doses of phytoestrogens at the feto-maternal interphase could play a negative role in maintenance of pregnancy. Stimulation of estrogen production by genistein and daidzein in trophoblast cells is probably due to estrogen receptor blocking effects of both phytoestrogens. Trophoblast cells seem to compensate blocking of its estrogen receptors by higher estrogen production.

  13. Native CB1 receptor affinity, intrinsic activity and accumbens shell dopamine stimulant properties of third generation SPICE/K2 cannabinoids: BB-22, 5F-PB-22, 5F-AKB-48 and STS-135.