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Sample records for 17beta-estradiol e2 plasmatico

  1. Removal of 17beta-estradiol (E2) and its chlorination by-products from water and wastewater using non-imprinted polymer (NIP) particles.

    PubMed

    Murray, Audrey; Ormeci, Banu; Lai, E P C

    2011-01-01

    Endocrine disrupting compounds and their chlorination by-products are two classes of emerging contaminants. Surface water and wastewater treatment technologies have limitations in removing these contaminants. This study evaluated the ability of non-imprinted polymer particles (NIP) to remove the endocrine disruptor 17beta-estradiol (E2) and its chlorination by-products from water and wastewater. NIP effectively removed 98% of 10 mg/L E2 from wastewater. NIP were also effective in removing chlorination by-products of E2 by 84.9% after 10 mg/L E2 in water was chlorinated at 5 mg/L. In the presence of 5 mg/L humic acid, NIP were able to achieve removal of 10 mg/L E2 by greater than 99.9%. Furthermore, after chlorination of 10 mg/L E2 and 5 mg/L humic acid at 10 mg/L chlorine, NIP were also able to remove the chlorination by-products formed as well as the remaining E2 by greater than 99.9%. The presence of 5 mg/L humic acid did not adversely affect the adsorption efficiency. The results of this research indicate that NIPs have good potential as a final treatment step for surface water and wastewater treatment. PMID:22214083

  2. [Biometric parameters of the uterus, ovaries and levels of 17 beta-estradiol (E2) after delivery in sheep].

    PubMed

    Krajnicáková, M; Elecko, J; Bekeová, E; Maracek, I; Hendrichovský, V

    1990-12-01

    Biometric changes of uterus, ovaries, follicles and 17 beta-oestradiol (E2) concentrations were investigated in 15 lambing ewes of the Slovak Merino breed in the puerperal period. The sex organs were excised immediately after bleeding from ewes slaughtered on days 1, 7, 17, 25 and 34 post partum (p. p.). Biometric parameters of the body and horns of uterus were measured by a calliper. The ovaries were weighed on an analytical balance, their length, width and height were measured at the same time. The size and number of follicles were determined on the ovary surface. The blood for E2 detection was collected from vena jugularis three and one day before delivery (days -1, -3). Blood samples were also collected after delivery on days 1, 7, 17, 25 and 34. E2 concentrations in the blood serum of ewes were determined by RIA-test-ESTRA kits, designed in one institute at Kosice. The highest weight of uterus body in the test ewes was recorded on day 1 p. p. In the following days the weight of uterus body had a decreasing trend. There were significant differences in the weight of uterus body from day 17 to day 34 p. p., in comparison with the first day after lambing (P less than 0.01). A significant decrease in the length of uterus body was observed from day 17 to day 34 of observation (P less than 0.01; P less than 0.001). An increase in the length of a nongravid horn, observed on day 7 p. p., was followed by a gradual decrease until day 34, similarly like in its weight. No statistically significant differences were found out in the ovary length, width and height. Neither were any greater changes recorded in the weight of ovaries from day 1 to day 34 after delivery. The highest number of small structures (28) observed on day 7 p. p. in the ipsilateral ovary was decreasing in the course of puerperium and the number of follicles larger than 2, 4 and 5 mm was increasing. The highest concentrations of E2 were not recorded on day -1 before delivery. The significantly lowest concentrations of E2 were recorded on day 25 p. p. The above-mentioned results are preliminary and they enlarge the knowledge of biometric parameters of uterus, ovaries, follicles and E2 concentrations after delivery in ewes. PMID:2100427

  3. Effects of endocrine disruptors on genes associated with 17beta-estradiol metabolism and excretion.

    PubMed

    Hanet, Nathalie; Lancon, Allan; Delmas, Dominique; Jannin, Brigitte; Chagnon, Marie-Christine; Cherkaoui-Malki, Moustapha; Latruffe, Norbert; Artur, Yves; Heydel, Jean-Marie

    2008-11-01

    In order to provide a global analysis of the effects of endocrine disruptors on the hormone cellular bioavailability, we combined 17beta-estradiol (E2) cellular flow studies with real-time PCR and Western blot expression measurements of genes involved in the hormone metabolism and excretion. Three endocrine disruptors commonly found in food were chosen for this study, which was conducted in the estrogen receptor (ER) negative hepatoblastoma HepG2 cell line: bisphenol A (BPA), genistein (GEN) and resveratrol (RES). We showed that 24 h after a single dose treatment with genistein, resveratrol or bisphenol A, the expression of ATP-binding cassette transporters (the multidrug resistance or MDR, and the multidrug resistance associated proteins or MRP) uridine diphosphate-glucuronosyltransferases (UGT) and/or sulfotransferases (ST) involved in 17beta-estradiol elimination process were significantly modulated and that 17beta-estradiol cellular flow was modified. Resveratrol induced MDR1 and MRP3 expressions, bisphenol A induced MRP2 and MRP3 expressions, and both enhanced 17beta-estradiol efflux. Genistein, on the other hand, inhibited ST1E1 and UGT1A1 expressions, and led to 17beta-estradiol cellular retention. Thus, we demonstrate that bisphenol A, genistein and resveratrol modulate 17beta-estradiol cellular bioavailability in HepG2 and that these modulations most probably involve regulations of 17beta-estradiol phase II and III metabolism proteins. Up to now, the estrogenicity of environmental estrogenic pollutants has been based on the property of these compounds to bind to ERs. Our results obtained with ER negative cells provide strong evidence for the existence of ER-independent pathways leading to endocrine disruption. PMID:18634814

  4. Occurrence and Pathways of Manure-borne 17beta-Estradiol in Vadose Zone Water

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reproductive hormones, such as 17beta-estradiol (E2), can cause physiological and reproductive disorders in numerous species at low part per trillion concentrations. The persistence and transport pathways of manure-borne E2 in agricultural soils were determined by comparing the occurrence of E2 in t...

  5. Sexual behavior and impregnation success of adult male mosquitofish following exposure to 17beta-estradiol.

    PubMed

    Doyle, Christopher J; Lim, Richard P

    2005-07-01

    The effects of 17beta-estradiol (E2) on the sexual activity of adult male mosquitofish (Gambusia holbrooki) were assessed. Sexually mature males were exposed to nominal concentrations of 20, 100, and 500 ng/L of E2 and a solvent control (0.00001% ethanol v/v) for 84 days under continuous flow conditions. Following exposure, males exposed to E2 at concentrations of 20 ng/L and above displayed a lower level of sexual activity, measured as the number of approaches and copulation attempts made toward nonexposed females, than the control males. In addition, E2-exposed males were less capable of impregnating females than the control males. Exposure to E2 had no significant effect on spermatozeugmata production or the secondary sexual characteristics, indicating that the reduced impregnation efficiency of the E2-exposed males was most likely due to the decline in sexual activity. PMID:15922805

  6. Modeling of Coupled Degradation, Sorption, and Transport of 17beta-Estradiol in Undisturbed Soil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence of 17 beta-estradiol in the environment, even at part-per trillion concentrations, may raise significant concern regarding the health of aquatic organisms. Once 17 beta-estradiol is released into the environment from human and animal sources, its fate and transport is controlled by fact...

  7. Regulation of cytoplasmic dihydrotestosterone binding in dog prostate by 17 beta-estradiol.

    PubMed Central

    Moore, R J; Gazak, J M; Wilson, J D

    1979-01-01

    17 beta-estradiol enchances androgen-induced prostate growth in the castrate dog to a degree comparable to that seen in spontaneous prostatic hypertrophy. To investigate the mechanism of this synergism, cytosol androgen binding was measured by a density gradient technique in prostates of control and 17 beta-estradiol-treated castrate dogs. [3H]Dihydrotestosterone was bound principally to a moiety that averaged 8.6S in size. Approximately twofold enhancement of this binding by 17 beta-estradiol was demonstrable after 1 wk of treatment with 750 microgram/wk and after 3 wk with 75 microgram/wk. Under conditions in which binding in the 8S region was demonstrable with dihydrotestosterone and testosterone no binding of 3 alpha-androstanediol or progesterone was detectable. Thus, enhancement by 17 beta-estradiol of a prostate cytosol androgen-binding protein occurs under circumstances in which 17 beta-estradiol enhances androgen-mediated prostatic growth. PMID:219036

  8. O dealkylation and aliphatic and aromatic hydroxylation of 3-methoxy-17 beta-estradiol by Aspergillus alliaceus.

    PubMed Central

    Williamson, J S; Van Orden, D E; Rosazza, J P

    1989-01-01

    Aspergillus alliaceus UI 315 was examined for its ability to metabolize 3-methoxy-17 beta-estradiol. Preparative-scale incubations with this substrate afforded good yields of 6 beta-hydroxy-17 beta-estradiol, 4-hydroxy-17 beta-estradiol, and 4,6 beta-dihydroxy-17 beta-estradiol, which were identified by high-pressure liquid chromatography, 1H and 13C nuclear magnetic resonance, and high-resolution mass spectrometry. PMID:2624469

  9. Low-dosage micronized 17 beta-estradiol prevents bone loss in postmenopausal women

    NASA Technical Reports Server (NTRS)

    Ettinger, B.; Genant, H. K.; Steiger, P.; Madvig, P.

    1992-01-01

    With the use of a double-blind, randomized, dose-ranging design, we tested during an 18-month period the degree of protection against postmenopausal bone loss afforded by micronized 17 beta-estradiol in dosages of 0.5, 1.0, and 2.0 mg. All subjects received supplementation to ensure a minimum of 1500 mg calcium daily. Fifty-one subjects completed at least 1 year of follow-up bone density measurements by quantitative computed tomography and by single- and dual-photon absorptiometry. In the placebo group spinal trabecular bone density decreased 4.9% annually (p less than 0.001), whereas in those taking micronized 17 beta-estradiol bone density tended to increase (annual increases of 0.3% in the 0.5 mg micronized 17 beta-estradiol group, 1.8% in the 1.0 mg micronized 17 beta-estradiol group, and 2.5% in the 2.0 mg micronized 17 beta-estradiol group). After completing the double-blind phase, 41 subjects completed an additional 18 months of follow-up while taking 1.0 mg micronized 17 beta-estradiol. During this time one third of the subjects were randomly assigned to discontinue calcium supplements. Among those who previously received placebo, trabecular bone density increased 4.3% annually, whereas among those who had used micronized 17 beta-estradiol, trabecular bone density response was inversely related to the dosage previously used. Additionally and independently, the level of calcium intake showed a statistically significant correlation with the change in spinal trabecular bone density (r = 0.37, p = 0.02). We conclude that micronized 17 beta-estradiol has a continuous skeletal dose-response effect in the range of 0.5 to 2.0 mg and that calcium intake positively modifies the skeletal response to 1.0 mg micronized 17 beta-estradiol.

  10. Influence factor of 17beta-estradiol photodegradation by heterogeneous Fenton reaction.

    PubMed

    Zhao, Yaping; Huang, Minsheng; Ge, Ming; Tang, Xiaochun; Liu, Lu

    2010-01-01

    The photocatalytic degradation of 17beta-estradiol (E2), by Fenton like reaction was investigated as a function of E2 concentrations, organic co-solvents and co-existing estrogens, humic acid (HA) and other background anions. E2 degradation was effectively achieved by hydroxyl radicals that were generated in the heterogeneous photo-Fenton process. The degradation kinetics were fitted to Langmuir-Hinshelwood model with kr = 0.3140 microM/h and Kads = 2.2146L/micromol. The removal kinetics of E2 were initiated by a rapid decay and then followed by a much slower one in acetonitrile-water solutions while in methanol-water solutions they followed the first-kinetic model for the diffusion-control of hydroxyl radicals and competition between E2 and co-solvents. In addition, the lower level of co-existing substances did not significantly influence the oxidation efficiency of E2. The degradation rates of E2 were found to depend not only on the concentrations of hydrogen peroxide and iron content as reported before but also on pH, E2 concentrations and composition of co-solvents. Thus it is very important to look for the optimum conditions for the purpose of most efficiently eliminating E2 from drinking water. PMID:20082022

  11. Concentration of 17beta-estradiol using an immunoaffinity porous hollow-fiber membrane.

    PubMed

    Nishiyama, S; Goto, A; Saito, K; Sugita, K; Tamada, M; Sugo, T; Funami, T; Goda, Y; Fujimoto, S

    2002-10-01

    We describe attempts to achieve high throughput of 17beta-estradiol (E2) analysis, including the development of an immunocleanup membrane using polyclonal antibodies and an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies. An epoxy-group-containing monomer, glycidyl methacrylate (GMA), was graft-polymerized onto a porous hollow-fiber membrane. Subsequently, anti-estrogen (ES) antibody, as a ligand, was coupled with the epoxy group. The ligand density ranged from 3.1 to 5.8 mg/g of the GMA-grafted porous hollow-fiber membrane. A 1.0 microg/L E2 solution was forced to permeate through pores rimmed by the anti-ES-antibody-immobilized polymer chains, at a constant permeation rate. A breakthrough curve, that is, the change in the E2 concentration of the effluent penetrating the outside of the hollow fiber with a change of the effluent volume, was determined. Bound E2 in amounts ranging from 0.42 to 0.80 microg was quantitatively eluted with 3-5 mL of methanol in the permeation mode. The higher permeation rate of the E2 solution resulted in the higher overall binding rate of E2 to the anti-ES-antibody-immobilized porous hollow-fiber membrane because of the negligible diffusional mass-transfer resistance of E2 to the antibody. PMID:12380815

  12. Degradation and metabolite formation of 17beta-estradiol-3-sulphate in New Zealand pasture soils.

    PubMed

    Scherr, Frank F; Sarmah, Ajit K; Di, Hong J; Cameron, Keith C

    2009-02-01

    Estrogens-sulphates such as 17beta-estradiol-3-sulphate and estrone-3-sulphate are excreted by livestock in the urine. These conjugates are precursors to the free counterparts 17beta-estradiol and estrone, which are endocrine disrupting chemicals. In this study microcosm laboratory experiments were conducted in three pasture soils from New Zealand to study the aerobic degradation and metabolite formation kinetics of 17beta-estradiol-3-sulphate at three different incubation temperatures. The degradation of 17beta-estradiol-3-sulphate followed a first-order kinetic and the temperature dependence of the rate constants was sufficiently described by the Arrhenius equation. Degradation was different between the three investigated soils and the rate constants across the soils were significantly correlated to the arylsulphatase activity at 7.5 and 15 degrees C. Estrone-3-sulphate and 17beta-estradiol were identified as primary metabolites and estrone as a secondary metabolite. Results suggest arylsulphatase activity originating from soil microbial biomass is the main driver for the degradation of 17beta-estradiol-3-sulphate. PMID:18694598

  13. Comparison of three enzyme immunoassays for measuring 17beta-estradiol in flushed dairy manure wastewater.

    PubMed

    Hanselman, Travis A; Graetz, Donald A; Wilkie, Ann C

    2004-01-01

    Natural steroidal estrogens are an environmental concern because low nanogram per liter concentrations in water can adversely affect aquatic vertebrate species by disrupting the normal function of their endocrine systems. There is a critical need to accurately measure estrogens in dairy wastes, a potential source of estrogens such as 17beta-estradiol, to assess the risk of estrogen contamination of agricultural drainage waters resulting from land application. Commercially available enzyme immunoassay (EIA) kits have been used for measuring 17beta-estradiol in livestock manure, but it is not known if different EIAs provide similar results. We compared three EIAs by measuring 17beta-estradiol in two samples of flushed dairy manure wastewater (FDMW). The measured concentrations of 17beta-estradiol in FDMW differed according to the immunoassay used. The differences were attributed to a matrix interference associated with coextracted humic substances. Future research should develop methods that enable routine measurement of 17beta-estradiol in livestock wastes by more conclusive analytical techniques such as gas chromatography-mass spectrometry. PMID:15356254

  14. Biodegradation of endocrine disrupters: case of 17beta-estradiol and bisphenol A.

    PubMed

    Stavrakakis, C; Hequet, V; Faur, C; Andres, Y; Le Cloirec, P; Colin, R

    2008-03-01

    The biodegradation of 17 beta-estradiol (E2) and bisphenol A (BPA) was compared to that of a reference pollutant, sodium benzoate (SB), known for its high biodegradability. The biodegradation was measured using the Sturm test (ISO 9439 modified Sturm test). The susceptibility of the target pollutants to be degraded by microorganisms of activated sludge from a wastewater treatment plant (WWTP) was evaluated by the production of carbon dioxide (CO2). Sorption experiments onto inactivated sludge were carried out to assess the contribution of sorption in E2 and BPA removal during biological treatment in a WWTP. E2 was more adsorbed than BPA onto inactivated sludge, probably making it less accessible to assimilation by microorganisms. In fact, E2 was less biodegradable than BPA with 66% and 74% of theoretical CO2 formation (Th(co2)) in 28 days, respectively. However, E2 showed faster biodegradation than BPA due to the shorter adaptation time of the microorganisms to start the assimilation. Final concentrations were measured and revealed that, under Sturm test conditions, E2 was totally removed from the aqueous phase while some traces of BPA were detected. This result could be explained by the lower adsorbability of BPA observed in adsorption experiments onto inactivated sludge. To investigate competition in a bi-component solution, Sturm tests were carried out with BPA/SB and E2/SB. Moreover, the biodegradation curves obtained did not indicate a toxicity of the target compounds towards microorganisms, which rapidly degraded SB. In the case of BPA/SB, an inflection in the curve confirmed the adaptation time of 4-5 days for BPA to be degraded. PMID:18610788

  15. Toxicity of 17 {beta}-estradiol and dibutyl-n-phthalate to Japanese medaka (Oryzias latipes)

    SciTech Connect

    Patvna, P.J.; Cooper, K.R.

    1995-12-31

    Phthalate esters are ubiquitous environmental contaminants that are hypothesized to cause developmental toxicity in aquatic organisms via an estrogenic mechanism. Japanese medaka embryos and larvae provide an excellent model for the study of toxicant effects on embryonic development. The following groups were examined (N = 10--20): a non-treatment control, a vehicle control, 17 {beta}-estradiol and Dibutyl-n-phthalate, in individual glass vials. The medaka embryos were treated beginning at the blastula stage, for ten days. At day 10, embryos were changed into fresh rearing solution. The embryos were observed daily, until three days post-hatching, for toxic developmental effects. Exposure to 17 {beta}-estradiol caused urinary bladder lesions at the lowest doses tested. At concentrations {le} 3 {micro}M/0.82 ppm, 17 {beta}-estradiol caused inhibition of swim bladder inflation, pericardial edema, and marked cachexia. Dibutyl-n-phthalate caused pronounced enlargement of the urinary bladder. No other gross lesions were observed. Both 17 {beta}-estradiol and Dibutyl-n-phthalate caused effects on the urinary tract which will be characterized at the light microscopic level. The lesions observed in the embryo medaka following Dibutyl-n-phthalate exposure were at or below water solubility and are in agreement with previously reported toxic levels.

  16. Persistence and Fate of 17beta-estradiol and testosterone in agricultural soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Steroidal hormones are constantly released into the environment by man-made and natural sources. The goal of this study was to examine the persistence and fate of 17beta-estradiol and testosterone, the two primary natural hormones. Incubation experiments were conducted under aerobic and anaerobic co...

  17. 17Beta-estradiol epoxidation as the molecular basis for breast cancer initiation and prevention.

    PubMed

    Yu, Fu-Li

    2002-01-01

    Epidemiological and animal studies have indicated that 17beta-estradiol (E2) is involved in breast cancer; however, the mechanism is unclear. We found that E2 could be activated by epoxidation, resulting in its ability to inhibit nuclear DNA-dependent RNA synthesis, and to bind DNA, forming DNA adducts both in vitro and in vivo. Because epoxidation is required for the activation of many chemical carcinogens, including benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene and aflatoxins, we proposed previously that E2 epoxidation is the underlying mechanism for the initiation of breast cancer. The first part of this review is to present the experimental evidence obtained from this laboratory in support of this hypothesis. Based on these newly discovered insights on E2 epoxidation and its initiation role in breast cancer carcinogenesis, a method to screen chemopreventive agents against breast cancer has been developed. This constitutes the second part of the review. Two examples will be used to illustrate the utility of this screening technique. The effect of fat on breast cancer has been a longstanding but unresolved issue. Epidemiological studies provide conflicting results regarding the association of dietary fat and breast cancer. Because vegetable oils contain various amount of mono- and polyunsaturated fatty acids, they are potential antioxidants. Data are presented to show that commercial vegetable oils, independent of their mono- or polyunsaturated fatty acid content, are all able to prevent the formation of E2 epoxide, as measured by the loss of the ability of E2 to inhibit nuclear RNA synthesis in vitro. Tamoxifen (TAM), an anti-estrogen used for breast cancer treatment, has recently been found to have a strong breast cancer preventive effect. The mechanism for this is unknown. Using the same screening technique, we found that when incubated together with E2 for epoxidation, TAM was able to prevent the formation of E2 epoxide, as evidenced by both the loss of the ability of E2 to inhibit nuclear RNA synthesis and the reduced binding of [3H]-labelled E2 to nuclear DNA in a dose-dependent manner. These experimental results suggest that the breast cancer preventive effect of TAM is to prevent the formation of E2 epoxide through a competitive epoxidation mechanism with E2. PMID:12492635

  18. An enriched environment and 17-beta estradiol produce similar pro-cognitive effects on ovariectomized rats.

    PubMed

    Ortiz-Pérez, A; Espinosa-Raya, J; Picazo, O

    2016-02-01

    Estrogen depletion due to aging, surgery or pathological events can cause a multitude of problems, including neurodegenerative alterations. In rodents without ovaries, 17-beta estradiol (E2) has been shown to produce beneficial effects on cognition, stimulating brain regions (e.g., the neocortex, hippocampus and amygdala) related to cognition and learning. Another treatment that stimulates these brain regions is an enriched environment (EE), which is a complex set of external factors in the immediate surroundings that facilitates greater stimulation of sensorial, cognitive and motor circuits of the brain. The aim of the present study was to test, using an animal model of ovariectomy-induced impairment of memory, the relative effect of E2 (with a time-released pellet; 1 μg/rat/day), EE exposure and a combination of both treatments. Experimental and control groups were submitted to two memory tests 18 weeks post-surgery: the autoshaping learning task (ALT) for measuring associative learning and the novel object recognition test (NORT) for evaluating short- and long-term memory. To assess potential motor impairments caused by treatments, all rats were tested after the ALT in an automatic activity counter. Results from ALT show that the ovariectomy blocked the conditioned responses displayed, an effect rescued by chronic treatment with estrogen or EE exposure. The combination of both treatments did not improve the results obtained separately. In the NORT, the exploration time for recognizing a novel object was similar in the short run with all groups, but greater in the long run with hormone administration or EE exposure. As with the ALT, in the NORT there was no improvement shown by the combination treatment. These data were not masked by changes in spontaneous activity because this parameter was not modified in the rats by either treatment. Possible action mechanisms are proposed, taking into account the role of corticosterone and BDNF on cognition. PMID:26872959

  19. 17Beta-estradiol enhances leptin expression in human placental cells through genomic and nongenomic actions.

    PubMed

    Gambino, Yésica P; Maymó, Julieta L; Pérez-Pérez, Antonio; Dueñas, José L; Sánchez-Margalet, Víctor; Calvo, Juan Carlos; Varone, Cecilia L

    2010-07-01

    The process of embryo implantation and trophoblast invasion is considered the most limiting factor in the establishment of pregnancy. Leptin was originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, it has been suggested that leptin is involved in other functions during pregnancy, particularly in the placenta, where it was found to be expressed. In the present work, we have found a stimulatory effect of 17beta-estradiol (E(2)) on endogenous leptin expression, as analyzed by Western blot, in both the BeWo choriocarcinoma cell line and normal placental explants. This effect was time and dose dependent. Maximal effect was achieved at 10 nM in BeWo cells and 1 nM in placental explants. The E(2) effects involved the estrogen receptor, as the antagonist ICI 182 780 inhibited E(2)-induced leptin expression. Moreover, E(2) treatment enhanced leptin promoter activity up to 4-fold, as evaluated by transient transfection with a plasmid construction containing the leptin promoter region and the reporter gene luciferase. This effect was dose dependent. Deletion analysis demonstrated that a minimal promoter region between -1951 and -1847 bp is both necessary and sufficient to achieve E(2) effects. Estradiol action involved estrogen receptor 1, previously known as estrogen receptor alpha, as cotransfection with a vector encoding estrogen receptor 1 potentiated the effects of E(2) on leptin expression. Moreover, E(2) action probably involves membrane receptors too, as treatment with an estradiol-bovine serum albumin complex partially enhanced leptin expression. The effects of E(2) could be blocked by pharmacologic inhibition of MAPK and the phosphoinositide-3-kinase (PI3K) pathways with 50 microM PD98059 and 0.1 microM Wortmannin, respectively. Moreover, cotransfection of dominant negative mutants of MAP2K or MAPK blocked E(2) induction of leptin promoter. On the other hand, E(2) treatment promoted MAPK1/MAPK3 and AKT phosphorylation in placental cells. In conclusion, we provide evidence suggesting that E(2) induces leptin expression in trophoblastic cells, probably through genomic and nongenomic actions via crosstalk between estrogen receptor 1 and MAPK and PI3K signal transduction pathways. PMID:20237333

  20. 17beta-estradiol enhances the response of plasmacytoid dendritic cell to CpG.

    PubMed

    Li, Xiaoxi; Xu, Yixin; Ma, Ling; Sun, Lingyun; Fu, Gengfeng; Hou, Yayi

    2009-01-01

    Gender differences in immune capabilities suggest that sex hormones such as estrogens were involved in the regulation of the immunocompetence. Numerous studies also suggest that plasmacytoid dendritic cells (PDCs) play a pathogenic role in SLE. However, it is unclear whether estrogen can modulate the function of PDCs to influence the development of SLE. In the present study, PDCs from murine spleens were treated with 17beta-estradiol (E2) and CpG respectively or both in vitro, then cell viability, costimulatory molecule expression, cytokine secretion of PDCs, as well as stimulatory capacity of PDCs to B cells were analyzed. Results showed that E2 and CpG increased the cell viability and costimulatory molecule expression on PDCs synergistically. Moreover, the intracellular and extracellular secretion of IFN-alpha was increased by E2 or E2 plus CpG. In addition, E2 and CpG also increased the stimulatory capacity of PDCs to B cells, and the viability of B cells was decreased after neutralizing IFN-alpha significantly. In the experiments in vivo, mice received daily s.c. injections of E2 and CpG respectively or both, then we found that the plasma concentration of IgM were elevated by E2 and CpG synergistically and the expression of IFN-alpha/beta in spleens were noticeably increased by CpG plus E2 compared with the treatment of E2 or CpG only. This study indicates that E2 could exacerbate PDCs' activation with CpG, which further activates B cells to upregulate susceptibility to autoantigens. IFN-alpha plays an important role in the stimulatory effect of PDCs on B cells. E2 stimulation of IFN-alpha production may result in female prevalence in autoimmune diseases such as SLE through activation of PDCs. This study provides novel evidence of relationship between estrogen and SLE and also sheds light on gender biases among SLE patients. PMID:20037646

  1. Regioselective 2-hydroxylation of 17{beta}-estradiol by rat cytochrome P4501B1

    SciTech Connect

    Rahman, Mostafizur; Hayes Sutter, Carrie; Emmert, Gary L.; Sutter, Thomas R. . E-mail: tsutter@memphis.edu

    2006-11-01

    Previous work demonstrated that human cytochrome P4501B1 (CYP1B1) forms predominantly 4-hydroxyestradiol (4-OHE2), a metabolite which is carcinogenic in animal models. Here, we present results from kinetic studies characterizing the formation of 4-OHE2 and 2-hydroxyestradiol (2-OHE2) by rat CYP1B1 using 17{beta}-estradiol (E2) as a substrate. K {sub m} and K {sub cat} values were estimated using the Michaelis-Menten equation. For rat CYP1B1, the apparent K {sub m} values for the formation of 4-OHE2 and 2-OHE2 were 0.61 {+-} 0.23 and 1.84 {+-} 0.73 {mu}M; the turnover numbers (K {sub cat}) were 0.23 {+-} 0.02 and 0.46 {+-} 0.05 pmol/min/pmol P450; and the catalytic efficiencies (K {sub cat}/K {sub m}) were 0.37 and 0.25, respectively. For human CYP1B1, the apparent K {sub m} values for the formation of 4-OHE2 and 2-OHE2 were 1.22 {+-} 0.25 and 1.10 {+-} 0.26; the turnover numbers were 1.23 {+-} 0.06 and 0.33 {+-} 0.02; and the catalytic efficiencies were 1.0 and 0.30, respectively. The turnover number ratio of 4- to 2-hydroxylation was 3.7 for human CYP1B1 and 0.5 for rat CYP1B1. These results indicate that, although rat CYP1B1 is a low K {sub m} E2 hydroxylase, its product ratio, unlike the human enzyme, favors 2-hydroxylation. The K {sub i} values of the inhibitor 2,4,3',5'-tetramethoxystilbene (TMS) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.69 and 0.78 {mu}M, respectively. The K {sub i} values of 7,8-benzoflavone ({alpha}-NF) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.01 and 0.02 {mu}M, respectively. The knowledge gained from this study will support the rational design of CYP1B1 inhibitors and clarify results of CYP1B1 related carcinogenesis studies performed in rats.

  2. Effect of 17beta-estradiol on the in vitro maturation of bovine oocytes.

    PubMed

    Beker, A R C L; Colenbrander, B; Bevers, M M

    2002-12-01

    Although 1 microg/ml of 17beta-estradiol (E2) is often used in routine in vitro maturation (IVM) and in vitro fertilization (IVF), its effect remains controversial. The objective of our study was to investigate the effects of E2 on bovine oocyte IVM and subsequent embryo development, using a defined medium. Bovine cumulus oocyte complexes (COCs), aspirated from 2 to 8 mm follicles of slaughterhouse ovaries, were matured in TCM199 in the presence of 1 microg/ml E2 with or without 0.05 IU/ml recombinant hFSH. Cultures without E2, FSH or both served as controls. COCs were matured for 22 h at 39 degrees C in a humidified atmosphere of 5% CO2 in air. To investigate the effect of E2 with and without FSH on nuclear maturation, COCs were fixed after maturation and the nuclear stage was assessed following DAPI staining. Similarly, denuded oocytes (DO) were matured in the presence of E2 and the nuclear stage assessed after 22 h. To investigate the effect of E2 with and without FSH during IVM on subsequent embryo development, in vitro matured COCs were fertilized in vitro and after removal of the cumulus cells, the presumed zygotes were cocultured on BRL monolayer for 11 days. At Day 4, the number of cleaved embryos, and at Days 9 and 11, the number of blastocysts, were assessed. Addition of 1 microg/ml E2 to TCM199 significantly decreased the percentage of Metaphase II (MII) compared to control (56.3 and 74.0%, respectively), and increased the percentage of nuclear aberrations compared to control (13.3 and 2.1%, respectively). The negative effect of E2 on nuclear maturation was stronger when DO were matured; 25.1 and 60.0% of the oocytes reached MII stage for the E2 and control groups, respectively. When COCs were matured in TCM199 supplemented with FSH, the addition of 1 microg/ml E2 did not influence the proportion of MII oocytes, although a higher percentage of nuclear aberrations as compared to control was observed. Presence of E2 during IVM also decreased the blastocyst rate (14.4 and 10.0% for control and E2 groups, respectively). However, when FSH was present, the addition of E2 had no effect on the cleavage rate and blastocyst formation (20.3 and 21.7% for control and E2 groups, respectively). In conclusion, supplementation of 1 microg/ml E2 to a serum free maturation medium negatively affects bovine oocyte nuclear maturation and subsequent embryo development. Although these effects are attenuated in the presence of FSH, we strongly suggest omission of E2 in routine maturation protocols of bovine oocytes. PMID:12472137

  3. Effect of agricultural antibiotics on the persistence and transformation of 17beta-estradiol in a Sequatchie loam.

    PubMed

    Chun, Soul; Lee, Jaehoon; Geyer, Roland; White, David C; Raman, D Raj

    2005-01-01

    A laboratory incubation study was conducted to investigate the effect of agricultural antibiotics (sulfamethazine, tylosin, and chlortetracycline) on the persistence and transformation of 17beta-estradiol in Sequatchie loam. We measured concentrations of 17beta-estradiol and its primary metabolite (estrone) in soils spiked with antibiotics and 17beta-estradiol. Dehydrogenase activity (DHA) was also measured as an indicator of the total microbial activity of the soils. The presence of antibiotics significantly decreased transformation of 17beta-estradiol to estrone. There was a positive correlation between the DHA and the concentrations of estrone in soil spiked with 17beta-estradiol only, implying that the reaction is mainly catalyzed by dehydrogenases. However, the positive correlation was weakened in soil spiked with 17beta-estradiol and antibiotics together. We recommend that any study evaluating the fate and transport of estrogenic hormones in soil should include the effect of agricultural antibiotics because antibiotics and estrogenic hormones are commonly excreted together in environmental samples. PMID:16190018

  4. Signal pathway of 17beta-estradiol-induced MUC5B expression in human airway epithelial cells.

    PubMed

    Choi, Hye Joung; Chung, Yoo-Sam; Kim, Hyun Jik; Moon, Uk Yeol; Choi, Yeon Ho; Van Seuningen, Isabelle; Baek, Seung Joon; Yoon, Ho-Geun; Yoon, Joo-Heon

    2009-02-01

    MUC5B is a major mucin of the human respiratory tract, and it is not clear how MUC5B expression is regulated in various airway diseases. The goal of this study was to determine the mechanisms by which 17beta-estradiol induces MUC5B gene expression in airway epithelial cells. It was found that E2, a sex hormone, stimulates MUC5B gene overexpression by interaction with estrogen receptor alpha (ERalpha) and by acting through extracellular signal-regulated kinase 1/2 (ERK1/2)-mitogen-activated protein kinase (MAPK). Pretreatment with ER antagonist ICI 182,780 blocked both E2-induced ERK1/2-MAPK activation and MUC5B gene expression. It was also found that the activation of p90 ribosomal S 6 protein kinase 1 (RSK1), cAMP-response element-binding protein (CREB), and cAMP-response element (CRE) (-956 region of the MUC5B promoter)-responsive signaling cascades via ERK1/2 MAPK are crucial aspects of the intracellular mechanisms that mediate MUC5B gene expression. Taken together, these studies give additional insights into the molecular mechanism of hormone-induced MUC5B gene expression and enhance our understanding of abnormal mucin secretion in response to hormonal imbalances. PMID:18688042

  5. Development of magnetic particle-based chemiluminescence enzyme immunoassay for the detection of 17beta-estradiol in environmental water.

    PubMed

    Xin, Tian-Bing; Wang, Xu; Jin, Hui; Liang, Shu-Xuan; Lin, Jin-Ming; Li, Zhen-Jia

    2009-09-01

    In the present work, a simple, fast, and highly sensitive chemiluminescence enzyme immunoassay for 17beta-estradiol (E2) in environmental water samples was developed, using magnetic particles (MPs) labeled with secondary antibody as both the immobilization matrix and the separation tools. The specific anti-E2 polyclonal antibody (PcAb) was produced against a conjugate of estradiol-bovine serum albumin. The specificity of the anti-E2 antibody was studied. The results showed that the antibody did not cross-react with the structurally related endocrine-disrupting compounds, including estrone, ethinyl E2, estriol, E2-17-glucuronide, E2-3-sulfate-17-glucuronide, androstenedione, and dihydrotestosterone. The water samples were pretreated with solid-phase extraction using C18 cartridges for the removal of matrix effects. Several physicochemical parameters including the dilution ratios of E2-6-horseradish peroxidase conjugate and anti-E2 PcAb, immunoreaction time, volume of chemiluminescent substrate and MPs, chemiluminescence reaction time, and pH of assay solution were studied and optimized. At optimal experimental conditions, it was found that the proposed method exhibited high performance with detection limit of 2.0 pg/mL, linear range of 20-1,200 pg/mL, and total assay time of 45 min. Both inter- and intra-assay coefficient of variation were less than 10%. The average recoveries of three different spiked concentration samples ranged from 86.3% to 108%. The method was successfully applied to the determination of E2 in river, waste, and tap water, and showed a good correlation with the commercially available radioimmunoassay kit. PMID:18841499

  6. Effects of 17beta-estradiol and 4-nonylphenol on osmoregulation and hepatic enzymes in gilthead sea bream (Sparus auratus).

    PubMed

    Carrera, Erkuden Pérez; García-López, Angel; Martín del Río, María del Pilar; Martínez-Rodríguez, Gonzalo; Solé, Montserrat; Mancera, Juan Miguel

    2007-03-01

    Sexually immature Sparus auratus were injected intraperitoneally with coconut oil either alone (control) or containing 17beta-estradiol (E2, 10 microg/g body mass) or 4-nonyphenol (4-NP, 100 and 200 microg/g body mass) and sampled 10 days later. Gill and kidney Na(+),K(+)-ATPase activities, plasma levels of E2 and cortisol, plasma osmolites (osmolality, sodium and chloride) and metabolites (glucose, lactate, proteins and triglycerides) were examined. Livers were used for measuring hepatosomatic index (HSI) and determinations of the activities of antioxidant defences catalase (CAT) and total glutatione peroxidase (t-GPX), the CYP1A-dependent, 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST). HSI and plasma levels of E2 were significantly increased in E2 -treated fish. E2 treatment enhanced plasma osmolality, glucose, triglycerides and proteins, but had no effect on plasma cortisol, and gill and kidney Na(+),K(+)-ATPase activities. Hepatic activities of EROD, GST and CAT were significantly decreased after E2 administration, whereas t-GPX remained unaffected. Treatment with 200 microg/g 4-NP caused a slight increase in plasma E2 relative to the control group. Plasma glucose and protein levels were not affected by 4-NP, while triglycerides were increased. Fish treated with the higher dose of 4-NP displayed a clear reduction in kidney Na(+),K(+)-ATPase activity, together with increases in plasma osmolality, relative to the control group. High 4-NP also caused a significant decrease in EROD and an increase in GST activity. Our results confirm the regulation of the natural estrogen E2 and the weak xenoestrogen 4-NP on osmoregulation and biotransformation enzymes in a partially similar manner. The actions of xenoestrogens on critical physiological processes may have an ecological significance as it can reduce adaptability and capacity to metabolise xenobiotics under stressful conditions. PMID:17251064

  7. 17beta-estradiol induces both up-regulation and processing of cyclin E in a calpain-dependent manner in MCF-7 breast cancer cells.

    PubMed

    Hou, Jianmei; Wang, Xudong; Li, Yang; Liu, Xiaohong; Wang, Zhuting; An, Jing; Yang, Li; He, Yan

    2012-03-23

    In the current study, we investigated whether 17beta-estradiol (E2) induces cyclin E expression and triggers cyclin E processing via calpain in MCF-7 breast cancer cells. We found that E2 induced increased expression of cyclin E in a slow and persistent manner, and a rapid yet sustained processing of cyclin E. In addition, estrogenic ethanol was able to stimulate cyclin E truncation. Calpeptin or ALLN greatly suppressed the E2-triggered cyclin E processing and its expression, suggesting a calpain-mediated action for E2. Finally, the E2-induced effects could also be significantly suppressed by BAPTA or U0126, indicating involvement of calcium/ERK signaling. Taken together, these results show that estrogen may contribute to both up-regulation and proteolysis of cyclin E through calpain in MCF-7 cells. PMID:22449977

  8. 17beta-estradiol enhances heparin-binding epidermal growth factor-like growth factor production in human keratinocytes.

    PubMed

    Kanda, Naoko; Watanabe, Shinichi

    2005-04-01

    Heparin-binding epidermal growth factor-like growth factor (HB-EGF) enhances reepithelialization in wounds. Estrogen is known to promote cutaneous wound repair. We examined the in vitro effects of 17beta-estradiol (E2) on HB-EGF production by human keratinocytes. E2 or membrane-impermeable BSA-conjugated E2 (E2-BSA) increased HB-EGF secretion, mRNA level, and promoter activity in keratinocytes. E2 or E2-BSA enhanced in vitro wound closure in keratinocytes, and the closure was suppressed by anti-HB-EGF antibody. Activator protein-1 (AP-1) and specificity protein 1 (Sp1) sites on HB-EGF promoter were responsible for the E2- or E2-BSA-induced transactivation. Antisense oligonucleotides against c-Fos, c-Jun, and Sp1 blocked E2- or E2-BSA-induced HB-EGF transactivation. E2 or E2-BSA enhanced DNA binding and transcriptional activity of AP-1 and generated c-Fos/c-Jun heterodimers by inducing c-Fos expression. E2 or E2-BSA enhanced DNA binding and transcriptional activity of Sp1 in parallel with the enhancement of Sp1 phosphorylation. These effects of E2 or E2-BSA were not blocked by the nuclear estrogen receptor antagonist ICI-182,780 or anti-estrogen receptor-alpha or -beta antibodies but were blocked by inhibitors of G protein, phosphatidylinositol-specific PLC, PKC-alpha, and MEK1. These results suggest that E2 or E2-BSA may enhance HB-EGF production via activation of AP-1 and Sp1. These effects of E2 or E2-BSA may be dependent on membrane G protein-coupled receptors different from nuclear estrogen receptors and on the receptor-mediated activities of phosphatidylinositol-specific PLC, PKC-alpha, and MEK1. E2 may enhance wound reepithelialization by promoting HB-EGF production in keratinocytes. PMID:15761212

  9. 17beta-estradiol accelerates tumor onset and decreases survival in a transgenic mouse model of ovarian cancer.

    PubMed

    Laviolette, Laura A; Garson, Kenneth; Macdonald, Elizabeth A; Senterman, Mary K; Courville, Kerri; Crane, Colleen A; Vanderhyden, Barbara C

    2010-03-01

    Epithelial ovarian cancer is thought to be derived from the ovarian surface epithelium (OSE) but often goes undetected in the early stages, and as a result, the factors that contribute to its initiation and progression remain poorly understood. Epidemiological studies have suggested that the female steroid hormones are involved in ovarian carcinogenesis and that women who use hormone replacement therapy are at increased risk of developing the disease. A novel transgenic mouse model of ovarian cancer (tgCAG-LS-TAg) was developed to examine the role of the female reproductive steroid hormones [17beta-estradiol (E(2)) and progesterone (P(4))] on the initiation, progression, and pathology of ovarian cancer. The mouse model uses the Cre-LoxP system to induce expression of the simian virus 40 large and small T antigens (SV40 TAg). After targeted induction of the oncogene in the OSE, mice develop poorly differentiated ovarian tumors, tumor dissemination to tissues within the abdominal cavity, and a subset develops hemorrhagic ascites. Treatment with P(4) had no impact on the disease, but E(2) altered the pathophysiology, resulting in an earlier onset of tumors, decreased overall survival time, and a distinctive papillary histology. Normal ovaries collected from mice treated with E(2), but lacking expression of SV40 TAg, displayed an increase in the areas of columnar and hyperplastic OSE cells compared to placebo-treated controls. A better understanding of the mechanisms by which E(2) alters the morphology of normal OSE cells and reduces survival in this mouse model may translate into improved prevention and treatment options for women using hormone replacement therapy. PMID:20056833

  10. 4-n-Nonylphenol and 17-beta estradiol may induce common DNA effects in developing barnacle larvae.

    PubMed

    Atienzar, Franck A; Billinghurst, Zoë; Depledge, Michael H

    2002-01-01

    There is a growing concern over the potential effects of environmental endocrine disrupters on both human and wildlife populations. However, to date, minimal research has been conducted to determine the effect of estrogens and xenoestrogens at the DNA level. In this study, we used the random amplified polymorphic DNA (RAPD) assay to evaluate the effects on the genomic DNA of barnacle larvae that had been exposed to 17beta-estradiol (E2) and low concentrations of 4-n-nonylphenol (NP). DNA effects include DNA damage as well as mutations and possibly other effects at the DNA level that can be induced by chemical or physical agents that directly and/or indirectly interact with genomic DNA. Not only did exposure to NP and E2 induce changes in RAPD profiles in the exposed barnacle larvae when compared to control patterns, but also, and more importantly, there were similarities in the RAPD modifications in the exposed populations that had been treated to either chemical. We propose that NP and E2 induced some common DNA effects in barnacle larvae and that these specific modifications in RAPD patterns may arise as a consequence of hot spot DNA damage (e.g. DNA adducts) and/or mutations (point mutations or genomic rearrangements). This could help to explain how xenoestrogens mimic the effects produced by natural estrogens. In conclusion, in the field of endocrine disruption, the study of DNA effects induced by estrogens and/or xenoestrogens warrants further investigation. Indeed, changes at the DNA levcl may be the precursors of some of the numerous effects reported at higher levels of biological organisation such as the feminization of males, developmental abnormalities, and infertility. PMID:12442797

  11. Dual derivatization-stir bar sorptive extraction--thermal desorption--gas chromatography-mass spectrometry for determination of 17beta-estradiol in water sample.

    PubMed

    Kawaguchi, Migaku; Ito, Rie; Sakui, Norihiro; Okanouchi, Noriya; Saito, Koichi; Nakazawa, Hiroyuki

    2006-02-10

    A novel method for the trace analysis of 17beta-estradiol (E2) in river water sample was developed, which involved stir bar sorptive extraction (SBSE) with in situ acylation (first derivatization) and thermal desorption (TD) with quartz wool assisted (QWA) in tube silylation (second derivatization), followed by gas chromatography-mass spectrometry (GC-MS), and is called the "dual derivatization method." The optimum conditions for SBSE with in situ acylation, such as the volume of acetic acid anhydride and the extraction time, were investigated. In addition, the optimum conditions for TD with QWA in tube silylation, such as the volume of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and the TD temperature and hold time, were investigated as well. The detection limit (S/N = 3) and the quantitation limit (S/N>10) of E2 in the river water sample were 0.5 and 2 pg ml(-1) (ppt), respectively, by the dual derivatization method. In addition, the detection limit was 0.1 pg ml(-1) by using dual derivatization method with multi-shot mode. The calibration curve for E2 was linear in the range of 0.002-10 ng ml(-1) with correlation coefficients >0.999. The average recoveries of E2 (n = 6) at the concentrations of 0.05 and 1.0 ng ml(-1) from the river water sample were 93.1 (RSD: 1.4%) and 98.4% (RSD: 0.8%), respectively, with correction using the added surrogate standard, 17beta-estradiol-(13)C(4). This simple, accurate, sensitive and selective analytical method may be applicable to the determination of trace amounts of E2 in water samples. PMID:16439260

  12. Fathead minnow vitellogenin: Complementary DNA sequence and messenger RNA and protein expression after 17{beta}-estradiol treatment

    SciTech Connect

    Korte, J.J.; Kahl, M.D.; Jensen, K.M.; Pasha, M.S.; Parks, L.G.; LeBlanc, G.A.; Ankley, G.T.

    2000-04-01

    Induction of vitellogenin (VTG) in oviparous animals has been proposed as a sensitive indicator of environmental contaminants that activate the estrogen receptor. In the present study, a sensitive ribonuclease protection assay (RPA) for VTG messenger RNA (mRNA) was developed for the fathead minnow (Pimephales promelas), a species proposed for routine endocrine-disrupting chemical (EDC) screening. The utility of this method was compared with an enzyme-linked immunosorbent assay (ELISA) specific for fathead minnow VTG protein. Assessment of the two methods included kinetic characterization of the plasma VTG protein and hepatic VTG mRNA levels in male fathead minnows following intraperitoneal injections of 17{beta}-estradiol (E2) at two dose levels (0.5, 5.0 mg/kg). Initial plasma E2 concentrations were elevated in a dose-dependent manner but returned to normal levels within 2 d. Lover VTG mRNA was detected within 4 h, reached a maximum around 48 h, and returned to normal levels in about 6 d. Plasma VTG protein was detectable within 16 h of treatment reached maximum levels at about 72 h. and remained near these maximum levels for at least 18 d. While the RPA was about 1,000 times more sensitive than the ELISA, the ELISA appears superior for routine screening tests. The ELISA method is relatively simple to perform and, because males lack a clearance mechanism for VTG, the protein remains at relatively high concentrations in the plasma for an extended period of time. As part of the development of the RPA, the complementary DNA (cDNA) sequence for fathead minnow VTG was determined and the deduced amino acid sequence compared with VTG sequences for other fish species.

  13. Elimination kinetic of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate ester metabolites in calves' urine.

    PubMed

    Pinel, Gaud; Rambaud, Lauriane; Cacciatore, Giuseppe; Bergwerff, Aldert; Elliott, Chris; Nielen, Michel; Le Bizec, Bruno

    2008-05-01

    Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C(18) SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CCalpha) for steroids were below 0.1 microg L(-1), except for 19-norandrosterone (CCalpha=0.7 microg L(-1)) and estrone (CCalpha=0.3 microg L(-1)). Kinetics of elimination of the administered 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate were established by monitoring 17beta-estradiol, 17alpha-estradiol, estrone and 17beta-nandrolone, 17alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17alpha-estradiol and 17alpha-nandrolone (>20 and 2 mg L(-1), respectively after 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate administration) whereas 17beta-estradiol, estrone, 17beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the microg L(-1) level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17beta form. PMID:18356042

  14. 17beta-estradiol potently suppresses cAMP-induced insulin-like growth factor-I gene activation in primary rat osteoblast cultures

    NASA Technical Reports Server (NTRS)

    McCarthy, T. L.; Ji, C.; Shu, H.; Casinghino, S.; Crothers, K.; Rotwein, P.; Centrella, M.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) is a key factor in bone remodeling. In osteoblasts, IGF-I synthesis is enhanced by parathyroid hormone and prostaglandin E2 (PGE2) through cAMP-activated protein kinase. In rats, estrogen loss after ovariectomy leads to a rise in serum IGF-I and an increase in bone remodeling, both of which are reversed by estrogen treatment. To examine estrogen-dependent regulation of IGF-I expression at the molecular level, primary fetal rat osteoblasts were co-transfected with the estrogen receptor (hER, to ensure active ER expression), and luciferase reporter plasmids controlled by promoter 1 of the rat IGF-I gene (IGF-I P1), used exclusively in these cells. As reported, 1 microM PGE2 increased IGF-I P1 activity by 5-fold. 17beta-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE2 by up to 90% (ED50 approximately 0.1 nM). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17alpha-estradiol was 100-300-fold less effective. By contrast, 17beta-estradiol stimulated estrogen response element (ERE)-dependent reporter expression by up to 10-fold. 17beta-Estradiol also suppressed an IGF-I P1 construct retaining only minimal promoter sequence required for cAMP-dependent gene activation, but did not affect the 60-fold increase in cAMP induced by PGE2. There is no consensus ERE in rat IGF-I P1, suggesting novel downstream interactions in the cAMP pathway that normally enhances IGF-I expression in skeletal cells. To explore this, nuclear extract from osteoblasts expressing hER were examined by electrophoretic mobility shift assay using the atypical cAMP response element in IGF-I P1. Estrogen alone did not cause DNA-protein binding, while PGE2 induced a characteristic gel shift complex. Co-treatment with both hormones caused a gel shift greatly diminished in intensity, consistent with their combined effects on IGF-I promoter activity. Nonetheless, hER did not bind IGF-I cAMP response element or any adjacent sequences. These results provide new molecular evidence that estrogen may temper the biological effects of hormones acting through cAMP to regulate skeletal IGF-I expression and activity.

  15. Effect of cadmium on the interaction of 17beta-estradiol with the rainbow trout estrogen receptor.

    PubMed

    Nesatyy, Victor J; Ammann, Adrian A; Rutishauser, Barbara V; Suter, Marc J F

    2006-02-15

    The widely reported negative effects of xenoestrogens on the endocrine system of aquatic organisms gave raise to public concern and led to a number of screening and testing initiatives on the international level. Recent studies indicated that not only organic chemicals but also certain heavy metals, including cadmium, can mimic the effects of the endogenous estrogen receptor agonist 17beta-estradiol (E2) and lead to estrogen receptor activation. While the effects of cadmium on the endocrine system and its potential to harm living organisms are no longer in doubt, the exact mode of action is still essentially unknown. In the present study we utilized the rainbow trout ER alpha ligand binding domain (rtER-LBD) fused to glutathione-S-transferase, to study noncovalent interactions between cadmium and the rtER-LBD. ICP-MS data showed that the Cd uptake by the rtER-LBD was strongly pH-dependent. Previous results showing that Cd shields Cys residues of the rtER-LBD against chemical modification, and competitive binding experiments reported here provide insights into the specificity of the interaction of cadmium with the ER hormone binding cavity. It could, for instance, be shown that most of the cadmium adsorbed to the protein could be released into solution either under denaturing conditions, or by stripping from the protein surface using EDTA at physiological conditions. Competitive binding experiments using radio-labeled estradiol showed that, in contrast to previously published data, E2 has an affinity an order of magnitude higher for the ER than for Cd. ICP-MS experiments showed that, despite its higher affinity, increasing E2 concentrations were unable to replace Cd from the rtER-LBD that had been preequilibrated with Cd. These findings were independently confirmed by the [3H]-E2 binding assay. At the same time both ICP-MS and the [3H]-E2 binding assay showed that increasing Cd concentrations not only lead to a decrease in the specific estradiol binding, but also to the release of E2 from the preequilibrated rtER-LBD. The gradual release of [3H]-E2 from the rtER-LBD following incubation with increasing Cd concentrations indicates either direct competition for the same binding site or Cd-induced conformational changes resulting in the release of estradiol. PMID:16572797

  16. Antagonistic effects of 17 beta-estradiol, progesterone, and testosterone on Ca2+ entry mechanisms of coronary vasoconstriction.

    PubMed

    Crews, J K; Khalil, R A

    1999-04-01

    The clinical observation that coronary artery disease is more common in men and postmenopausal women than in premenopausal women has suggested cardioprotective effects of female sex hormones including hormone-mediated coronary vasodilation. The purpose of this study was to investigate whether the sex hormone-induced coronary relaxation is caused by inhibition of Ca2+ mobilization into coronary smooth muscle. The effects of 17beta-estradiol, progesterone, and testosterone on vascular reactivity and 45Ca2+ influx were tested in deendothelialized coronary artery strips isolated from castrated male pigs. Prostaglandin F2alpha (PGF2alpha) (10(-5) mol/L) caused significant, maintained contraction of coronary artery strips. Caffeine (25 mmol/L), an activator of Ca2+ release from intracellular stores, caused transient contraction in Ca2+-free solution whereas membrane depolarization by 96 mmol/L KCl, an activator of Ca2+ entry, caused maintained contraction in the presence of external Ca2+. The 3 sex hormones caused significant and concentration-dependent relaxation of PGF2alpha- and 96 mmol/L KCl-induced contractions with 17beta-estradiol being the most effective. The sex hormones did not significantly affect the transient caffeine contraction in Ca2+-free solution. In contrast, the sex hormones significantly inhibited the PGF2alpha- and KCl-induced 45Ca2+ influx. 17beta-Estradiol caused similar inhibition of PGF2alpha- and KCl-induced contractions, suggesting inhibition of the same Ca2+ entry mechanism. However, progesterone and testosterone caused greater relaxation of PGF2alpha-induced contraction than of KCl-induced contraction. We conclude that in coronary arteries of castrated male pigs, sex hormones inhibit Ca2+ entry from extracellular space but not Ca2+ release from intracellular stores. 17beta-Estradiol mainly inhibits Ca2+ entry, whereas progesterone and testosterone cause coronary relaxation by inhibiting other mechanisms in addition to Ca2+ entry. PMID:10195933

  17. Sorption and transport of 17beta-estradiol and testosterone in undisturbed soil columns.

    PubMed

    Sangsupan, H A; Radcliffe, D E; Hartel, P G; Jenkins, M B; Vencill, W K; Cabrera, M L

    2006-01-01

    Land-applied domestic animal wastes contain appreciable amounts of 17beta-estradiol (henceforth, estradiol) and testosterone. These sex hormones may be transported through soil to groundwater and streams, where they may adversely affect the environment. Previous column transport studies with these hormones used repacked soil and did not consider preferential flow. We, therefore, determined the sorption and transport characteristics of estradiol and testosterone in undisturbed soil columns (15-cm i.d. by 32-cm height). In the sorption experiment, isotherms for estradiol and testosterone were nonlinear with Freundlich exponents (n) less than one. Sorption of both hormones decreased with soil depth, and estradiol sorbed more strongly than testosterone. Average estradiol Freundlich sorption coefficients (K(f)) values were 36.9 microg(1 - n) mL(n) g(-1) for the 0- to 10-cm soil depth and 25.7 microg(1 - n) mL(n) g(-1) for the 20- to 30-cm soil depth. Average testosterone K(f) values were 26.7 microg(1 - n) mL(n) g(-1) for the 0- to 10-cm soil depth and 14.0 microg(1 - n) mL(n) g(-1) for the 20- to 30-cm soil depth. In the transport experiment, 27% of the estradiol and 42% of the testosterone leached through the soil columns. Approximately 50% of the remaining soil-bound hormones were sorbed in the top 10 cm of soil. In almost all instances, breakthrough concentrations of estradiol, testosterone, and a chloride tracer peaked simultaneously. Simultaneous breakthrough and HYDRUS-1D transport parameters indicated both chemical and physical nonequilibrium processes affected hormone transport. This suggests hormones placed on soil surfaces may contaminate groundwater under conditions of preferential flow. PMID:17071897

  18. Rapid vascular escape of arterially injected 16alpha-radioiodo, 17beta-estradiol

    SciTech Connect

    Scharl, A.; Holt, J.A. )

    1993-03-20

    The authors undertook this study because confirmation of a rapid vascular escape and slow release back into the circulatory system suggests that arterial injection of radiohalogenated steroid receptor ligands might provide an efficacious route of administration for imaging or treatment of receptor-rich malignant tumors in peripheral tissues. The authors injected radiolabeled 16alpha-iodo, 17beta-estradiol ([I]-E) into the femoral artery of swine in a solution that contained [[sup 125]I]-E in a known ratio to [[sup 99]Tc]-labeled red blood cells. Fractions of femoral venous blood were collected at short intervals during 10 min. They looked for changes in the ratio of the radiolabeles. [[sup 99m]Tc]-labeled red blood cells are known to remain in the vascular system for an hour or more. After passage of the injectate through the capillary bed of the swine leg, a dramatic decrease of the initial [sup 125]I:[sup 99m]Tc ratio to only 10% was observed in the femoral venous blood. This ratio increased gradually during the next 10 min to approximately 30% of that in the injectate, indicating that a significant portion (approximately 90%) of the [[sup 125]I]-E was initially trapped in the limb and then slowly re-entered the vascular system. To obtain visual confirmation of the rapid vascular escape of iodo-estrogen, they injected either an imageable form of [I]-E ([[sup 123]I]-E) or [[sup 99m]Tc]-labeled red blood cells into the dorsal aorta of superovulated rabbits, whose smaller size allowed whole-body imaging. The biodistributions of these radiopharmaceuticals were surveyed continuously by real-time planar gamma imaging. A large fraction of [I]-E escapes from the vascular system during the first pass through an organ or limb, without regard to the estrogen receptor content of the tissue. 28 refs., 3 figs., 1 tab.

  19. Decreased [Ca(2+)](i) during inhibition of coronary smooth muscle contraction by 17beta-estradiol, progesterone, and testosterone.

    PubMed

    Murphy, J G; Khalil, R A

    1999-10-01

    The clinical observation that coronary heart disease is more common in men and postmenopausal women than in premenopausal women has suggested cardiovascular protective effects of female sex hormones including hormone-mediated coronary vasodilation. We investigated whether the sex hormones induced coronary relaxation is due to a decrease in [Ca(2+)](i) as measured in single coronary smooth muscle cells isolated from gonadectomized male and female pigs. In the presence of external Ca(2+), prostaglandin F(2alpha) (PGF(2alpha); 10(-5) M) and membrane depolarization by 51 mM KCl caused significant cell contraction and maintained increase in [Ca(2+)](i) to 297 +/- 4 and 341 +/- 20 nM, respectively. At 10(-9) to 6 x 10(-7) M, 17beta-estradiol, progesterone, and testosterone caused inhibition of PGF(2alpha)- and KCl-induced contraction and [Ca(2+)](i) with 17beta-estradiol being most effective. 17alpha-Estradiol did not affect PGF(2alpha)-induced contraction, and the inhibition of PGF(2alpha) contraction by 17beta-estradiol, progesterone, or testosterone was abolished by tamoxifen and ICI 182, 780, RU-486, or flutamide, respectively. 17beta-Estradiol caused similar inhibition of PGF(2alpha)- and KCl-induced contraction and [Ca(2+)](i). Progesterone and testosterone caused greater inhibition of PGF(2alpha)-induced cell contraction and [Ca(2+)](i) compared with the KCl responses. In Ca(2+)-free (2 mM EGTA) solution, caffeine (10 mM) and carbachol (10(-5) M), which activate Ca(2+) release from intracellular stores, caused small cell contraction and transiently increased [Ca(2+)](i) to 256 +/- 53 and 262 +/- 32 nM, respectively. Sex hormones did not significantly affect caffeine- or carbachol-induced contraction or [Ca(2+)](i). Thus, 17beta-estradiol, progesterone, and testosterone cause relaxation of coronary smooth muscle cells and decrease [Ca(2+)](i) mainly by inhibiting Ca(2+) entry from extracellular space but not Ca(2+) release from intracellular stores. The differences in potency of sex hormones in reducing cell contraction and [Ca(2+)](i) suggest differences in the sensitivity of the PGF(2alpha)- and depolarization-activated Ca(2+) entry pathways to inhibition by sex hormones. PMID:10490885

  20. Upregulation of estrogen receptor subtypes and vitellogenin mRNA in cinnamon clownfish Amphiprion melanopus during the sex change process: profiles on effects of 17beta-estradiol.

    PubMed

    Kim, Na Na; Jin, Deuk-Hee; Lee, Jehee; Kil, Gyung-Suk; Choi, Cheol Young

    2010-10-01

    In the present study, we investigated the expression pattern of estrogen receptors (esr) and vitellogenin (vtg) mRNA in the gonads and liver during sex change in cinnamon clownfish by using quantitative polymerase chain reaction. We divided gonadal development during the sex change from male to female into 3 stages (mature male, male at 90days after removing female, and mature female) and investigated esr and vtg mRNA expressions during the sex change. With female, the esr and vtg mRNA expressions increased. In western blot analysis, Esr1 protein was detected only in the ovaries of female cinnamon clownfish. Also, to understand the effect of 17beta-estradiol (E(2)), we investigated the esr and vtg mRNA expression patterns in the gonads and liver, and the changes in plasma E(2) level after E(2) injection. E(2) treatment increased both mRNA expression levels of esr and vtg and plasma E(2) levels. The present study describes the molecular characterization of esr subtypes and the interactions between esr and vtg after E(2) treatment in cinnamon clownfish. PMID:20601066

  1. Oxidation products of stigmasterol interfere with the action of the female sex hormone 17beta-estradiol in cultured human breast and endometrium cell lines.

    PubMed

    Newill, Heike; Loske, Renate; Wagner, Jörg; Johannes, Christian; Lorenz, Reinhard L; Lehmann, Leane

    2007-07-01

    Phytosterols are constituents of plant membranes and are thus contained in low concentrations in vegetable products as well as at high concentrations in functional food designed to reduce serum cholesterol levels. Similar to ChOL, phytosterols are oxidized chemically in food and by biotransformation in vivo. Although oxyphytosterols have been detected in the serum of healthy human subjects, little is known of their biological activity. Therefore, the estrogenic and antiestrogenic activities of a mixture of six oxidation products of stigmasterol (oxy-StOL) were determined at the following endpoints: (i) the affinity to isolated human estrogen receptors (ER), (ii) the basal and 17beta-estradiol (E2)-induced expression of the alkaline phosphatase (AlP) in human endometrial adenocarcinoma (Ishikawa) cells, and (iii) the basal and E2-induced proliferation of human breast adenocarcinoma (MCF-7) cells. Oxy-StOL was able to replace E2 from human ERalpha and ERbeta and induced a weak estrogenic response in MCF-7 cells. Moreover, the E2-induced activity of the AlP in Ishikawa cells as well as the E2-induced proliferation of MCF-7 cells were decreased at noncytotoxic concentrations (up to 10 microM), indicating that at least one component of oxy-StOL represents an estrogen-active compound which might interfere with endogenous estrogens. PMID:17579897

  2. Arctic char (Salvelinus alpinus) metallothionein: cDNA sequence, expression, and tissue-specific inhibition of cadmium-mediated metallothionein induction by 17{beta}-estradiol, 4-OH-PCB 30, and PCB 104

    SciTech Connect

    Gerpe, M.; Kling, P.; Berg, A.H.; Olsson, P.E.

    2000-03-01

    In this study, the authors have examined the basal level expression and tissue-specific expression patterns of metallothionein (MT) in Arctic char following metal and E2 (17{beta}-estradiol) treatment. To study the gene regulation in Arctic char, the two MT isoforms were isolated from a lambda-ZAP hepatic cDNA library and characterized. Determination of basal MT and mRNA and MT expression for 10 different tissues revealed a lack of correlation between MT and mRNA and MT levels. The inducibility of MT mRNA and the correlation to resulting MT levels were then determined for liver and kidney. They found a more rapid and stronger induction of MT mRNA in liver than in kidney at day 1 and 3 postinjection, whereas the MT protein quantification showed higher MT levels in kidney than in liver at days 3 and 7 postinjection. These discrepancies indicate that differences in metal handling or posttranscriptional regulation of MT exists between tissues. Whereas metals induce MT synthesis, E2 inhibit the hepatic MT expression. To examine the tissue specificity of this inhibition, the authors determined the effect of 17{beta}-estradiol (E2) and two estrogenic PCBs (4{prime}-OH-PCB 30 and PCB 104) on Cd-mediated MT induction in liver and kidney. Although E2 and the estrogenic PCBs inhibited cadmium-mediated hepatic MT induction, these compounds did not interfere with renal MT induction.

  3. Individual, temporal, and population-level variations in circulating 11-ketotestosterone and 17beta-estradiol concentrations in the oyster toadfish Opsanus tau.

    PubMed

    Maruska, Karen P; Korzan, Wayne J; Mensinger, Allen F

    2009-04-01

    Sex steroid hormones are important for reproduction in all vertebrates, but few studies examine inter-individual, temporal, and population-level variations, as well as environmental influences on circulating steroid levels within the same species. In this study we analyzed plasma 11-ketotoestosterone (11-KT) and 17beta-estradiol (E(2)) levels in the oyster toadfish to test for 1) individual and temporal variations by serially sampling the same individuals during the reproductive and post-reproductive period, 2) variations in steroid levels among toadfish obtained from different sources or maintained under different holding conditions, and 3) correlations with environmental parameters. Results from serial sampling showed marked inter-individual variations in male 11-KT levels in two separate groups of toadfish, but no temporal differences from June to September. Females also showed inter-individual variations in E(2) concentrations, but most had elevated levels late in the reproductive season coincident with oocyte growth prior to winter quiescence. E(2) concentration, but not 11-KT, was positively correlated with water temperature, and negatively correlated with daylength and lunar phase. Maricultured toadfish held under constant conditions had elevated levels of E(2) and 11-KT that should be considered when using these fish for experimentation. This study provides important comparative information on the relationship between individual variations in steroid levels, and how they relate to physiological and environmental correlates in a model marine teleost. PMID:19167510

  4. Gender-related effects of 17-{beta}-estradiol and B-hexachlorocyclohexane on liver tumor formation in medaka (Oryzias latipes)

    SciTech Connect

    Cooke, J.B.; Hinton, D.E.

    1994-12-31

    When medaka were acutely exposed to diethylnitrosamine (DEN), greater incidence of hepatocarcinoma was seen in female versus male fish. This is possibly related to elevated female endogenous estrogens, which increase liver weight and production of vitellogenin. To examine roles of estrogens in tumor modulation, 21-day old medaka were exposed to DEN (200 ppm for 24 hr.), then fed purified diets containing the estrogenic compound {beta}-hexachlorocyclohexane ({beta}-HCH) or 17-{beta}estradiol (E2) for 6 months. Incidences of basophilic preneoplastic foci of cellular alteration in females receiving DEN and 0.01, 0.1, or 1.0 ppm E2 were three times the incidences in similarly-treated males. Also, incidences of basophilic foci in DEN + 0.1 ppm E2 males were significantly increased over DEN-only males and were equal to incidences in DEN-only females. Liver weights and hepatosomatic indices of males given 0.1 ppm E2 were not significantly different than females fed control diet. Females fed 0.01-10.0 ppm {beta}-HCH after DEN had 4--5 times greater incidences of basophilic foci as males. Gender-related effects on kinetics of growth rates and volumes of foci are being examined.

  5. Gender determination in the Paiche or Pirarucu (Arapaima gigas) using plasma vitellogenin, 17beta-estradiol, and 11-ketotestosterone levels.

    PubMed

    Chu-Koo, F; Dugu, R; Alvn Aguilar, M; Casanova Daza, A; Alcntara Bocanegra, F; Chvez Veintemilla, C; Duponchelle, F; Renno, J-F; Tello, Salvador; Nuez, J

    2009-03-01

    Arapaima gigas is an air-breathing giant fish of Amazonian rivers. Given its great economic and cultural importance, the aquaculture development of this species represents an evident solution to face the decline of wild populations. In captivity, reproduction occurs generally in large earthen ponds where stocks of a few tens of brooders are maintained together at the beginning of the rainy season (December-March in the Peruvian Amazon). Fry production relies on the spontaneous formation of male and female pairs, which build a nest, delimit a territory and guard the offspring for at least 20 days from other congeners and predators. However, as sex determination of A. gigas is not possible by morphological criteria, it is very difficult to optimize reproduction conditions and fry production in each pond, which seriously hampers the culture of this species. This situation prompted us to develop sexing methodologies based on (1) the detection of female specific plasma Vitellogenin (Vtg) using an enzyme immuno assay (EIA), and (2) the determination of plasma 17beta-estradiol and 11-ketotestosterone levels for immature specimens. The Vtg purification was performed by electro-elution after polyacrilamide gel electrophoresis (PAGE) from plasma of 17beta-estradiol treated A. gigas juveniles. Two different Vtg molecules were isolated, (Vtg(1) and Vtg(2)) with 184 and 112 kDa apparent molecular masses, respectively, and two antibodies were raised in rabbits for each Vtg molecule. Adult fish were 100% accurately sexed by Vtg EIA, while 100% of immature fish and 95% of adults were accurately sexed by 17beta-Estradiol and 11-Ketestosterone ratios. We also observed different color pattern development in male and female adult fish (6-year-olds) around the reproductive period. PMID:19189239

  6. Effect of 17beta-estradiol or alendronate on the bone densitometry, bone histomorphometry and bone metabolism of ovariectomized rats.

    PubMed

    da Paz, L H; de Falco, V; Teng, N C; dos Reis, L M; Pereira, R M; Jorgetti, V

    2001-08-01

    The objective of the present study was to evaluate the effect of 17beta-estradiol or alendronate in preventing bone loss in 3-month-old ovariectomized Wistar rats. One group underwent sham ovariectomy (control, N = 10), and the remaining three underwent double ovariectomy. One ovariectomized group did not receive any treatment (OVX, N = 12). A second received subcutaneous 17beta-estradiol at a dose of 30 microg/kg for 6 weeks (OVX-E, N = 11) and a third, subcutaneous alendronate at a dose of 0.1 mg/kg for 6 weeks (OVX-A, N = 8). Histomorphometry, densitometry, osteocalcin and deoxypyridinoline measurements were applied to all groups. After 6 weeks there was a significant decrease in bone mineral density (BMD) at the trabecular site (distal femur) in OVX rats. Both alendronate and 17beta-estradiol increased the BMD of ovariectomized rats, with the BMD of the OVX-A group being higher than that of the OVX-E group. Histomorphometry of the distal femur showed a decrease in trabecular volume in the untreated group (OVX), and an increase in the two treated groups, principally in the alendronate group. In OVX-A there was a greater increase in trabecular number. An increase in trabecular thickness, however, was seen only in the OVX-E group. There was also a decrease in bone turnover in both OVX-E and OVX-A. The osteocalcin and deoxypyridinoline levels were decreased in both treated groups, mainly in OVX-A. Although both drugs were effective in inhibiting bone loss, alendronate proved to be more effective than estradiol at the doses used in increasing bone mass. PMID:11471040

  7. 17beta-Estradiol responsiveness of MCF-7 laboratory strains is dependent on an autocrine signal activating the IGF type I receptor.

    PubMed

    Hamelers, Irene HL; Van Schaik, Richard FMA; Sussenbach, John S; Steenbergh, Paul H

    2003-07-11

    BACKGROUND: Human MCF-7 cells have been studied extensively as a model for breast cancer cell growth. Many reports have established that serum-starved MCF-7 cells can be induced to proliferate upon the sole addition of 17beta-estradiol (E2). However, the extent of the mitogenic response to E2 varies in different MCF-7 strains and may even be absent. In this study we compared the E2-sensitivity of three MCF-7 laboratory strains. RESULTS: The MCF-7S line is non-responsive to E2, the MCF-7 ATCC has an intermediate response to E2, while the MCF-7 NKI is highly E2-sensitive, although the levels and activities of the estrogen receptor (ER) are not significantly different. Both suramin and IGF type I receptor blocking antibodies are able to inhibit the mitogenic response to E2-treatment in MCF-7 ATCC and MCF-7 NKI cells. From this we conclude that E2-induced proliferation is dependent on IGF type I receptor activation in all three MCF-7 strains. CONCLUSIONS: The results presented in this article suggest that E2-responsiveness of MCF-7 cells is dependent on the secretion of an autocrine factor activating the IGF-IR. All three strains of MCF-7 breast cancer cells investigated do not respond to E2 if the IGF-RI-pathway is blocked. Generally, breast cancer therapy is targeted at inhibiting estrogen action. This study suggests that inhibition of IGF-action in combination with anti-estrogen-treatment may provide a more effective way in treatment or even prevention of breast cancer. PMID:12890289

  8. Organochlorine compounds in liver and concentrations of vitellogenin and 17beta-estradiol in plasma of sea bass fed with a commercial or with a natural diet.

    PubMed

    Navas, J M; Merino, R; Jiménez, B; Rivera, J; Abad, E; Zanuy, S; Carrillo, M

    2005-11-30

    Results from previous experiments directed to determine the effect of different nutritional factors or the effect of xenobiotics on hormonal control of reproduction, lead to the hypothesis that hormonal perturbations repeatedly observed in sea bass (Dicentrarchus labrax) broodstock feeding commercial diets could have been caused by the presence of aryl hydrocarbon receptor (AhR) ligands, such as dioxins, furans and polychlorinated biphenyls (PCBs) in the diet. To evaluate this hypothesis, dioxins and related compounds were analysed in liver of female sea bass fed with a commercial or with a natural diet consisting of trash fish (bogue, Boops boops), and concentrations of vitellogenin (VTG) and 17beta-estradiol (E2) were determined in plasma obtained previously in monthly samplings of these animals. As observed in other experiments, females fed with a commercial diet exhibited lower VTG and higher E2 plasma levels than females fed with the natural diet. In liver, sea bass fed with the commercial diet exhibited a profile clearly dominated by high-chlorinated dioxins while in fish fed with the natural diet this profile was dominated by low chlorinated furans. However, typical AhR ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin showed no differences between groups or, as is the case of planar PCBs, showed higher concentrations in the liver of fish fed with the natural diet. These results do not permit to explain the observed hormonal alterations by a possible antiestrogenic effect caused by dioxins and related compounds. PMID:16213605

  9. A RIKILT yeast estrogen bioassay (REA) for estrogen residue detection in urine of calves experimentally treated with 17beta-estradiol.

    PubMed

    Divari, S; De Maria, R; Cannizzo, F T; Spada, F; Mulasso, C; Bovee, T F H; Capra, P; Leporati, M; Biolatti, B

    2010-01-01

    17beta-Estradiol is one of the most powerful sex steroids illegally used in bovine production. The objective of this study was to evaluate the application and the specificity of the RIKILT yeast estrogen bioassay (REA) for the detection of molecules with estrogenic activities in the urine of calves experimentally treated with anabolics. Four groups of six calves each received an injection of 17beta-estradiol intramuscularly (group B), androsterone and gliburide (group A), and testosterone (group C) molecules at different dosage for 40 days. Group D was the control. The ability of the REA test to detect estrogenic activity in urine samples from all animals was assessed. All estrogen-treated animals (group B) showed as being positive up to 7 days after administration of the highest dosage of 17beta-estradiol, while the other three groups showed as being negative. The identity of estrogenic molecules in the urine of group B (17beta-estradiol, 17alpha-estradiol) was confirmed by gas chromatography-mass spectrometry (GC/MS). This is the first time the REA test has been applied to detect 17beta-estradiol in the urine of calves treated with the hormone in vivo. The technique may offer an advantageous laboratory method for the veterinary surveillance of illegal steroid use. PMID:19763989

  10. Antioxidants J811 and 17beta-estradiol protect cerebellar granule cells from methylmercury-induced apoptotic cell death.

    PubMed

    Dar, E; Gtz, M E; Zhivotovsky, B; Manzo, L; Ceccatelli, S

    2000-11-15

    Cerebellar granule cells (CGC) have provided a reliable model for studying the toxicity of methylmercury (MeHg), a well-known neurotoxicant contaminating the environment. In the present study we report that doses of MeHg ranging from 0.1 microM to 1.5 microM activated apoptosis, as shown by cell shrinkage, nuclear condensation, and formation of high-molecular-weight DNA fragments. Nevertheless, caspase-3-like activity was not significantly induced, and the broad caspase inhibitor Z-VAD-FMK was not capable of protecting the cells. This argues for a minor role of caspases in the intracellular pathways leading to MeHg-induced cell death in CGC. Instead, proteolytic fragments obtained by specific calpain cleavage of procaspase-3 and alpha-fodrin were increased consistently in samples exposed to MeHg, pointing to a substantial activation of calpain. Notably, two antioxidants, 17beta-estradiol (10 microM) and the Delta(8,9)-dehydro derivative of 17alpha-estradiol J811 (10 microM), protected from MeHg damage, preventing morphological alterations, chromatin fragmentation, and activation of calpain. These findings underscore the key role of oxidative stress in MeHg toxicity, placing it upstream of calpain activation. The shielding effect of the 17beta-estradiol and the radical scavenger J811 is potentially relevant for the development of therapeutic strategies for MeHg intoxication. PMID:11070499

  11. Juvenile sea bass biotransformation, genotoxic and endocrine responses to beta-naphthoflavone, 4-nonylphenol and 17 beta-estradiol individual and combined exposures.

    PubMed

    Teles, M; Gravato, C; Pacheco, M; Santos, M A

    2004-10-01

    Juvenile sea bass, Dicentrarchus labrax L., were exposed during 2, 4, 8, and 24 h to 0.9 microM beta-naphthoflavone (BNF), 131 nM 17 beta-estradiol (E(2)), 4.05 microM 4-nonylphenol (NP), as well as to BNF combined either to E(2) or NP (maintaining the previous concentrations). Liver cytochrome P450 content (P450), ethoxyresorufin-O-deethylase (EROD), and glutathione S-transferase (GST) activities were measured in order to evaluate biotransformation responses. Genotoxicity was assessed as erythrocytic nuclear abnormalities (ENA) frequency. The effects on endocrine function were evaluated as plasma cortisol and glucose. Cortisol was not affected by xeno/estrogens tested, either in single exposure or mixed with BNF. Nevertheless, the intermediary metabolism was affected since glucose concentration increased after 4 h exposure to E(2), and after all BNF+NP exposure lengths. Moreover, a synergism between BNF and NP was thoroughly demonstrated, whereas a sporadic antagonistic interaction was found at 4 h BNF + E(2) exposure. Liver EROD and GST activities were not significantly altered by single E(2) or NP exposure. However, both compounds were able to induce EROD activity in the presence of BNF. NP single exposure was able to significantly increase liver P450 content, while its mixture with BNF displayed an antagonistic interference. Considering the xeno/estrogens single exposures, only NP induced an ENA increase; however, both mixtures (BNF + E(2) and BNF + NP) displayed genotoxic effects. Fish responses to mixtures of xenobiotics are complex and the type of interaction (synergism/potentiation or antagonism) in a particular mixture can vary with the evaluated biological response. PMID:15294438

  12. Modulation of 17{beta}-estradiol-induced responses in fish by cytochrome P4501A1 inducing compounds

    SciTech Connect

    Anderson, M.J.; Hinton, D.E.

    1995-12-31

    Some compounds which induce cytochrome P4501A1 (CYP1A1) are antiestrogenic in mammalian bioassay, and this effect is linked to aryl hydrocarbon (Ah) receptor. Liver of fish synthesizes estrogen-inducible egg yolk precursor protein vitellogenin (Vg) which is critical for oocyte maturation and ovarian development. To determine if Ah receptor-linked endocrine modulation could occur in fish liver, primary cultures of juvenile rainbow trout (Oncorhynchus mykiss) liver cells were co-administered 17{beta}-estradiol and CYP1A1 inducing compounds. Vitellogenin and albumin, estimated by ELISA measurement of concentration in the media 48 hrs after treatment, formed the basis for the test. Cellular CYP1A1 protein content and catalytic activity was estimated by ELISA and ethoxyresorufin-O-deethylase (EROD) activity assays respectively. Equivalent viability (mitochondrial dehydrogenase activity) and secretary functional capacity (albumin synthesis) were estimated and correlated with other results. In descending order, 2,3,4,7,8 pentachlorodibenzofuran (10{sup {minus}12} to 10{sup {minus}8} M) > 2,3,7,8 tetrachlorodibenzo-p-dioxin {approx_equal} 2,3,7,8 tetrachlorodibenzofuran (10{sup {minus}11} to 10{sup {minus}8} M) > {beta}-naphthoflavone (10{sup {minus}7} to 10{sup {minus}6} M) inhibited Vg synthesis in 17{beta}-estradiol treated liver cells. Potency of inhibition directly related to strength as an inducer of CYP1A1 protein. At 10-8 M, PCB congeners 77, 126, and 156 did not inhibit Vg synthesis and induced no or only moderate CYP1A1 protein. At 10-8 M, PCB congener 114, a weak CYP1A1 inducer, potentiated Vg synthesis relative to cells treated with 17{beta}-estradiol alone. This study increases their understanding of the consequences of hepatic CYP1A1 induction, forewarns of reproductive impairment of sexually maturing fishes exposed to CYP1A1 inducing compounds and argues for further, more detailed in vivo investigation.

  13. The effect of 17beta-estradiol on the development of modified hemal spines in early-life stage Gambusia holbrooki.

    PubMed

    Rawson, Christopher A; Lim, Richard P; Warne, Michael St J; Doyle, Christopher J

    2006-08-01

    The morphologic development of the gonopodium of male mosquitofish, Gambusia holbrooki, is essential for proper reproductive function and has previously been used as a biomarker for the presence of endocrine-disrupting chemicals (EDCs) in freshwater systems. The development of the gonopodium is accompanied by the modification of the hemal spines on the 14th, 15th, and 16th vertebrae. These form an anchor for the movement of the gonopodium, and their normal development is therefore critical. This development is under hormonal control. It has been hypothesized that the modification of the hemal spines may be used as an end point for understanding the effects of exposure of early-life stage fish to EDCs and to add to the understanding of the differential sensitivities of different life stages. Larval fish were exposed to concentrations (0, 20, 100, and 500 ng/L) of 17beta-estradiol (E(2)) in two experiments using a flow-through system for 8 and 12 weeks. There was a general delay in the development of the three hemal spines when exposed to high concentrations of E(2). However, this delay was not observed for all end points on a particular spine nor across all spines. Fish examined at 8 weeks postpartum showed a more extensive delay at high concentrations than those examined at 12 weeks postpartum, suggesting that later in the life cycle, high levels of exogenous E(2) may be overcome by increasing levels of endogenous androgen. There was a decrease in the number of phenotypic male fish present in the 500-ng/L treatment at 12 weeks postpartum and an apparent decrease in the degree of sexual differentiation at high levels of E(2) exposure. The results suggest that effects of EDC exposure begin very early in a fish's life cycle. PMID:16783623

  14. Effect of 17beta-estradiol and progesterone on the expression of FeLV in chronically infected cells.

    PubMed

    Tejerizo, German; Domenech, Ana; Illera, Juan C; Collado, Victorio M; Gomez-Lucia, Esperanza

    2005-08-30

    In a previous study, it was found that even though more male cats were infected by feline leukaemia virus (FeLV), females seemed to progress easier to overt disease. To study the effect of female hormones, 17beta-estradiol and progesterone were added in different concentrations (10(-3) M to 10(-12) M) to a culture of persistently FeLV-infected cells. The effect of both hormones was very similar. After 24 h the cell viability was very low at 10(-3) M and 10(-4) M but similar to controls at the remaining concentrations. Liberation of viral particles was estimated by the reverse transcriptase activity (RT), which was the lowest also at 10(-3) M and 10(-4) M. However, low viability could not account for this low RT, as when cells were lysed with lysis buffer RT was high. Thus, cells were dying without freeing viral particles, suggestive of apoptosis. This possibility was confirmed by staining hormone-treated cells with annexin V and propidium iodide. The FeLV antigen p27 measured in the cultures had a maximum at 10(-3) M and 10(-4) M, higher than controls and lysed cells, so the presence of p27 in the supernatant was not only due to cell lysis but a consequence of hormone effect. In conclusion, 17beta-estradiol and progesterone induce death of FeLV-infected cells at high concentrations, probably through a process of apoptosis, which might limit the spread of the infection, as infective viral particles would be hampered from budding. PMID:16023797

  15. Progesterone and 17beta-estradiol enhance regulatory responses to human papillomavirus type 16 virus-like particles in peripheral blood mononuclear cells from healthy women.

    PubMed

    Marks, Morgan A; Gravitt, Patti E; Burk, Robert D; Studentsov, Yevgeniy; Farzadegan, Homayoon; Klein, Sabra L

    2010-04-01

    Human papillomavirus (HPV) virus-like particle (VLP) vaccines are highly effective at preventing viral infections and the development of precancerous lesions through the induction of high-titer neutralizing antibodies and strong cell-mediated immune responses. Women taking combined oral contraceptives (COCs), however, show large variabilities in the magnitudes of their antibody responses. The goal of the present study was to determine the effects of 17beta-estradiol (E2) and progesterone (P4) alone and in combination on the cellular immune response to HPV type 16 (HPV-16) VLPs in vitro. Peripheral blood mononuclear cells (PBMCs) from healthy donor women were stimulated in vitro with HPV-16 VLPs (2.5 microg/ml) in the presence of E2 and P4 administered either alone or in combination; and lymphoproliferation, cytokine production, transcription factor expression, and steroid hormone receptor expression were analyzed. HPV-16 VLPs significantly increased the levels of lymphoproliferation, proinflammatory cytokine (gamma interferon [IFN-gamma], interleukin-1beta [IL-1beta], IL-2, IL-6, IL-8, IL-12p70, IL-17, tumor necrosis factor alpha [TNF-alpha]) production, anti-inflammatory cytokine (IL-1ra, IL-10) production, and the expression of Eralpha and Erbeta but decreased the levels of Foxp3 expression and production of transforming growth factor beta (TGF-beta). Exposure of PBMCs to E2 and P4 either alone or in combination significantly decreased the levels of lymphoproliferation and production of proinflammatory cytokines (IFN-gamma, IL-12p70, TNF-alpha) but increased the levels of production of IL-10 and TGF-beta and the expression of Foxp3 in response to HPV-16 VLPs. Treatment of cells with biologically relevant concentrations of sex steroid hormones suppressed the inflammatory response and enhanced the regulatory response to HPV-16 VLPs, which may have implications for predicting the long-term efficacy of HPV vaccines, adverse events, and cross-protection among women taking COCs. PMID:20130130

  16. Effect of neonatal exposure of 17beta-estradiol and tamoxifen on hepatic CYP3A activity at developmental periods in rats.

    PubMed

    Murakami, Teruo; Sato, Akiko; Inatani, Michiyasu; Sakurai, Hanao; Yumoto, Ryoko; Nagai, Junya; Takano, Mikihisa

    2004-04-01

    We evaluated hepatic CYP3A activity during development in male and female rats and the effect of neonatal exposure of 17beta-estradiol and tamoxifen. In untreated and olive oil-treated (control) rats, hepatic CYP3A activities evaluated by erythromycin metabolism in vitro increased several-fold from age 2 to 9 weeks in males. In contrast, activity in females remained at a low and constant level from 2 to 15 weeks. Exposure of 17beta-estradiol to neonates at a dose of 10 micromol/kg daily for 3 days on day 1-3 (approximately) or 4-6 (approximately) after birth significantly increased hepatic CYP3A activity during the developmental period in both males and females, and a greater influence was observed in females exposed during days 4-6 (approximately). Pubertal exposure of 17beta-estradiol (7-weeks old, 10 micromol/kg daily for 3 days) also increased hepatic CYP3A activity, but only in females. Neonatal exposure to tamoxifen (10 micromol/kg daily for 3 days) showed no appreciable effect in either males or females. In conclusion, a marked sex-difference was observed in hepatic CYP3A activity, and exposure of 17beta-estradiol to neonates increased hepatic CYP3A activity during the developmental period, especially in female rats. PMID:15499175

  17. Levels of 17beta-estradiol receptors expressed in embryonic and adult zebrafish following in vivo treatment of natural or synthetic ligands.

    PubMed

    Chandrasekar, Gayathri; Archer, Amena; Gustafsson, Jan-Ake; Andersson Lendahl, Monika

    2010-01-01

    The nuclear receptors encompass a group of regulatory proteins involved in a number of physiological processes. The estrogen receptors (ERs), of which one alpha and one beta form exist in mammals function as transcription factors in response to 17beta-estradiol (E2). In zebrafish there are three gene products of estrogen receptors and they are denoted esr1 (ERalpha), esr2a (ERbeta2) and esr2b (ERbeta1). Total RNA of zebrafish early life stages (<3, 6, 12, 24, 48, 72, 96 and 120 hours post fertilization) and of adult fish (liver, intestine, eye, heart, brain, ovary, testis, gill, swim bladder and kidney) were isolated following in vivo exposures. Using specific primers for each of the three zebrafish ERs the expression levels were quantified using real time PCR methodology. It was shown that in absence of exposure all three estrogen receptors were expressed in adult fish. The levels of expression of two of these three ER genes, the esr1 and esr2a were altered in organs such as liver, intestine, brain and testis in response to ligand (E2, diethylstilbestrol or 4-nonylphenol). During embryogenesis two of the three receptor genes, esr1 and esr2b were expressed, and in presence of ligand the mRNA levels of these two genes increased. The conclusions are i) estrogen receptor genes are expressed during early development ii) altered expression of esr genes in response to ligand is dependent on the cellular context; iii) the estrogenic ligand 4-nonylphenol, a manufactured compound commonly found in sewage of water treatment plants, acts as an agonist of the estrogen receptor during development and has both agonist and antagonist properties in tissues of adult fish. This knowledge of esr gene function in development and in adult life will help to understand mechanisms of interfering mimicking endocrine chemicals in vivo. PMID:20300630

  18. 17beta-estradiol protects SH-SY5Y Cells against HIV-1 gp120-induced cell death: evidence for a role of estrogen receptors.

    PubMed

    Russo, Rossella; Navarra, Michele; Maiuolo, Jessica; Rotiroti, Domenicantonio; Bagetta, Giacinto; Corasaniti, Maria Tiziana

    2005-10-01

    Despite the large body of experimental evidence demonstrating the neuroprotective properties of 17beta-estradiol (17beta-E2) both in vitro and in vivo experimental models of neuronal injury, the exact mechanisms implicated in neuroprotection have not been fully delineated. Some experimental evidence highlight a role for the antioxidant properties of 17beta-E2 in mediating protection against oxidative injury. Parallel to these, evidence also exist which point to alternative mechanisms involving estrogen receptors (ER). The HIV-1 coat protein, gp120, has been implicated in the progression of central nervous system damage caused by HIV-1 infection. The neurotoxic effects induced by gp120 are triggered via an excitotoxic mechanism of cell death which implicates alteration of calcium homeostasis, activation of calcium-dependent pathways, mitochondrial uncoupling and membrane lipid peroxidation. In the present study, we demonstrate that 17beta-E2 protects human SH-SY5Y neuroblastoma cells from cell death elicited by gp120. Tamoxifen and ICI 182,780, two ER antagonists, both antagonized 17beta-E2-mediated inhibition of cell death. Exposure of SH-SY5Y cells to gp120 for 30min caused a significant accumulation of intracellular reactive oxygen species (ROS) and this was abrogated by 17beta-E2; however, the ability of 17beta-E2 to counteract ROS generation induced by gp120 does not account for the reported prevention of cell death because ICI 182,780 failed to revert intracellular ROS reduction caused by 17beta-E2 though it was able to revert prevention of cell death. Furthermore, by using 17alpha-E2, the isomer unable to stimulate ER which, however, retains the antioxidant effects, we observed that a pre-treatment with 17alpha-E2 was effective in preventing gp120-induced accumulation of ROS but it failed to affect cell death caused by the viral protein. Collectively, these data demonstrate that neuroprotection afforded by 17beta-E2 is receptor-mediated and ROS scavenging effects may not be implicated. PMID:15899520

  19. Transport of 17beta-estradiol and testosterone in a field lysimeter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    17ß-estradiol (E2) and testosterone (T) are present in sources such as waste treatment effluent and manures, and can potentially disrupt aquatic organisms at low concentrations. Laboratory studies consistently indicate limited mobility and rapid attenuation of E2 and T in soils; however, these hormo...

  20. Transformation of 17beta-estradiol mediated by lignin peroxidase: the role of veratryl alcohol.

    PubMed

    Mao, Liang; Lu, Junhe; Gao, Shixiang; Huang, Qingguo

    2010-07-01

    Lignin peroxidases (LiPs) are a group of extracellular enzymes excreted by certain fungi, e.g., Phanerochaete chrysosporium. These fungi also produce veratryl alcohol (VA) as a secondary metabolite to regulate the performance of LiP. 17ss-Estradiol (E2) is a natural female hormone that is strongly endocrine disruptive when released to the natural environment. The widespread occurrence of E2 and related hormonal chemicals in soil and water environments has been identified, representing an emerging contamination of concern. We report in this study that E2 can be effectively transformed and removed through reactions mediated by LiP and such reactions are significantly enhanced in the presence of VA. We systematically investigated LiP activity and enzymatic reaction kinetics in systems having VA absent or present. The results suggest that VA enhanced the transformation and removal of E2 by the combination of two effects: (i) mitigating LiP inactivation and (ii) modifying the enzyme catalytic kinetics. These findings provide insights into an important pathway that may govern the environmental transformation of E2 and other emerging endocrine-disrupting contaminants of similar nature in the environment, and provide a basis for potential development and optimization of enzyme-based processes for remediation and removal of these contaminants. PMID:20035325

  1. Tissue expression of glandular kallikrein and its response to 17 beta-estradiol in the acclimatized carp.

    PubMed

    Haussmann, Denise; Vidal, Rene; Figueroa, Jaime

    2006-06-01

    Cyprinus carpio skeletal muscle kallikrein was isolated to apparent homogeneity, and a polyclonal antiserum against the purified protein was generated. Glandular kallikrein expression and tissue distribution were assessed using both Western blots and immunohistochemistry. A 39-kDa protein was detected in skeletal muscle, the gill, kidney, and pituitary gland, where an additional 72-kDa immunoreactive band was observed. Immunohistochemistry revealed immunoreactive kallikrein in the intermuscle tissue, epithelial gill cells, apical portion of distal and proximal tubular cells in the kidney, mucus and epithelial cells of the skin, intestinal tube, and prolactin-producing cells of the pituitary gland. In addition, the effect of 17beta-estradiol on kallikrein expression was analyzed in three different tissues of winter- and summer-acclimatized male carps. A 2.5-fold (p<0.05) increase in kallikrein immunoreactivity due to estrogen treatment was observed in winter-acclimatized carp muscle, but not in summer-acclimatized fish. In contrast, the gill responded differently, since a 2-fold (p<0.05) increase was found only in summer-acclimatized carps. Kallikrein immunoreactivity in the kidney increased both in summer- (2.5 fold) and in winter-acclimatized carps (1.5 fold). The signals obtained demonstrate the existence of tissue-specific variable responses to estrogen treatment in vivo, between winter and summer-acclimatized carp. PMID:16849838

  2. 17Beta-estradiol expands IgA-producing B cells in mice deficient for the mu chain.

    PubMed

    Lagerquist, M K; Erlandsson, M C; Islander, U; Svensson, L; Holmdahl, R; Carlsten, H

    2008-01-01

    Oestrogen is not only a sex hormone but also an important regulator of the immune system. Expression of the heavy chain of IgM (mu) is essential for B-cell differentiation. However, a small number of IgA-positive B cells can be found in mice lacking the mu chain (muMT-/-). The aim of this study was to investigate the effects of oestrogen on this alternative B-cell pathway in muMT-/- mice. Our results clearly demonstrate that oestrogen increases the frequency of IgA-producing B cells in muMT-/- mice in both bone marrow and spleen cells. We also show that mature IgM-producing B cells are not required for oestrogen-mediated suppression of granulocyte-mediated inflammation or thymic involution. In conclusion, we demonstrate that 17beta-estradiol benzoate increases the frequency of IgA-producing B cells in muMT-/- mice, suggesting that oestrogen can influence the alternative B-cell pathway found in muMT-/- mice. PMID:18021189

  3. Contrasting effects of increased and decreased dopamine transmission on latent inhibition in ovariectomized rats and their modulation by 17beta-estradiol: an animal model of menopausal psychosis?

    PubMed

    Arad, Michal; Weiner, Ina

    2010-06-01

    Women with schizophrenia have later onset and better response to antipsychotic drugs (APDs) than men during reproductive years, but the menopausal period is associated with increased symptom severity and reduced treatment response. Estrogen replacement therapy has been suggested as beneficial but clinical data are inconsistent. Latent inhibition (LI), the capacity to ignore irrelevant stimuli, is a measure of selective attention that is disrupted in acute schizophrenia patients and in rats and humans treated with the psychosis-inducing drug amphetamine and can be reversed by typical and atypical APDs. Here we used amphetamine (1 mg/kg)-induced disrupted LI in ovariectomized rats to model low levels of estrogen along with hyperfunction of the dopaminergic system that may be occurring in menopausal psychosis, and tested the efficacy of APDs and estrogen in reversing disrupted LI. 17beta-Estradiol (50, 150 microg/kg), clozapine (atypical APD; 5, 10 mg/kg), and haloperidol (typical APD; 0.1, 0.3 mg/kg) effectively reversed amphetamine-induced LI disruption in sham rats, but were much less effective in ovariectomized rats; 17beta-estradiol and clozapine were effective only at high doses (150 microg/kg and 10 mg/kg, respectively), whereas haloperidol failed at both doses. Haloperidol and clozapine regained efficacy if coadministered with 17beta-estradiol (50 microg/kg, an ineffective dose). Reduced sensitivity to dopamine (DA) blockade coupled with spared/potentiated sensitivity to DA stimulation after ovariectomy may provide a novel model recapitulating the combination of increased vulnerability to psychosis with reduced response to APD treatment in female patients during menopause. In addition, our data show that 17beta-estradiol exerts antipsychotic activity. PMID:20237462

  4. Maternally derived testosterone and 17beta-estradiol in the eggs of Arctic-breeding glaucous gulls in relation to persistent organic pollutants.

    PubMed

    Verboven, Nanette; Verreault, Jonathan; Letcher, Robert J; Gabrielsen, Geir W; Evans, Neil P

    2008-08-01

    It is largely unknown if and how persistent organic pollutants (POPs) affect the transfer of maternal hormones to eggs. This occurs despite an increasing number of studies relating environmental conditions experienced by female birds at the time of egg formation to maternal hormonal effects. Here we report the concentrations of maternal testosterone, 17beta-estradiol and major classes of POPs (organochlorines, brominated flame retardants and metabolically-derived products) in the yolk of unincubated, third-laid eggs of the glaucous gull (Larus hyperboreus), a top-predator in the Arctic marine environment. Controlled for seasonal and local variation, positive correlations were found between the concentrations of certain POPs and testosterone. Contaminant-related changes in the relative concentrations of testosterone and 17beta-estradiol were also observed. In addition, yolk steroid concentrations were associated with contaminant profiles describing the proportions of different POPs present in the yolk. Eggs from nests in which two sibling eggs hatched or failed to hatch differed in POP profiles and in the relative concentrations of testosterone and 17beta-estradiol. Although the results of this correlative study need to be interpreted with caution, they suggest that contaminant-related changes in yolk steroids may occur, possibly affecting offspring performance over and above toxic effects brought about by POPs in eggs. PMID:18550446

  5. Affinity chromatography of human estrogen receptor-alpha expressed in Saccharomyces cerevisiae. Combination of heparin- and 17beta-estradiol-affinity chromatography.

    PubMed

    Feng, W; Graumann, K; Hahn, R; Jungbauer, A

    1999-08-01

    Estrogen receptor-alpha is a member of the nuclear hormone receptor superfamily and is considered as a very important regulatory protein. Human estrogen receptor-alpha has been cloned into Saccharomyces cerevisiae as a fusion to ubiquitin and expression is controlled by a metallothionin promotor. Pilot scale quantities of receptor have been produced by a yeast strain transformed with expression plasmid YEpE13 [Graumann et al., J. Steroid Biochem. Mol. Biol. 57 (1996) 293] in a 14 l stirred tank reactor. The yeast extract contained 2-4 pmol of receptor protein per mg total protein. A purification scheme has been developed using heparin-affinity chromatography combined with affinity chromatography with immobilized 17beta-estradiol 17-hemisuccinate. Heparin-affinity chromatography was very efficient to remove host cell protein. Accompanying proteins that stabilize unoccupied receptor have not been dissociated during elution. The receptor could be purified 5-10-fold in ligand-free state. In contrast to previous reports, we did not find a difference of the binding affinity of liganded and unliganded receptor for heparin immobilized onto Sepharose. The unoccupied receptor could be further purified 100-fold with ligand-affinity chromatography using 17beta-estradiol 17-hemisuccinate-bovine serum albumin-Sepharose. The receptor could be kept in its native state, although saturated with 17beta-estradiol. The purification sequence allows an efficient production of receptor. Further improvement of productivity can be only accomplished by increasing the expression level. PMID:10480241

  6. The metabolism of 17 beta-estradiol by lactoperoxidase: a possible source of oxidative stress in breast cancer.

    PubMed

    Sipe, H J; Jordan, S J; Hanna, P M; Mason, R P

    1994-11-01

    Electron spin resonance (ESR) spectroscopy and oxygen consumption measurements using a Clark-type oxygen electrode have been used to study the metabolism of the estrogen 17 beta-estradiol by lactoperoxidase. Evidence for a one-electron oxidation of estradiol to its reactive phenoxyl radical intermediate is presented. The phenoxyl radical metabolite abstracts hydrogen from reduced glutathione generating the glutathione thiyl radical, which is spin trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and subsequently detected by ESR spectroscopy. In the absence of DMPO, molecular oxygen is consumed by a sequence of reactions initiated by the glutathione thiyl radical. Similarly, the estradiol phenoxyl radical abstracts hydrogen from reduced beta-nicotinamide-adenine dinucleotide (NADH) to generate the NAD. radical. The NAD. radical is not spin trapped by DMPO, but instead reduces molecular oxygen to the superoxide radical, which is then spin-trapped by DMPO. The superoxide generated may either spontaneously dismutate to form hydrogen peroxide or react with another NADH to form NAD., thus propagating a chain reaction leading to oxygen consumption and hydrogen peroxide accumulation. Ascorbate inhibits oxygen consumption when estradiol is metabolized in the presence of either glutathione or NADH by reducing radical intermediates back to their parent molecules and forming the relatively stable ascorbate radical. These results demonstrate that the futile metabolism of micromolar quantities of estradiol catalyzes the oxidation of much greater concentrations of biochemical reducing cofactors, such as glutathione and NADH, with hydrogen peroxide produced as a consequence. The accumulation of intracellular hydrogen peroxide could explain the hydroxyl radical-induced DNA base lesions recently reported for female breast cancer tissue. PMID:7955118

  7. Evaluation of the primary humoral immune response following exposure of male rats to 17beta-estradiol or flutamide for 15 days.

    PubMed

    Ladics, G S; Smith, C; Nicastro, S C; Loveless, S E; Cook, J C; O'Connor, J C

    1998-11-01

    There is a concern that certain industrial chemicals found in the environment may mimic or antagonize endogenous hormones and adversely affect the endocrine as well as the immune system. The objective of this study was to determine if exposure of Crl:CD (SD)BR male rats to 17beta-estradiol (17beta-E2), an estrogen receptor agonist, or flutamide (FLUT), an androgen receptor antagonist, would significantly alter the primary IgM humoral immune response to sheep red blood cells (SRBC). This study was conducted in the context of a male in vivo Tier I battery designed to identify endocrine-active compounds (EACs). The Tier I male battery consists of organ weights coupled with a comprehensive hormonal assessment. Rats were dosed by the intraperitoneal route for 15 days with vehicle or 0.001, 0.0025, 0.0075, or 0.050 mg/kg/day 17beta-E2 or 0.25, 1, 5, or 20 mg/kg/day FLUT. Six days prior to termination, selected rats were injected intravenously with SRBC for assessment of humoral immune function. Spleen cell number and spleen and thymus weights were obtained. Serum was analyzed for anti-SRBC IgM antibody by using an enzyme-linked immunosorbent assay. At 0.050 mg/kg/day 17beta-E2, mean final body and absolute thymus weights were significantly decreased to 84 and 65% of control, respectively. 17beta-E2 did not significantly alter spleen weight, spleen cell number, or the primary IgM humoral immune response to SRBC. The no-observed-adverse-effect level (NOAEL) for immune system alteration was 0.050 mg/kg/day 17beta-E2 since the decrease in absolute thymus weight was judged to be secondary to the decrements in body weight. In the Tier I male battery, responses to 17beta-E2 included decreased absolute testis and epididymis weights, decreased relative accessory sex gland unit weights, hormonal alterations (decreased serum testosterone (T), dihydrotestosterone (DHT), and luteinizing hormone (LH), and increased serum prolactin and E2 levels). The lowest-observed-adverse-effect level (LOAEL) for the reproductive indices was 0.001 mg/kg/day 17beta-E2 based on the hormonal alterations seen at this level; no NOAEL was established. Exposure to FLUT did not significantly alter mean final body, spleen, or absolute thymus weights, spleen cell number, or the primary IgM humoral immune response to SRBC. A significant increase (118% of control) in relative thymus weight was observed at 20 mg/kg/day FLUT. The NOAEL for immune system alteration was 5 mg/kg/day FLUT based on the increased relative thymus weights that were judged to be compound-related. In the Tier I male battery, responses to FLUT included decreased absolute epididymis and relative accessory sex gland unit weights and hormonal alterations (increased serum T, DHT, E2, and LH, and decreased follicle stimulating hormone levels). The LOAEL for the reproductive indices was 0.25 mg/kg/day FLUT based on the hormonal alterations seen at this level; no NOAEL was established. Based on these data, the reproductive and not the immune system appears to be the primary target organ of toxicity in young adult male rats treated with either 17beta-E2 or FLUT. PMID:9928670

  8. Effects of 17beta-estradiol, 4-nonylphenol and PCB 126 on the estrogenic activity and phase 1 and 2 biotransformation enzymes in male sea bass (Dicentrarchus labrax).

    PubMed

    Vaccaro, Emilia; Meucci, Valentina; Intorre, Luigi; Soldani, Giulio; Di Bello, Domenica; Longo, Vincenzo; Gervasi, Pier Giovanni; Pretti, Carlo

    2005-11-30

    The endocrine system of wildlife is exposed to a wide variety of natural and man-made chemicals which may lead to damage to the reproductive system and other adverse effects, including alteration of drug-metabolizing enzymes. In the present study, the effects of in vivo exposure to a natural (17beta-estradiol: E2) or a xenoestrogen (4-nonylphenol: NP) estrogen or an anti-estrogen (3,3',4,4',5-pentachlorobiphenyl: PCB 126) upon vitellogenin (VTG) synthesis and hepatic phase 1 and 2 enzymes have been investigated in adult male sea bass. By means of ELISA analysis with the use of polyclonal antibodies prepared against VTG purified from E2-treated sea bass, we assessed the time course and sensitivity of VTG induction in the plasma of sea bass treated with E2 at 0.1, 0.5, 2.5 and 5.0 mg/kg doses or NP at 5.0 or 50 mg/kg doses, respectively. Sea bass sensitivity to this induction was found to be similar to that of other fish species, but with a delay in maximal response. E2 treatment also caused a selective time- and dose-dependent inhibition of hepatic CYP1A-linked EROD and phase 2 glutathione S-transferase (GST) activities, without affecting the activity of CYP3A-linked 6beta-testosterone hydroxylase, (omega)- and (omega-1)-lauric acid hydroxylases or phase 2 DT-diaphorase. A similar selective inhibition on CYP1A was also observed in fish treated with 50 mg/kg NP. The results regarding CYP1A and CYP3A were also confirmed by Western blot analysis. When the sea bass were treated with either 10 or 100 microg/kg PCB 126, an AhR ligand not yet tested in vivo in fish to assess its anti-estrogenicity, a modest and selective induction of EROD and DT-diaphorase activities was observed. Interestingly, both these activities were recovered to their control levels in sea bass co-treated with 0.5 mg/kg E2 and 10 or 100 microg/kg PCB 126, probably through a cross-talk mechanism between the estrogen receptor and AhR or other transcription factors that regulate the expression of these enzymes. Furthermore, it was demonstrated that PCB 126 possesses a potent anti-estrogenic activity in the sea bass in vivo as it inhibited the E2-induced VTG synthesis with an IC50 of 28 microg/kg. The results of this study suggest that the exposure of fish to xenoestrogens or anti-estrogens may alter, in addition to various physiological processes, the expression of specific CYPs and phase 2 enzymes, thereby reducing the capability of their detoxication system. PMID:16219370

  9. 17 beta-estradiol inhibits tumor necrosis factor-alpha-induced nuclear factor-kappa B activation by increasing nuclear factor-kappa B p105 level in MCF-7 breast cancer cells.

    PubMed

    Hsu, S M; Chen, Y C; Jiang, M C

    2000-12-01

    Tumor necrosis factor-alpha (TNF-alpha) exerts many cytological effects on a wide range of cells. TNF-alpha can activate nuclear factor-kappa B (NF-kappa B). Activation of NF-kappa B by TNF-alpha mediates many functions of TNF-alpha. The NF-kappa B inhibitor, I kappa B alpha, negatively regulates the activity of NF-kappa B. In MCF-7 cells (an estrogen and TNF-alpha receptor positive cell line), treatment with 17 beta-estradiol (E(2)) inhibited TNF-alpha-induced NF-kappa B DNA binding activity in the gel retardation assays. But, the level of the I kappa B alpha and the TNF-alpha receptor, TNF-R1, were not obviously affected. The NF-kappa B precursor, NF-kappa B p105, has been shown to be associated with NF-kappa B in the cytoplasm and efficiently blocks its nuclear translocation and activation. Treatment of MCF-7 cells with E(2) increased the level of NF-kappa B p105 protein. The anti-estrogen, 4OH-tamoxifen, treatment inhibited E(2)-induced NF-kappa B p105 expression. Our findings indicate that NF-kappa B p105 plays a role in modulating the functions of TNF-alpha in the estrogen receptor positive breast cancer cells. PMID:11112416

  10. Use of biotinylated 17beta-estradiol in enzyme-immunoassay development: spacer length and chemical structure of the bridge are the main determinants in simultaneous streptavidin-antibody binding.

    PubMed

    Lacorn, Markus; Fleischer, Kerstin; Willig, Stephanie; Gremmel, Sabine; Steinhart, Hans; Claus, Rolf

    2005-02-01

    17beta-estradiol (E2) concentrations are in the low pg/ml range in plasma. To develop a sensitive enzyme immunoassay (EIA) for E2-determination a highly specific antibody raised against a 6-carboxymethyl (CMO)-E2-bovine serum albumine conjugate was used. Based on 6-CMO-E2 and 6-amino-E2, four biotinylated tracers with two different spacer lengths between E2 and biotin were synthesized using biotinylation reagents in one step reactions. All amino-based tracers were unsuitable for assay development because the antibody binding was too weak compared to the analyte E2. For 6-CMO-based tracers the simultaneous binding of the tracer to the antibody and streptavidin seems to be the determining step in the procedure depending on incubation temperature and spacer lengths. While a short spacer of 9 carbon atoms was susceptible to room temperature, a longer spacer of 16 carbon atoms showed nearly the same results for incubation at 4 degrees C or at room temperature. The absolute detection limit of this system was 0.63 pg/well. For sample clean-up, porcine plasma was solvent-extracted and depending on the initial plasma volume further purified by solvent partition. Determination of reproducibility resulted in intraassay coefficients of variation of 13% and 5.3% for samples with E2-levels of 15 pg/ml and 236 pg/ml, respectively. Measurement of E2-spiked blood plasma revealed recoveries of 83% up to 100% for E2 concentrations between 50 pg/ml and 1000 pg/ml. Only for the lowest concentration (20 pg/ml) a recovery of 58% was observed. Correlation of the EIA with an established radio immunoassay resulted in r=0.991 using the same antibody. PMID:15777945

  11. Novel estrogens and their radical scavenging effects, iron-chelating, and total antioxidative activities: 17 alpha-substituted analogs of delta 9(11)-dehydro-17 beta-estradiol.

    PubMed

    Römer, W; Oettel, M; Menzenbach, B; Droescher, P; Schwarz, S

    1997-11-01

    Antioxidant effects of N,N-dimethyl-p-toluidine, p-cresol, and p-(hydroxy)thioanisol 17 alpha-substituted analogs of 17 beta-estradiol and their delta 9(11)-dehydro homologs were investigated using four different in vitro models: rat synaptosomal lipid peroxidation induced by Fenton's reagent, Fe(II)-chelating activities, the formation of superoxide anion radicals, and total antioxidative activity. Whereas the classical estrogen 17 beta-estradiol as well as selected phenolic compounds was only moderately inhibiting iron-dependent lipid peroxidation and stimulating total antioxidative activity, besides delta 9(11)-dehydro-17 beta-estradiol (J 1213), novel estrogens such as C-17-oriented side chain analogs of 17 beta-estradiol (J 843, J 872, and J 897) and delta 9(11)-dehydro homologs (J 844, J 864, and J 898) directly altered the iron redox chemistry and diminished the formation of superoxide anion radicals generated by a xanthine/xanthine oxidase-dependent luminescence reaction to a great extent. These results suggest that definite modifications in the chemical structure of 17 beta-estradiol, e.g., the introduction of a delta 9(11)-double bond and/or p-cresol as well as p-(hydroxy)thioanisol C-17 substitution, may result in substantial changes in their antioxidant behavior. These compounds may be drug candidates for treating pathologies related to free radical formation. PMID:9366006

  12. [Biological and clinical safety of nomegestrol acetate administered alone then associated in inverse sequence with transdermal 17 beta estradiol, in women at risk of dyslipoproteinemia type IIa].

    PubMed

    Zartarian, M; Chevallier, T; Micheletti, M C; Leber, C; Jamin, C

    1998-12-01

    In this study including 26 patients with dyslipoproteinemia classified IIa, we evaluated biochemical and clinical safety of Nomegestrol acetate (Lutenyl) used for its antigonadotrophin property. It was administered alone, during 3 cycles at the dose of 5 mg/d for 21 days by cycle and then it was associated (at the same sequence and dose), without any wash out, for the next 6 cycles, with a 17 beta estradiol patch (Estraderm TTS 50), 50 micrograms/d from the 11th to the 21st day of each cycle. Nomegestrol acetate, alone, had no significant effect on glycemia, antithrombin III, triglycerides, total cholesterol, apoprotein A1, and LpA1 values compared to those at baseline but apoprotein B and Lp (a) values tended to decrease slightly. Serum progesterone levels were collapsed, and FSH values were low. Weight and blood pressure remained constant. Adding 17 beta estradiol enabled to significantly decrease and normalize the apoprotein B values after the first 3 cycles compared to the baseline values, then these values remained constant during the next 3 cycles. There was no effect on the other parameters (except for a significant increase in plasmatic estradiol values) on the antigonadotrophin property of Nomegestrol acetate, nor on weight and blood pressure which remained constant. Moreover, we observed an important decrease in the rate of amenorrheic cycles compared to those with Nomegestrol acetate alone. PMID:9949893

  13. 17Beta-estradiol protects against oxidative stress-induced cell death through the glutathione/glutaredoxin-dependent redox regulation of Akt in myocardiac H9c2 cells.

    PubMed

    Urata, Yoshishige; Ihara, Yoshito; Murata, Hiroaki; Goto, Shinji; Koji, Takehiko; Yodoi, Junji; Inoue, Satoshi; Kondo, Takahito

    2006-05-12

    The GSH/glutaredoxin (GRX) system is involved in the redox regulation of certain enzyme activities, and this system protects cells from H2O2-induced apoptosis by regulating the redox state of Akt (Murata, H., Ihara, Y., Nakamura, H., Yodoi, J., Sumikawa, K., and Kondo, T. (2003) J. Biol. Chem. 278, 50226-50233). Estrogens, such as 17beta-estradiol (E2), play an important role in development, growth, and differentiation and appear to have protective effects on oxidative stress mediated by estrogen receptor alpha (ERalpha). However, the role of the ERbeta-mediated pathway in this cytoprotection and the involvement of E2 in the redox regulation are not well understood. In the present study, we demonstrated that E2 protected cardiac H9c2 cells, expressing ERbeta from H2O2-induced apoptosis concomitant with an increase in the activity of Akt. E2 induced the expression of glutaredoxin (GRX) as well as gamma-glutamylcysteine synthetase, a rate-limiting enzyme for the synthesis of GSH. Inhibitors for both gamma-glutamylcysteine synthetase and GRX and ICI182,780, a specific inhibitor of ERs, abolished the protective effect of E2 on cell survival as well as the activity of Akt, suggesting that ERbeta is involved in the cytoprotection and redox regulation by E2. Transcription of the GRX gene was enhanced by E2. The promoter activity of GRX was up-regulated by an ERbeta-dependent element. These results suggest that the GRX/GSH system is involved in the cytoprotective and genomic effects of E2 on the redox state of Akt, a pathway that is mediated, at least in part, by ERbeta. This mechanism may also play an antiapoptotic role in cancer cells during carcinogenesis or chemotherapy. PMID:16549430

  14. Effects of o,p'-DDE, heptachlor, and 17beta-estradiol on vitellogenin gene expression and the growth hormone/insulin-like growth factor-I axis in the tilapia, Oreochromis mossambicus.

    PubMed

    Davis, Lori K; Visitacion, Nancy; Riley, Larry G; Hiramatsu, Naoshi; Sullivan, Craig V; Hirano, Tetsuya; Grau, E Gordon

    2009-05-01

    Effects of two endocrine disruptors, o,p'-DDE and heptachlor, and 17beta-estradiol (E(2)) on vitellogenin (Vg) and the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis were examined in male tilapia. In the first experiment, fish were given 5 weekly injections of either E(2), o,p'-DDE or heptachlor (5 microg/g). E(2) treatment increased plasma Vg and hepatic expression of three Vg genes (Vgs A, B, and C) and estrogen receptor alpha (ERalpha), while reducing plasma levels of IGF-I and suppressing the expression of IGF-I, the GH receptor (GHR2) and the putative somatolactin receptor (GHR1). Neither pesticide greatly affected the other parameters examined, except for a significant reduction in expression of GHR2 and increased plasma IGF-I. In the second experiment, fish were given a single injection of o,p'-DDE or heptachlor (100 microg/g), or E(2) (5 microg/g) and sacrificed 5 days post-injection. Treatment with E(2) stimulated expression of all three Vg genes. Both o,p'-DDE and heptachlor increased expression of VgB, whereas only o,p'-DDE increased VgA expression. There was no effect of o,p'-DDE or heptachlor on VgC expression or plasma Vg levels. Treatment with o,p'-DDE and heptachlor as well as E(2) increased ERalpha and ERbeta transcript levels. Similarly, both pesticides increased GHR1 and IGF-I expression, whereas no significant effect of E(2) was observed on GHR1, GHR2 or IGF-I expression. These results indicate that o,p'-DDE and heptachlor have varying temporal and dose effects on modulation of Vg and the GH/IGF-I axis that are distinct from E(2). PMID:19101654

  15. Up-regulation of PI3K/Akt signaling by 17{beta}-estradiol through activation of estrogen receptor-{alpha}, but not estrogen receptor-{beta}, and stimulates cell growth in breast cancer cells

    SciTech Connect

    Lee, Young-Rae; Park, Jinny; Kim, Jong-Suk; Youn, Hyun Jo; Jung, Sung Hoo . E-mail: shjung@chonbuk.ac.kr

    2005-11-04

    Estrogen stimulates cell proliferation in breast cancer. The biological effects of estrogen are mediated through two intracellular receptors, estrogen receptor-{alpha} (ER{alpha}) and estrogen receptor-{beta} (ER{beta}). However, the role of ERs in the proliferative action of estrogen is not well established. Recently, it has been known that ER activates phosphatidylinositol-3-OH kinase (PI3K) through binding with the p85 regulatory subunit of PI3K. Therefore, possible mechanisms may include ER-mediated phosphoinositide metabolism with subsequent formation of phosphatidylinositol-3,4,5-trisphosphate (PIP{sub 3}), which is generated from phosphatidylinositol 4,5-bisphosphate via PI3K activation. The present study demonstrates that 17{beta}-estradiol (E2) up-regulates PI3K in an ER{alpha}-dependent manner, but not ER{beta}, and stimulates cell growth in breast cancer cells. In order to study this phenomenon, we have treated ER{alpha}-positive MCF-7 cells and ER{alpha}-negative MDA-MB-231 cells with 10 nM E2. Treatment of MCF-7 cells with E2 resulted in a marked increase in PI3K (p85) expression, which paralleled an increase in phospho-Akt (Ser-473) and PIP{sub 3} level. These observations also correlated with an increased activity to E2-induced cell proliferation. However, these effects of E2 on breast cancer cells were not observed in the MDA-MB-231 cell line, indicating that the E2-mediated up-regulation of PI3K/Akt pathway is ER{alpha}-dependent. These results suggest that estrogen activates PI3K/Akt signaling through ER{alpha}-dependent mechanism in MCF-7 cells.

  16. Analysis of gene expression profiles in largemouth bass exposed to 17-beta-estradiol and to anthropogenic contaminants that behave as estrogens.

    PubMed

    Larkin, P; Sabo-Attwood, T; Kelso, J; Denslow, N D

    2003-12-01

    Novel molecular based methods are being developed to study changes in gene expression in wildlife exposed to anthropogenic chemicals. Gene arrays, in particular, are useful tools that can be used to simultaneously monitor hundreds to thousands of genes within a single experiment, giving an investigator the ability to determine how exposure affects multiple metabolic pathways. These methods are thought to be both sensitive and able to reveal biochemical mechanisms of action. A largemouth bass (LMB) array containing 132 genes has been designed to study the impact of gene expression in male fish exposed to 17-beta estradiol or to the compounds 4-nonylphenol (4-NP) or 1,1-dichloro-2, 2-bis (p-chlorophenyl) ethylene (p,p'-DDE). The results of these experiments demonstrate distinct gene expression patterns in LMB exposed to these compounds. PMID:14680325

  17. 17beta-estradiol affords protection against 4-vinylcyclohexene diepoxide-induced ovarian follicle loss in Fischer-344 rats.

    PubMed

    Thompson, Kary E; Sipes, I Glenn; Greenstein, Ben D; Hoyer, Patricia B

    2002-03-01

    Repeated dosing with 4-vinylcyclohexene diepoxide (VCD) accelerates atresia via apoptosis in primordial and primary follicles in ovaries of rats. The mechanisms that control atresia and VCD-induced toxicity are unknown; however, they could involve 17beta-E2. Atresia slows as animals enter puberty, whereas circulating E2 levels increase with the the onset of cyclicity. This inverse relationship suggests that E2 may be involved in the control of atresia. Therefore, this study was designed to determine whether treatment of immature rats with E2 could protect follicles normally destroyed by VCD-induced apoptosis. Female F344 rats were treated daily with E2, ER analogs, and/or VCD for 15 d. VCD alone caused a 50% reduction in primordial and primary follicles. Coinjection of E2 (0.1 mg/kg) and VCD (80 mg/kg) selectively protected primary follicles from VCD-induced follicle loss. This protection was mimicked by an ER agonist, genistein (0.1 mg/kg), and prevented by an ER antagonist, 4-hydroxytamoxifen (2 mg/kg). VCD treatment increased caspase-3-like activity, whereas concurrent treatment with genistein and VCD restored caspase-3-like activity to control levels. VCD treatment had no effect on circulating E2 levels, uterine weight, or E2 binding to the ER, nor could it directly displace E2 from ERbeta. These observations support the idea that ER-mediated protection against VCD-induced follicle toxicity is obtained by reducing apoptosis in small preantral follicles, although VCD does not appear to directly interact with ER. PMID:11861533

  18. Regulation of leptin expression by 17beta-estradiol in human placental cells involves membrane associated estrogen receptor alpha.

    PubMed

    Gambino, Yésica P; Pérez Pérez, Antonio; Dueñas, José L; Calvo, Juan Carlos; Sánchez-Margalet, Víctor; Varone, Cecilia L

    2012-04-01

    The placenta produces a wide number of molecules that play essential roles in the establishment and maintenance of pregnancy. In this context, leptin has emerged as an important player in reproduction. The synthesis of leptin in normal trophoblastic cells is regulated by different endogenous biochemical agents, but the regulation of placental leptin expression is still poorly understood. We have previously reported that 17β-estradiol (E(2)) up-regulates placental leptin expression. To improve the understanding of estrogen receptor mechanisms in regulating leptin gene expression, in the current study we examined the effect of membrane-constrained E(2) conjugate, E-BSA, on leptin expression in human placental cells. We have found that leptin expression was induced by E-BSA both in BeWo cells and human placental explants, suggesting that E(2) also exerts its effects through membrane receptors. Moreover E-BSA rapidly activated different MAPKs and AKT pathways, and these pathways were involved in E(2) induced placental leptin expression. On the other hand we demonstrated the presence of ERα associated to the plasma membrane of BeWo cells. We showed that E(2) genomic and nongenomic actions could be mediated by ERα. Supporting this idea, the downregulation of ERα level through a specific siRNA, decreased E-BSA effects on leptin expression. Taken together, these results provide new evidence of the mechanisms whereby E(2) regulates leptin expression in placenta and support the importance of leptin in placental physiology. PMID:22310000

  19. PLASMA CLEARANCE OF VITELLOGENIN IN SHEEPSHEAD MINNOWS AFTER CESSATION OF EXPOSURE TO 17BETA-ESTRADIOL AND PARA-NONYLPHENOL

    EPA Science Inventory

    Two experiments were performed to determine the rate of vitellogenin plasma accumulation and clearance in male sheepshead minnows (Cyprinodon variegatus) during and after exposure to either 17b-estradiol (E2) or para-nonylphenol (p-NP). Adult fish were continuously exposed to aqu...

  20. Glandular kallikreins in the teleost Cyprinus carpio: tissue distribution, possible involvement in prolactin processing, and effect of 17 beta-estradiol in vivo.

    PubMed

    Figueroa, J; Fernández, K; Haussmann, D; Richards, G; Barra, V; Kausel, G

    2002-09-01

    We examined glandular kallikrein (GK), a putative prolactin processing protease, in the teleost Cyprinus carpio. When employing an anti-Centropristis striata GK antibody proteins of 39 kDa in muscle, 52 kDa in gill, 52 kDa in kidney, and two proteins of 46 and 72 kDa in pituitary gland were detected. Immunoreactive kallikreins were recognized in intermuscle cell tissue, epithelial gill cells, apical region of tubular cells, and prolactin producing lactotrophs in pituitary gland, suggesting a osmoregulatory role for this enzyme. We found three prolactin (PRL) variants using anti-tilapia PRL antibodies, in pituitary gland 23 and 16 kDa, and in plasma 23 and 22 kDa forms. Clearly co-localization of GK and PRL in lactotrophs could be demonstrated. In winter-acclimatized male carp, where the pituitary PRL level is low, 17beta-estradiol treatment increased PRL but not GK immunoreactivity. In contrast to GK and PRL co-regulation by estrogen in mammalian pituitary gland, no similar effect on immunoreactive PRL and GK was observed in the ichtyc pituitary. No changes in GK immunostaining occurred in gill or muscle tissue in response to estrogen treatment. These results, taken with the observation of significantly increased GK immunoreactivity in the apical region of kidney tubular cells in estrogen treated male carp, indicate that the regulation of GK expression in pituitary and kidney could be different in fish with respect to mammals. PMID:12392686

  1. Rise of testosterone, nortestosterone, and 17beta-estradiol concentrations in peripheral blood plasma of pigs after sublingual application in vivo.

    PubMed

    Claus, Rolf; Häussler, Susanne; Lacorn, Markus

    2007-02-01

    Application of endogenous anabolic steroids to meat producing animals is not allowed in the EU. In other countries application is practised due to a low oral activity based on an efficient first liver passage. This contrasts with pharmacological investigations where steroids were readily absorbed by the buccal and sublingual mucosa using absorption enhancers. An in vivo study was performed to clarify possible absorption after sublingual applications of one milligram portions of either testosterone (T), 17beta-estradiol (E), or nortestosterone (NT) in sesame oil to castrated male pigs (n=5) without specific delivery systems during anaesthesia. Blood samples were drawn using jugular vein catheters for 15 min before and 3h after application. Hormone concentrations were determined by Radioimmunoassay for T and E or Enzymeimmunoassay for NT. For all steroids a slight increase was measurable one minute after application. Maximal values for T, E, and NT were 2.5 ng/ml, 1.5 ng/ml and 4.2 ng/ml, respectively, and were observed after 10 min. The concentrations of the three steroids decreased slowly thereafter but were still significantly elevated 1-3h after application. Oral absorption of steroids without enhancers should be considered in risk analysis. PMID:17010491

  2. Beneficial effect of 17{beta}-estradiol on hyperglycemia and islet {beta}-cell functions in a streptozotocin-induced diabetic rat model

    SciTech Connect

    Yamabe, Noriko; Kang, Ki Sung; Zhu Baoting

    2010-11-15

    The modulating effect of estrogen on glucose homeostasis remains a controversial issue at present. In this study, we sought to determine the beneficial effect of 17{beta}-estradiol (E{sub 2}) on hyperglycemia and islet {beta}-cell functions in streptozotocin (STZ)-induced diabetic rats. Male Sprague-Dawley rats were injected i.p. with STZ to induce a relatively mild diabetic condition. The rats were then treated with E{sub 2} orally at 500 {mu}g/kg body weight/day for 15 days to evaluate the modulating effect on hyperglycemia, insulin secretion, and islet {beta}-cell proliferation. E{sub 2} administration for 10 days significantly lowered plasma glucose levels, increased plasma insulin levels, and improved glucose tolerance by attenuating insulin response to oral glucose loading. These beneficial effects of E{sub 2} were accompanied by increases in islet number and volume, rate of islet cell proliferation, and the amount of insulin secreted. The growth-stimulatory effect of E{sub 2} on islet cells was linked to the functions of the estrogen receptor {alpha}. Notably, these protective effects of E{sub 2} on diabetic conditions were basically not observed when the STZ-treated rats had a more severe degree of islet damage and hyperglycemia. Taken together, we conclude that E{sub 2} can promote the regeneration of damaged pancreatic islets by stimulating {beta}-cell proliferation in diabetic rats, and this effect is accompanied by improvements in glucose tolerance and a decrease in plasma glucose levels. These findings suggest that oral administration of E{sub 2} may be beneficial in diabetic patients with an accelerated loss of islet {beta}-cells.

  3. Hepatic estrogen receptor and plasma 17{beta}-estradiol concentrations as biomarkers of 2,3,7,8-TCDD exposure in avian hatchlings

    SciTech Connect

    Janz, D.M.; Bellward, G.D.

    1995-12-31

    The authors have been investigating the sensitivity of various toxicologically relevant endpoints as environmental biomarkers in avian hatchlings exposed in ovo to 2,3,7,8-TCDD. Potential biomarkers included various endocrine endpoints such as plasma 17{beta}-estradiol (E{sub 2}), hepatic estrogen receptor (ER) affinities and concentrations, and plasma thyroid hormones, which were compared to hepatic ethoxyresorufin O-deethylase (EROD) induction. The animal models used were domestic chickens and pigeons, and great blue herons. An experiment conducted in pigeon hatchlings compared ``early`` (embryonic day 4; E4) vs. ``late`` (E14) in ovo exposure to 1 {micro}g/kg and 3 {micro}g/kg of TCDD, respectively. Birds were sacrificed on day of hatch (H) and day 7 after hatch (D7). In the late exposure experiment, plasma E{sub 2} concentrations were reduced at H and elevated at D7 in the TCDD-exposed birds (p < 0.05). Hepatic ER concentrations were elevated at H (p < 0.01). Although EROD was half-maximally induced at H and D7 in the early exposure experiment in pigeons, there was no effect of TCDD treatment on E, or ER levels. The nominal TCDD concentration in these pigeons (1 {micro}g/kg egg) was within the range observed in wild piscivorous bird eggs collected from aquatic systems contaminated with TCDD and related chemicals (approx. 0.5--2 ng TEQ/g egg). In herons exposed to 2 {micro}g/kg of TCDD at the midpoint of incubation, hepatic ER affinities (Kd) and concentrations (Bmax) were elevated in treated birds at H (p < 0.05); however there was no effect on plasma E, levels. Liver [{sup 3}H]-TCDD concentrations were 11.3 {+-} 0.8 ng/g at H, and 0.8 {+-} 0.1 ng/g at D7, representing 9.9% and 4.9% of the nominal TCDD dose, respectively.

  4. Long-term dysregulation of circadian and 17-beta estradiol-induced LH, prolactin and corticosterone secretion after dimethylbenz (a) anthracene administration in the Sprague-Dawley female rat.

    PubMed

    Yon de Jonage-Canonico, M Beau; Lenoir, V; Scholler, R; Kerdelhué, B

    2005-07-01

    A single intragastric administration of 7,12-dimethylbenz (a) anthracene (DMBA) has been shown, when given at 55-60 days of age, to induce mammary tumors in young cycling female Sprague-Dawley rats. The appearance of the tumors is preceded by a series of neuroendocrine disturbances of the hypothalamo-pituitary-gonadal (HPG) axis, including attenuation of the preovulatory Luteinizing Hormone (LH) and Gonadotropin-Releasing Hormone (GnRH) release and amplification of the preovulatory 17beta-Estradiol (E(2)) surge. In this study, we examined the hypothesis that a single administration of DMBA could also, in the long range, induce disturbances of others neuroendocrine axis, like the Hypothalamic-Pituitary-Adrenal (HPA) axis and/or the Lactotroph axis. Sprague-Dawley rats, 55-60 days of age, received, on the day of Estrous of the Estrous cycle, a single administration of 15 mg of DMBA delivered by intragastric intubation. Then, they were ovariectomized 5 days later. One month later, (1) Two groups of animal were sacrificed by decapitation at 09:00 a.m. and 05:00 p.m. to record the circadian rhythm of plasma LH, Prolactin (PRL) and corticosterone, (2) Three other groups of animal were sacrificed by decapitation at three different times after a morning subcutaneous administration of 50 microg/kg of Estradiol Benzoate (EB), to induce a negative and positive feed-back of the secretion of LH. Then, plasma LH, PRL and corticosterone concentrations were measured. After DMBA administration, (1) the negative--but not the positive--LH feed-back was seen, (2) the PRL circadian rhythm was blunted and the corticosterone circadian rhythm was almost absent, (3) the increase in PRL or Corticosterone plasma concentration was significantly reduced. In conclusion, a single administration of DMBA provokes a long-term dysregulation of not only the HPG axis but also of the lactotroph and HPA axis. These dysregulations, along with the already evidenced long-term inhibition of DMBA upon Melatonin secretion from the pineal gland, might accelerate the promotion of mammary tumors induced by the mammary carcinogen. PMID:15980990

  5. Effect of light irradiation and sex hormones on jurkat T cells: 17beta-estradiol but not testosterone enhances UVA-induced cytotoxicity in Jurkat lymphocytes.

    PubMed

    Cohly, Hari H P; Graham-Evans, Barbara; Ndebele, Kenneth; Jenkins, John K; McMurray, Robert; Yan, Jian; Yu, Hongtao; Angel, Michael F

    2005-04-01

    In Eastern cultures, such as India, it is traditionally recommended that women but not men cover their heads while working in the scorching sun. The purpose of this pilot study was to determine whether there was any scientific basis for this cultural tradition. We examined the differential cytotoxic effects of ultraviolet A light (UVA) on an established T cell line treated with female and male sex hormones. CD4+ Jurkat T cells were plated in 96 well plates at 2 x 106 cells/ml and treated with 17beta-estradiol (EST) or testosterone (TE). These cells were irradiated by UVA light with an irradiance of 170 J/cm2 for 15min at a distance of 6 cm from the surface of the 96-well plate. Controls included cells not treated with hormones or UVA. The effects of EST and TE were investigated between 1 and 20 ng/mL. Cytotoxicity by fluorescein-diacetate staining and COMET assay generating single strand DNA cleavage, tail length and tail moment measurements were examined. The effect of estrogen (5ng/mL) on apoptosis and its mediators was further studied using DNA laddering and western blotting for bcl-2 and p53. We found that EST alone, without UVA, enhanced Jurkat T cell survival. However, EST exhibited a dose-related cytotoxicity in the presence of UVA; up to 28% at 20 ng/ml. TE did not alter UVA-induced cytotoxicity. Since TE did not alter cell viability in the presence of UVA further damaging studies were not performed. COMET assay demonstrated the harmful effects of EST in the presence of UVA while EST without UVA. had no significant effect on the nuclear damage. Apoptosis was not present as indicated by the absence of DNA laddering on agarose gel electrophoresis at 5ng/ml EST or TE +/- UVA. Western blot showed that estrogen down regulated bcl-2 independently of UVA radiation while p53 was down regulated in the presence of UVA treatment. EST and TE have differential effects on UVA-induced cytotoxicity in Jurkat T-lymphocyte which suggested that women may be more susceptible to the harmful effects of solar irradiation than men. PMID:16705813

  6. Effects of 17beta-estradiol and testosterone on hepatic mRNA/protein levels and catalytic activities of CYP2M1, CYP2K1, and CYP3A27 in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Buhler, D R; Miranda, C L; Henderson, M C; Yang, Y H; Lee, S J; Wang-Buhler, J L

    2000-10-15

    There is growing concern that exposure to chemicals in the environment can disrupt the endocrine systems of wildlife and humans, causing reproductive problems or other adverse effects. The expression of many cytochrome P450s (CYPs) is under hormonal control, hence, levels of these enzymes can be affected by exposure to endocrine-disrupting chemicals. Previous research has reported that treatment of fish and other animals with the estrogenic and androgenic hormones 17beta-estradiol (E2) and testosterone (T) alters the P450 content or enzyme activities in the treated animals. However, the results of many of these studies are either incomplete or in disagreement and in most cases the effect on specific P450 forms has not been determined. Therefore, to better understand the effects of gonadal hormones on the expression of P450s and their associated enzyme activities, it was of interest to undertake a comprehensive investigation of the transcriptional and translational expression of three constitutive hepatic P450s in the rainbow trout (Oncorhynchus mykiss) following hormone exposure. Accordingly, juvenile trout were injected intraperitoneally with propylene glycol vehicle and the most active estrogenic and androgenic hormones E2 (3 mg/kg) or T (3 mg/kg) on days 1, 4, 7, 13, and 15 and euthanized on day 19. After treatment with E2, hepatic microsomes showed significantly lower levels (percentage of control) in total P450 contents (52%), lauric acid hydroxylase (32%), and 6beta-progesterone hydroxylase activities (27%), [(3)H]aflatoxin-DNA binding (31%), and the protein levels of individual cytochrome P450s (CYPs) LMC1 (CYP2M1), LMC2, (CYP2K1), and LMC5 (CYP3A27) (average for three isoforms a reduction to 29% of control values) with only minor differences between sexes. Treatment with T had either no effect or resulted in small increases in total P450 in males (42%), in lauric acid hydroxylase in females (24%), and in 6beta-progesterone hydroxylase activity in males (21%). Biological variabilities among fish were high and a polymorphic or new LMC2-like form was detected at about 52 kDa in some liver microsomal samples after exposure of fish to either hormone. Female liver RNAs were analyzed through Northern blots and an average decrease of 94% in CYP2 M1, CYP2K1, and CYP3A27 mRNA levels occurred in the E2-treated trout. In livers from T-treated trout, the changes of mRNA levels of CYP2M1 and CYP3A27 were negligible, but CYP2K1 mRNA level decreased by about 60%. Additional CYP2K1 cDNA hybridizable mRNAs were seen in some fish as faint bands at about 2.8 kb for both hormone treatments. Results of this study, therefore, indicated that E2 down-regulated while T produced small but variable effects on the hepatic mRNA/protein levels of CYP2K1, CYP2M1, and CYP3A27 in juvenile rainbow trout. This study, therefore, suggests that exposure of fish and other wildlife to environmental endocrine disruptors, especially estrogen mimics, can adversely affect a number of physiological processes through mechanisms involving altered levels of expression of specific P450 isozymes. PMID:11032764

  7. Environmental Technology Verification Report for Abraxis 17β-Estradiol (E2) Magnetic Particle Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits

    EPA Science Inventory

    The EPA's National Risk Management Research Laboratory (NRMRL) and its verification organization partner, Battelle, operate the Advanced Monitoring Systems (AMS) Center under ETV. The AMS Center recently evaluated the performance of the Abraxis 17(beta)-estradiol (E2) magnetic p...

  8. Divergent effects of 17-{beta}-estradiol on human vascular smooth muscle and endothelial cell function diminishes TNF-{alpha}-induced neointima formation

    SciTech Connect

    Nintasen, Rungrat; Multidisciplinary Cardiovascular Research Center , University of Leeds, Leeds LS2 9JT; Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University ; Riches, Kirsten; Mughal, Romana S.; Multidisciplinary Cardiovascular Research Center , University of Leeds, Leeds LS2 9JT ; Viriyavejakul, Parnpen; Chaisri, Urai; Maneerat, Yaowapa; Turner, Neil A.; Multidisciplinary Cardiovascular Research Center , University of Leeds, Leeds LS2 9JT ; Porter, Karen E.

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} augments neointimal hyperplasia in human saphenous vein. Black-Right-Pointing-Pointer TNF-{alpha} induces detrimental effects on endothelial and smooth muscle cell function. Black-Right-Pointing-Pointer Estradiol exerts modulatory effects on TNF-induced vascular cell functions. Black-Right-Pointing-Pointer The modulatory effects of estradiol are discriminatory and cell-type specific. -- Abstract: Coronary heart disease (CHD) is a condition characterized by increased levels of proinflammatory cytokines, including tumor necrosis factor-{alpha} (TNF-{alpha}). TNF-{alpha} can induce vascular endothelial cell (EC) and smooth muscle cell (SMC) dysfunction, central events in development of neointimal lesions. The reduced incidence of CHD in young women is believed to be due to the protective effects of estradiol (E2). We therefore investigated the effects of TNF-{alpha} on human neointima formation and SMC/EC functions and any modulatory effects of E2. Saphenous vein (SV) segments were cultured in the presence of TNF-{alpha} (10 ng/ml), E2 (2.5 nM) or both in combination. Neointimal thickening was augmented by incubation with TNF-{alpha}, an effect that was abolished by co-culture with E2. TNF-{alpha} increased SV-SMC proliferation in a concentration-dependent manner that was optimal at 10 ng/ml (1.5-fold increase), and abolished by E2 at all concentrations studied (1-50 nM). Surprisingly, E2 itself at low concentrations (1 and 5 nM) stimulated SV-SMC proliferation to a level comparable to that of TNF-{alpha} alone. SV-EC migration was significantly impaired by TNF-{alpha} (42% of control), and co-culture with E2 partially restored the ability of SV-EC to migrate and repair the wound. In contrast, TNF-{alpha} increased SV-SMC migration by 1.7-fold, an effect that was completely reversed by co-incubation with E2. Finally, TNF-{alpha} potently induced ICAM-1 and VCAM-1 expression in both SV-EC and SV-SMC. However there was no modulation by E2 in either cell-type. In conclusion, TNF-{alpha} induced SV neointima formation, increased SMC proliferation and migration, impaired SV-EC migration and increased expression of adhesion molecules. E2 exerted distinct cell-type and function-specific modulation, the mechanisms underlying which are worthy of further detailed study.

  9. An inter-laboratory study on the variability in measured concentrations of 17Beta-estradiol, testosterone and 11-ketotestosterone in white sucker: implications and recommendations

    EPA Science Inventory

    Endocrine-disrupting chemicals (EDCs) are exogenous substances that can lead to impacts on the reproduction of fish sometimes by altering circulating concentrations of 17â-estradiol (E2), testosterone (T) and 11-ketotestosterone (11-KT). Common methods to measure steroids in pla...

  10. An inter-laboratory study on the variability in measured concentrations of 17Beta-estradiol, testosterone and 11-ketotestosterone in white sucker: implications and recommendations

    EPA Science Inventory

    Endocrine-disrupting chemicals (EDCs) are exogenous substances that can lead to impacts on the reproduction of fish sometimes by altering circulating concentrations of 17-estradiol (E2), testosterone (T) and 11-ketotestosterone (11-KT). Common methods to measure steroids in pla...

  11. FoxM1 influences embryo implantation and is regulated by 17 beta-estradiol and progesterone in mouse uteri and endometrium cells

    PubMed Central

    Xie, Yunpeng; Cui, Dan; Kong, Ying

    2014-01-01

    To be a successful implantation, endometrial receptivity should be established. Forkhead box M1 (FoxM1) is described as a major oncogenic transcription factor in tumor initiation, promotion, and progression. FoxM1 regulates the expression of lots of targeted genes important to cell differentiation, proliferation and apoptosis; cell-cycle progression; and tumor angiogenesis, migration, invasion, and metastasis. According to these functions, we believe that FoxM1 should also play an essential role in embryo implantation. To test our hypothesis, we observed the expression and distribution of FoxM1 during the early pregnancy of mouse. Then, we used Immunohistochemistry to examine the expression of FoxM1 induced by E2 and/or P4 in the ovariectomized mouse uterus and human endometrium cells. This study further investigated whether FoxM1 was an important factor in the implantation. Our results showed that FoxM1 expressed in the mouse uterus during early pregnancy (Day 1 to 5). The expression of FoxM1 gradually increased along pregnancy process; FoxM1 expression could be increased by E2. On the contrary, FoxM1 expression could be decreased by P4 and E2 plus P4. We also detected the proliferation of human endometrium cells. We found that E2 might promote cells proliferation, while P4 and E2 plus P4 inhibited cells proliferation; Inhibiting FoxM1 could interfere the embryo implantation of mouse. Amplification or inhibiting of FoxM1 in JAR cells can increase or decrease the adhesion rate to Rl95-2 and HEC-1A cells separately. Our data indicate that FoxM1 might play an important role during the process of mouse embryo implantation. PMID:25400737

  12. FoxM1 influences embryo implantation and is regulated by 17 beta-estradiol and progesterone in mouse uteri and endometrium cells.

    PubMed

    Xie, Yunpeng; Cui, Dan; Kong, Ying

    2014-01-01

    To be a successful implantation, endometrial receptivity should be established. Forkhead box M1 (FoxM1) is described as a major oncogenic transcription factor in tumor initiation, promotion, and progression. FoxM1 regulates the expression of lots of targeted genes important to cell differentiation, proliferation and apoptosis; cell-cycle progression; and tumor angiogenesis, migration, invasion, and metastasis. According to these functions, we believe that FoxM1 should also play an essential role in embryo implantation. To test our hypothesis, we observed the expression and distribution of FoxM1 during the early pregnancy of mouse. Then, we used Immunohistochemistry to examine the expression of FoxM1 induced by E2 and/or P4 in the ovariectomized mouse uterus and human endometrium cells. This study further investigated whether FoxM1 was an important factor in the implantation. Our results showed that FoxM1 expressed in the mouse uterus during early pregnancy (Day 1 to 5). The expression of FoxM1 gradually increased along pregnancy process; FoxM1 expression could be increased by E2. On the contrary, FoxM1 expression could be decreased by P4 and E2 plus P4. We also detected the proliferation of human endometrium cells. We found that E2 might promote cells proliferation, while P4 and E2 plus P4 inhibited cells proliferation; Inhibiting FoxM1 could interfere the embryo implantation of mouse. Amplification or inhibiting of FoxM1 in JAR cells can increase or decrease the adhesion rate to Rl95-2 and HEC-1A cells separately. Our data indicate that FoxM1 might play an important role during the process of mouse embryo implantation. PMID:25400737

  13. [CA 125, CA 19.9, MCA, CEA, 17 beta-estradiol and progesterone in functional and benign cysts and carcinoma of the ovary].

    PubMed

    Caione, C; Minelli, L; Angiolillo, M

    1994-06-01

    CA 125, CA 19.9, CEA, MCA, E2 and P levels have been assessed in the serum and in the ovarian mass fluid of 216 patients through echo-guided puncture to distinguish between functional, benign and malignant cystic neoplasm. Statistically evaluated data have been assessed through histological diagnosis in 122 cases: cyst fluid markers do not always differentiate ovarian carcinoma from benign cysts, while they have a significant concentration in persistent functional cysts (follicular and luteal) if compared with benign and malignant neoplasms. The following management is suggested: 1) Echo-guided puncture in case of simple, persistent ovarian cyst with low levels of CA 125 and CA 19.9 in serum; 2) Operation in case of fatty, chocolate or mucinous fluid. Wait and see if the fluid is either clear or hematic/chocolate with high P levels (luteal cyst); 3) Operation on simple cyst with high CA 125 ald low E2 and P levels (serous c.), in case of relapse or persistence after aspiration; 4) Wait and see if CA 125 is low and E2 and P levels are high (follicular c.). PMID:7936384

  14. Reconnaissance of 17 beta-estradiol, 11-ketotestosterone, vitellogenin, and gonad histopathology in common carp of United States streams; potential for contaminant-induced endocrine disruption

    USGS Publications Warehouse

    Goodbred, Steven L.; Gilliom, Robert J.; Gross, Timothy S.; Denslow, Nancy P.; Bryant, Wade B.; Schoeb, Trenton R.

    1997-01-01

    A reconnaissance of sex steroid hormones and other biomarkers in common carp was used to assess whether endocrine disruption may be occurring in fish in United States streams, to evaluate relations between endocrine disruption and contaminant levels, and to determine requirements for further studies. 17?-estradiol, 11-ketotestosterone, vitellogenin, and gonadal histopathology were measured in adult carp (usually 10--15 for each sex) at 25 sites (647 fish), representing a wide range of environmental settings typical of major regions of the nation. Fish were collected during August--December 1994, a period of gonadal maturation after spawning. Contaminants evaluated were organochlorine pesticides and polychlorinated biphenyls in tissue; phthalates, phenols, and polycyclic aromatic hydrocarbons in bed sediment; and dissolved pesticides in water. Mean site concentrations of steroid hormones spanned two orders of magnitude for both sexes. No significant regional differences in steroid hormones were detected for males, but females from the Northern and Southern Midcontinent were significantly different from other regions of the country in one or both hormones. Within all regions there were significant differences between sites in one or both hormones for both sexes. Most correlation coefficients between biomarkers and contaminants were negative. Contaminants that had significant (a=0.05) correlations with biomarkers were organochlorine pesticides, phenols, and dissolved pesticides. The strongest pattern common to both males and females was a negative correlation between the hormone ratio (E2/11-KT) and dissolved pesticides. The significant site-to-site differences in biomarkers, and the presence of significant correlations between biomarkers and contaminants, are evidence that fish in some streams may be experiencing endocrine disruption. Improved information is needed to evaluate whether endocrine disruption is actually occurring and if there are reproductive effects on individual or populations of carp or other species. Future studies should shift to more intensive study of fewer sites, including reference and contaminated sites, in order to address these additional questions.

  15. Land-cover effects on the fate and transport of surface-applied antibiotics and 17-beta-estradiol on a sandy outwash plain, Anoka County, Minnesota, 2008–09

    USGS Publications Warehouse

    Trost, Jared J.; Kiesling, Richard L.; Erickson, Melinda L.; Rose, Peter J.; Elliott, Sarah M.

    2013-01-01

    A plot-scale field experiment on a sandy outwash plain in Anoka County in east-central Minnesota was used to investigate the fate and transport of two antibiotics, sulfamethazine (SMZ) and sulfamethoxazole (SMX), and a hormone, 17-beta-estradiol (17BE), in four land-cover types: bare soil, corn, hay, and prairie. The SMZ, SMX, and 17BE were applied to the surface of five plots of each land-cover type in May 2008 and again in April 2009. The cumulative application rate was 16.8 milligrams per square meter (mg/m2) for each antibiotic and 0.6 mg/m2 for 17BE. Concentrations of each chemical in plant-tissue, soil, soil-water, and groundwater samples were determined by using enzyme-linked immunosorbent assay (ELISA) kits. Soil-water and groundwater sampling events were scheduled to capture the transport of SMZ, SMX, and 17BE during two growing seasons. Soil and plant-tissue sampling events were scheduled to identify the fate of the parent chemicals of SMZ, SMX, and 17BE in these matrices after two chemical applications. Areal concentrations (mg/m2) of SMZ and SMX in soil tended to decrease in prairie plots in the 8 weeks after the second chemical application, from April 2009 to June 2009, but not in other land-cover types. During these same 8 weeks, prairie plots produced more aboveground biomass and had extracted more water from the upper 125 centimeters of the soil profile compared to all other land-cover types. Areal concentrations of SMZ and SMX in prairie plant tissue did not explain the temporal changes in areal concentrations of these chemicals in soil. The areal concentrations of SMZ and SMX in the aboveground plant tissues in June 2009 and August 2009 were much lower, generally two to three orders of magnitude, than the areal concentrations of these chemicals in soil. Pooling all treatment plot data, the median areal concentration of SMZ and SMX in plant tissues was 0.01 and 0.10 percent of the applied chemical mass compared to 22 and 12 percent in soil, respectively. Furthermore, areal concentrations of SMZ and SMX in plant-tissue samples were variable, and did not differ significantly between control and treatment plots within each land-cover type. SMZ was detected in 23 percent of soil-water samples and in 16 percent of groundwater samples collected between October 2008 and October 2009 in treatment plots, indicating that surface-applied SMZ leached below the rooting zone and reached groundwater. SMX was detected in only 1 percent of soil-water and groundwater samples during this same time period. In contrast to the antibiotics, 17BE was not reliably detected in soil samples. Additionally, ELISA-determined 17BE concentrations in plant-tissue, soil-water, and groundwater samples indicated the presence of chemicals that were not applied as part of this experiment [17BE from an external source or other chemical(s) that interfered with the 17BE ELISA kits].

  16. Water-compatible magnetic imprinted nanoparticles served as solid-phase extraction sorbents for selective determination of trace 17beta-estradiol in environmental water samples by liquid chromatography.

    PubMed

    Hao, Yi; Gao, Ruixia; Shi, Lu; Liu, Dechun; Tang, Yuhai; Guo, Zengjun

    2015-05-29

    Endocrine disrupting compounds (EDCs) are a potential risk for wildlife and humans for their existence in water. The efficient extraction and clean-up steps are required before detection of low concentration levels of EDCs. In this work, a novel water-compatible magnetic molecularly imprinted nanoparticles is synthesized for the selective extraction of 17β-estradiol (E2) in environmental water samples. The preparation is carried out by introducing aldehyde groups to the surface of amino-functionalized magnetic nanoparticles through a simple one-step modification, followed by copolymerization of functional monomer gelatin and template E2 via surface imprinting technique. The gelatin with abundant active groups could not only act as functional monomer reacting with template, but also assemble covalently at the surface of magnetic nanoparticles. At the same time, gelatin would improve the water-compatibility of imprinted materials for attaining high extraction efficiency. To obtain high imprinting effect, the preparation conditions are optimized in detail using Central composite design-response surface methodology. The resultant polymers have uniform spherical shape with a shell thickness of about 8nm, stable crystalline form, and super-paramagnetic property. Meanwhile, the obtained polymers have high capacity of 12.87mgg(-1) and satisfactory selectivity to template molecule. To testify the feasibility of the magnetic imprinted polymers in sample pretreatment, a method for determination of trace E2 in environmental water samples was set up by combination of solid-phase extraction (SPE) using the prepared polymers as sorbents and HPLC for rapid isolation and determination of E2. The limit of detection of proposed method is 0.04ngmL(-1), the intra- and inter-day relative standard deviations (RSDs) are less than 4.6% and 5.7%, respectively. The recoveries of E2 from environmental water samples are in the range from 88.3% to 99.1% with the RSDs less than 7.2%. PMID:25890441

  17. Genetic mapping of Eutr1, a locus controlling E2-induced pyometritis in the Brown Norway rat, to RNO5.

    PubMed

    Gould, Karen A; Pandey, Jyotsna; Lachel, Cynthia M; Murrin, Clare R; Flood, Lisa A; Pennington, Karen L; Schaffer, Beverly S; Tochacek, Martin; McComb, Rodney D; Meza, Jane L; Wendell, Douglas L; Shull, James D

    2005-11-01

    In certain rat strains, chronic estrogen administration can lead to pyometritis, an inflammation of the uterus accompanied by infection and the accumulation of intraluminal pus. In this article, we report that the Brown Norway (BN) rat is highly susceptible to pyometritis induced by 17beta-estradiol (E2). The susceptibility of the BN rat to E2-induced pyometritis appears to segregate as a recessive trait in crosses to the resistant August x Copenhagen Irish (ACI) strain. In a (BN x ACI)F(2) population, we find strong evidence for a major genetic determinant of susceptibility to E2-induced pyometritis on rat chromosome 5 (RNO5). Our data are most consistent with a model in which the BN allele of this locus, designated Eutr1 (Estrogen-induced uterine response 1), acts in an incompletely dominant manner to control E2-induced pyometritis. Furthermore, we have confirmed the contribution of Eutr1 to E2-induced uterine pyometritis using an RNO5 congenic rat strain. In addition to Eutr1, we obtained evidence suggestive of linkage for five additional loci on RNO2, 4, 11, 17, and X that control susceptibility to E2-induced pyometritis in the (BN x ACI)F(2) population. PMID:16284801

  18. Assessing the effects of exposure timing on biomarker expression using 17beta-estradiol

    EPA Science Inventory

    Temporal and spatial variability in estrogenicity has been documented for many treated wastewater effluents with the consequences of this variability on the expression of biomarkers of endocrine disruption being largely unknown. Laboratory exposure studies usually utilize constan...

  19. Polymorphism of 17-beta estradiol in a transdermal drug delivery system.

    PubMed

    Variankaval, N E; Jacob, K I; Dinh, S M

    2002-03-01

    The inclusions in a typical transdermal drug delivery system (TDS) containing estradiol drug were characterized using microscopic, spectroscopic and thermal analytical techniques. Optical and scanning electron microscopy were used to determine the locations and morphologies of the crystals in the matrix. Two different types of crystals randomly distributed laterally inside the patch were observed. Solid aggregates were found surrounding needle-like inclusions. Optical imaging through the thickness of the patch and SEM sections of the patch revealed that these inclusions were found to occupy a single layer inside the adhesive matrix. No inclusions were observed either in the backing-matrix interface or the matrix-liner interface. The inclusions exhibited a wide range of sizes. The thickness of the crystals as determined by SEM ranged from 10-14 microm. Out of the four crystal forms of estradiol, two of which are solvates (EA and EM) and the other two are anhydrous (EC and ED). Forms EC and ED did not exhibit significant differences in the spectra. Thermal analysis revealed that this was due to the highly unstable nature of ED and its tendency to either convert spontaneously to EC or occur in mixtures with it. The Raman spectrum of the aggregates in the patch showed peaks that seemed characteristic of at least two different forms of estradiol. Only one of these forms is a completely hydrogen bonded system and therefore, was concluded to be estradiol hemihydrate. A splitting of the C17-O peak at 1284 cm(-1) and 1294 cm(-1) was attributed to the existence of at least two types of crystal forms - one that exhibits hydrogen bonding and one that does not. DSC on different concentrations of estradiol in acrylic adhesive showed a clear endotherm for 14 wt % estradiol and apparent endotherms for lower concentrations. The absence of crystallization exotherms is due to the extremely slow kinetics of crystals growth in the polymeric patch. PMID:15348624

  20. Biodistribution and metabolism of 16. cap alpha. -((/sup 18/F)-fluoro)-17. beta. -estradiol

    SciTech Connect

    Mathias, C.J.; Brodack, J.W.; Kilbourn, M.R.; Carlson, K.A.; Katzenellenbogen, J.A.; Welch, M.J.

    1985-05-01

    The uptake of receptor-mediated radiopharmaceuticals as measured by target to non-target uptake ratios depends upon many parameters. These include blood flow to the tissue, blood volume, receptor concentration as well as metabolism of the tracer. In a rat tumor model (DMBA) induced mammary tumors with high concentration of estrogen receptors) uptake of /sup 18/F-estradiol was studied while blood flow was measured with the use of /sup 125/I-iodoantipyrine, blood volume was measured with the use of /sup 99m/Tc-labeled red blood cells, and the receptor concentration by in vitro assay. The results demonstrate no correlation between blood flow and uptake of ligand, or between receptor concentration and uptake of ligand. No correlation existed between blood volume and uptake or /sup 18/F-estradiol, even though the blood volume varied by a factor of --20 in the tumors studied. The distribution of the fluorine-18 may depend upon metabolites of the ligand rather than the ligand itself. The authors have developed a technique to separate metabolites from the administered compound in blood and tissues. The distribution of the compound in the blood at times >30 mins after injection was primarily within the red blood cells in a chemical form that was not extractable even in lysed blood samples. By injecting blood from one rate into another the authors have shown that the activity in blood 2 hours after injection of /sup 18/F-estradiol is not available for uptake in receptor rich tissue but remains in the blood and non-target tissues.

  1. 17Beta-Estradiol Inhibits Calcium-Activated Potassium Channel Expressions in Rat Whole Bladder

    PubMed Central

    2016-01-01

    Purpose: To investigate the effect of estrogen on the expression of calcium-activated potassium (KCa) channels in an overactive bladder rat model. To this end, mRNA and protein levels of KCa channel subtypes in the bladder of ovariectomized rats were measured by reverse transcription polymerase chain reaction and western blotting, respectively. Methods: Ten-week-old female Sprague-Dawley rats were divided randomly into 3 groups: sham-operated control group (n=11), ovariectomy group (n=11), and the group treated with estrogen after ovariectomy (n=12). Rats in the last group were subcutaneously injected with 17β-estradiol (50 μg/kg) every other day for 2 weeks, whereas rats in the other 2 groups received vehicle (soybean oil) alone. Two weeks after treatment, the whole bladder was excised for mRNA and protein measurements. Results: Protein levels of the large-conductance KCa (BK) channels in the ovariectomy group were 1.5 folds higher than those in the sham-operated control group. However, the protein levels of the other KCa channel subtypes did not change significantly upon bilateral ovariectomy. Treatment with 17β-estradiol after ovariectomy restored BK channel protein levels to the control value. In contrast, BK channel mRNA levels were not significantly affected by either ovariectomy alone or 17β-estradiol treatment. The small-conductance KCa type 3 channel (SK3) mRNA and protein levels decreased to 75% of control levels upon 17β-estradiol treatment. Conclusions: These results suggest that 17β-estradiol may influence urinary bladder function by modulating BK and SK3 channel expression. PMID:27032553

  2. Lipocalin 2: a "sexy" adipokine that regulates 17Beta-estradiol and obesity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this article we review the findings of Guo et. al. (Endocrinology, 153: 1183-1193) that the protein, Lipocalin 2 is more highly expressed in subcutaneous adipose tissue than in gondal tissue of female mice. Of particular interest is that the paper by Guo et. al. observed that ablation of the Lip...

  3. PROMOTION BY 17BETA-ESTRADIOL AND BETA-HEXACHLOROCYCLOHEXANE OF HEPATOCELLULAR TUMORS IN MEDAKA, ORYZIAS LATIPES. (R825298)

    EPA Science Inventory

    Abstract

    A feature common to many laboratory and field studies with various fish species is a higher prevalence of hepatocellular neoplasia in females than in males. During female sexual maturation, endogenous estrogens stimulate substantial increases in synthetic acti...

  4. The effect of 17beta estradiol withdrawal on the level of brain and peripheral neurosteroids in ovarectomized rats.

    PubMed

    Maayan, Rachel; Strous, Rael D; Abou-Kaoud, Machmoud; Weizman, Abraham

    Dehydroepiandrosterone (DHEA), pregnenolone (P) and their sulfate derivatives are neuroactive neurosteroids synthesized endogenously in the brain and in steroidogenic organs and influence or are influenced by a variety of physiological processes. Since parturition is followed by a rapid drop in estrogen levels in serum and brain it may be hypothesized that the drastic drop in the brain exposure to estrogens may cause a disturbance in the neurosteroid-to-neurosteroid-sulfate equilibrium with clinical relevance. In order to develop a rat animal model for human postpartum rapid estrogen decline conditions, the present study investigated effects of sudden withdrawal of hyperphysiological estrogens levels on levels of DHEA, DHEAS, P and PS in peripheral blood and brain tissue as well as cortical sulfatase activity. Twenty-four 3-month-old female rats were ovarectomized followed by either no estrogen, high levels of estrogen alone, or followed by sudden withdrawal after high-administered estrogen levels. Results indicated elevated brain cortical DHEA-S and reduced cortical sulfatase in ovarectomized rats following sudden estrogen withdrawal. No significant alterations in DHEA, P or PS were noted. Study observations suggest the marked influence estrogen withdrawal states may have on cortical DHEA-S levels in particular, the precise mechanism of which remains unknown but which may be related to the paralleled decrease in sulfatase activity. This DHEA-S increase may lead to attenuated GABAergic tone and may be relevant to post-natal behavioral disturbances (e.g. depression, anxiety). PMID:15927368

  5. The effect of 17 beta-estradiol on cholesterol in human macrophages is influenced by the lipoprotein milieu

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Estrogen and testosterone are thought to modulate coronary heart disease (CHD) risk. To examine how these hormones affect human macrophage cholesterol transport, a key factor in atherogenesis, we obtained monocytes from healthy male and postmenopausal female donors (age 50-70 y). Cells were allowe...

  6. Effects of 17Beta-estradiol on cognitive performance of ovariectomized female rats exposed to 56Fe particles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    On exploratory class missions to other planets astronauts will be exposed to types and doses of radiation (HZE particles) that are not experienced in low earth orbit. While it is likely that the crew will consist of both male and female astronauts, there has been little research on the effects of ...

  7. 17beta-estradiol counteracts neuropathic pain: a behavioural, immunohistochemical, and proteomic investigation on sex-related differences in mice

    PubMed Central

    Vacca, Valentina; Marinelli, Sara; Pieroni, Luisa; Urbani, Andrea; Luvisetto, Siro; Pavone, Flaminia

    2016-01-01

    Sex differences play a role in pain sensitivity, efficacy of analgesic drugs and prevalence of neuropathic pain, even if the underlying mechanisms are far from being understood. We demonstrate that male and female mice react differently to structural and functional changes induced by sciatic nerve ligature, used as model of neuropathic pain. Male mice show a gradual decrease of allodynia and a complete recovery while, in females, allodynia and gliosis are still present four months after neuropathy induction. Administration of 17β-estradiol is able to significantly attenuate this difference, reducing allodynia and inducing a complete recovery also in female mice. Parallel to pain attenuation, 17β-estradiol treated-mice show a functional improvement of the injured limb, a faster regenerative process of the peripheral nerve and a decreased neuropathy-induced gliosis. These results indicate beneficial effects of 17β-estradiol on neuropathic pain and neuronal regeneration and focuses on the importance of considering gonadal hormones also in clinical studies. PMID:26742647

  8. SPE-LC/ESI/MS: a simple and reproducible method for detection and quantification of 17beta-estradiol in aqueous samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Steroid estrogens contained in wastewater discharge from sewage treatment plants and agricultural run-off can alter endocrine function in exposed wildlife at part per trillion (ng/L) levels. Detection and quantification of estrogens in the environment at these levels pose numerous analytical challen...

  9. Modulation of vitellogenin synthesis through estrogen receptor beta-1 in goldfish (Carassius auratus) juveniles exposed to 17-{beta} estradiol and nonylphenol

    SciTech Connect

    Soverchia, L.; Ruggeri, B.; Palermo, F.; Mosconi, G.; Cardinaletti, G.; Scortichini, G.; Gatti, G.; Polzonetti-Magni, A.M. . E-mail: alberta.polzonetti@unicam.it

    2005-12-15

    Many synthetic chemicals, termed xenoestrogens, have been shown to interact as agonists with the estrogen receptor (ER) to elicit biological responses similar to those of natural hormones. To date, the regulation of vitellogenesis in oviparous vertebrates has been widely used for evaluation of estrogenic effects. Therefore, Carassius auratus juveniles were chosen as a fish model for studying the effects of estradiol-17{beta} and different concentrations (10{sup -6} and 10{sup -7} M) of 4-nonylphenol (4-NP) on the expression of liver ER{beta}-1 subtype; plasma vitellogenin and sex steroids (androgens and estradiol-17{beta}) were also evaluated together with the bioaccumulation process, through mass-spectrometry. C. auratus is a species widespread in the aquatic environment and, on the toxicological point of view, can be considered a good 'sentinel' species. Juveniles of goldfish were maintained in tanks with only tap water or water with different concentrations (10{sup -6} and 10{sup -7} M) of 4-nonylphenol (4-NP), or 10{sup -7} M of estradiol-17{beta}. After 3 weeks of treatment, animals were anesthetized within 5 min after capture, and blood was immediately collected into heparinized syringes by cardiac puncture and stored at -70 deg. C; the gonads were fixed, then frozen and stored at -70 deg. C; the whole fish, liver, and muscle tissues were harvested and immediately stored at -70 deg. C for molecular biology experiments and bioaccumulation measurements. The estrogenic effects of 4-NP were evidenced by the presence of plasma vitellogenin in juveniles exposed both to estradiol-17{beta} and the two doses of 4-NP; moreover, exposure to 4-NP also increased aromatization of androgens, as suggested by decreasing androgens and increasing estradiol-17{beta} plasma levels. The changes of these parameters were in agreement with the increasing transcriptional rate of ER{beta}-1 mRNA in the liver, demonstrating that both estradiol-17{beta} and 4-NP modulate the vitellogenin rate through interaction with the ER{beta}-1 subtype. The present study also suggests that 4-NP at the concentration of 10{sup -6} M bioaccumulates in the liver.

  10. Identification of two Isoforms of Vitelline Envelope Protein as Complementary Biomarkers to Vitellogenin in the Plasma of Rainbow Trout Exposed to 17beta-estradiol

    EPA Science Inventory

    In the present study, protein markers of estrogenic exposure in rainbow trout (Oncorhynchus mykiss) were isolated and identified using innovative sample preparation techniques followed by advanced MS and bioinformatics approaches. Juvenile trout were administered 17ß-estradiol t...

  11. A Computational Model of the Hypothalamic-pituitary-gonadal Axis in Male Fathead Minnows Exposed to 17 | *alpha* | -ethinylestradiol and 17 | *beta* | -estradiol

    EPA Science Inventory

    Estrogenic chemicals in the aquatic environment have been shown to cause a variety of reproductive anomalies in fish including full sex reversal, intersex, and altered population sex ratios. Two estrogens found in the aquatic environment, 17-ethinylestradiol and 17â-estradiol, h...

  12. Activation of growth factor secretion in tumorigenic states of breast cancer induced by 17. beta. -estradiol or v-Ha-ras oncogene

    SciTech Connect

    Dickson, R.B.; Kasid, A.; Huff, K.K.; Bates, S.E.; Knabbe, C.; Bronzert, D.; Gelmann, E.P.; Lippman, M.E.

    1987-02-01

    The MCF-7 human breast cancer cell line responds to estrogen stimulation in vitro by increased secretion of growth factors and proliferation and in vivo by tumor formation in the nude mouse. To test a possible role of growth factor secretion in expression of the tumorigenic phenotype, the authors stably transfected MCF-7 cells with the v-Ha-ras oncogene to produce the MCF-7ras cell line. The MCF-7ras cell line was tumorigenic in the absence of estrogens and secreted 3- to 5-fold elevated levels of a high molecular weight form of a type ..cap alpha.. transforming growth factor-like growth factor, type ..beta.. transforming growth factor, and insulin-like growth factor I. MCF-7ras cells, in contrast to MCF-7, were less sensitive to further growth stimulation by estrogen, type ..cap alpha.. transforming growth factor, and insulin-like growth factor I and showed little change in receptor levels for these hormones. Conditioned medium from MCF-7ras cells as well as two of its component growth factors replaced estrogen in stimulating MCF-7 colony formation in vitro. A coordinate increase in growth factor secretion by human breast cancer may contribute to its escape from estrogen dependence.

  13. Mammary gland morphology and gene expression differ in female rats treated with 17 beta-estradiol or fed soy protein isolate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soy foods have been suggested to have both positive health benefits and potentially adverse effects as a result of their content of phytoestrogens. However, studies on the estrogenicity of soy foods are lacking. Here we directly compared the effects of soy protein isolate (SPI), the protein in soy i...

  14. Identification of two Isoforms of Vitelline Envelope Protein as Complementary Biomarkers to Vitellogenin in the Plasma of Rainbow Trout Exposed to 17beta-estradiol

    EPA Science Inventory

    In the present study, protein markers of estrogenic exposure in rainbow trout (Oncorhynchus mykiss) were isolated and identified using innovative sample preparation techniques followed by advanced MS and bioinformatics approaches. Juvenile trout were administered 17-estradiol t...

  15. Effect of ovariectomy and 17 beta-estradiol implantation on bone metabolism in female rats fed 1,25 dihydroxyvitamin D/sub 3/

    SciTech Connect

    Osborne, M.; Sherman, S.; Soares, J.H. Jr.

    1986-03-01

    Eight-week old Sprague-Dawley female rats were used in two experiments to test the effects of 17-B estradiol (E/sub 2/) via silastic tubing implants on /sup 3/H-labelled tetracycline (TC) incorporation into bone. 1,25(OH)/sub 2/D/sub 3/ was the dietary source of vitamin D in a low calcium (.2%) semipurified eggwhite diet. In experiment I, the Ovx + E/sub 2/ animals showed a significantly (p < .01) higher /sup 3/H TC uptake in scapula during a 2-week labelling experiment. An increase in /sup 3/H TC content resulted (p < .01) when E/sub 2/ was implanted in 1,25(OH)/sub 2/D/sub 3/ fed Ovx rats. However, the calcium content was not significantly different. The effect of dietary 1,25(OH)/sub 2/D/sub 3/ and E/sub 2/ implantation appears to be additive. Optimal action of the Vitamin D endocrine system may be dependent on presence of E/sub 2/.

  16. Uterine physiological responses and global gene expression in ovariectomized (ovx) rats treated with soy protein isolate (spi) or 17Beta-estradiol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Concerns regarding increased endometrial cancer risk have been raised in women who consume soy products as the result of the estrogenicity of phytochemical components such as the isoflavones genistein and daidzein. Female Sprague-Dawley rats (N = 20/group) were fed AIN-93G diets with casein or SPI a...

  17. A Computational Model of the Hypothalamic-pituitary-gonadal Axis in Male Fathead Minnows Exposed to 17 | *alpha* | -ethinylestradiol and 17 | *beta* | -estradiol

    EPA Science Inventory

    Estrogenic chemicals in the aquatic environment have been shown to cause a variety of reproductive anomalies in fish including full sex reversal, intersex, and altered population sex ratios. Two estrogens found in the aquatic environment, 17-ethinylestradiol and 17-estradiol, h...

  18. DNA ARRAYS TO MONITOR GENE EXPRESSION IN RAT BLOOD AND UTERUS FOLLOWING 17-BETA-ESTRADIOL EXPOSURE: BIOMONITORING ENVIRONMENTAL EFFECTS USING SURROGATE TISSUES

    EPA Science Inventory

    DNA arrays to monitor gene expression in rat blood and uterus following 17-b-estradiol exposure - biomonitoring environmental effects using surrogate tissues
    John C. Rockett, Robert J. Kavlock, Christy R. Lambright, Louise G. Parks, Judith E. Schmid, Vickie S. Wilson, Carmen W...

  19. Pathogenic infection confounds induction of the estrogenic biomarker vitellogenin in rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To examine the behavior of the estrogenic biomarker vitellogenin (VTG) under the combined impact of estrogens and pathogens, parasite-infected or noninfected rainbow trout were exposed to two doses of 17 beta-estradiol (E2). Infected and E2-exposed fish showed significantly lower hepatic VTG mRNA le...

  20. Differential effects of short term feeding of a soy protein isolate diet and estrogen treatment on bone in the pre-pubertal rat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beneficial effects of a soy diet on bone quality have been assumed to be due to the putative estrogenic actions of isoflavones. We studied the effects of soy protein isolate (SPI) on bone quality and compared these effects to 17 beta-estradiol (E2) in pre-pubertal rats. Female rats were weaned to a ...

  1. Sorption, fate, and transport of endogenous steroid hormones in soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The natural hormones 17 beta-estradiol (E2) and testosterone (T) are present in animal manures that are applied to agricultural land as fertilizer and, potentially, may act as endocrine disruptors. Laboratory incubation, batch, and column experiments have been conducted on a series of soils and wer...

  2. The Papillomavirus E2 proteins

    SciTech Connect

    McBride, Alison A.

    2013-10-15

    The papillomavirus E2 proteins are pivotal to the viral life cycle and have well characterized functions in transcriptional regulation, initiation of DNA replication and partitioning the viral genome. The E2 proteins also function in vegetative DNA replication, post-transcriptional processes and possibly packaging. This review describes structural and functional aspects of the E2 proteins and their binding sites on the viral genome. It is intended to be a reference guide to this viral protein. - Highlights: • Overview of E2 protein functions. • Structural domains of the papillomavirus E2 proteins. • Analysis of E2 binding sites in different genera of papillomaviruses. • Compilation of E2 associated proteins. • Comparison of key mutations in distinct E2 functions.

  3. Estrogen and inflammation modulate estrogen receptor alpha expression in specific tissues of the temporomandibular joint

    PubMed Central

    2009-01-01

    Background Estrogen is known to play role in temporomandibular joint (TMJ) disorders and estrogen effects can be mediated by estrogen receptor (ER) alpha present in the TMJ. Cells expressing the estrogen receptor ERalpha are present in the temporomandibular joint (TMJ) but changes in expression due to estrogen and inflammation have not been characterized. In this study, ERalpha protein content and the number of cells expressing ERalpha was measured in 17 beta-estradiol-treated rats after inflammation was induced in the TMJ. Methods Sixteen ovariectomized female rats were divided into two groups such that one group received 17 beta estradiol (E2) and the other was given vehicle (VEH). Groups were then subdivided further, one received injections of saline and the other received Complete Freund's adjuvant (CFA) within the superior joint space of the TMJ. Thus the four groups include no E2/saline, E2/saline, no E2/CFA and E2/CFA. After treatment, the rats were sacrificed, and the TMJ anterior, disc, retrodiscal and synovial tissues were analyzed by western blot and immunocytochemistry. Positive stained cells were counted using a Nikon epifluorescent microscope. Results The western blot showed that ERalpha protein significantly decreased with inflammation. The number of ERalpha-positive cells in the TMJ was not affected by inflammation or 17 beta-estradiol with exception of the retrodiscal tissue. In the retrodiscal tissue 17 beta-estradiol significantly decreased the number of ERalpha-positive cells but only in a non-inflamed joint. Conclusions In conclusion, inflammation and 17 beta-estradiol can modulate ERalpha expression in the TMJ but the effects are tissue specific. PMID:20043825

  4. DIFFERENTIAL EFFECTS OF 17-BETA-ESTRADIOL AND 1,25-DIHYDROXYVITAMIN D ON CAT1 AND CALBINDIN D GENE EXPRESSION AND TRANSEPITHELIAL CALCIUM TRANSPORT IN CACO2 CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Estrogen deficiency has been associated with low intestinal calcium (Ca) absorption and increased bone loss. Studies in experimental animals and humans have shown that estrogen treatment has a positive effect on calcium absorption. However, it is unclear whether estrogen has a direct effect on calci...

  5. Properties of the estrogen receptors contained in the MtTF4 tumor whose growth is inhibited by estradiol: 17 beta-estradiol, 17 alpha-estradiol and DNA bindings.

    PubMed

    Albaladejo, V; Pharaboz, M O; Morel, Y; Andre, J

    1986-07-01

    The steroid and the DNA bindings of the estrogen receptor of the MtTF4 tumor whose growth is inhibited by estradiol where characterized and compared to those of uterine estrogen receptors. In the tumor cytosol: E protects its binding sites against thermal denaturation, depending on the effects of sodium molybdate upon the dissociation rate of [3H]E at 20 degrees C and the ability of receptor to bind to DNA, the activation (or transformation) process, supposed to be necessary for the full action of estrogen ligand, occurs on estrogen receptor complexes and the calf thymus DNA interacts with estrogen receptor with an affinity similar to that of uterine estrogen receptor. Kinetic and equilibrium studies with 17 alpha-[3H]E both in uterus and tumor indicate that this ligand is fast-associating, fast-dissociating and that its affinity for ER is 2- to 4-fold lower than that of 17 beta-[3H]estradiol one. Competition experiments between 17 beta-[3H]estradiol and the unlabelled 17 alpha epimer reveal, in both uterus and tumor, a time-dependent decrease of the apparent potency of 17 alpha-E to inhibit the binding of [3H]E. It is concluded that the estrogen receptors are very similar in MtTF4 tumor and uterus and the diversity of the response of cell growth to E is due rather to differences at the post-receptor level. PMID:3747513

  6. 26 CFR 1.367(e)-2 - Distributions described in section 367(e)(2).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 4 2012-04-01 2012-04-01 false Distributions described in section 367(e)(2). 1.367(e)-2 Section 1.367(e)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (Continued) Effects on Corporation § 1.367(e)-2 Distributions described in section 367(e)(2)....

  7. 26 CFR 1.367(e)-2 - Distributions described in section 367(e)(2).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 4 2014-04-01 2014-04-01 false Distributions described in section 367(e)(2). 1.367(e)-2 Section 1.367(e)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Effects on Corporation § 1.367(e)-2...

  8. 26 CFR 1.367(e)-2 - Distributions described in section 367(e)(2).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 4 2013-04-01 2013-04-01 false Distributions described in section 367(e)(2). 1.367(e)-2 Section 1.367(e)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Effects on Corporation § 1.367(e)-2...

  9. 26 CFR 1.367(e)-2 - Distributions described in section 367(e)(2).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 4 2011-04-01 2011-04-01 false Distributions described in section 367(e)(2). 1.367(e)-2 Section 1.367(e)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Effects on Corporation § 1.367(e)-2 Distributions described in section 367(e)(2). (a) Purpose and...

  10. 26 CFR 1.367(e)-2 - Distributions described in section 367(e)(2).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 4 2010-04-01 2010-04-01 false Distributions described in section 367(e)(2). 1.367(e)-2 Section 1.367(e)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Effects on Corporation § 1.367(e)-2 Distributions described in section 367(e)(2). (a) Purpose and...

  11. Colicin E2 is DNA endonuclease.

    PubMed Central

    Schaller, K; Nomura, M

    1976-01-01

    Colicin E2 purified by conventional methods contains a tightly bound low-molecular-weight protein, as has been found with purified colicin E3 [Jakes,N.&Zinder,N.D.(1974) Proc. Natl. Acad. Sci. USA 71, 3380-3384]. Such E2 preparations do not cause DNA cleavage in vitro. After separation from the low-molecular-weight protein, colicin E2 retained the original in vivo killing activity, and in addition showed a high activity in vitro in cleaving various DNA molecules, such as a ColE1 hybrid plasmid and DNAs from Escherichia coli, lambda phage, chiX174 phage, and simian virus 40. The low-molecular-weight protein ("E2-immunity protein") specifically prevented this in vitro DNA cleavage reaction, i.e., had an "immunity function." The results demonstrate that colicin E2 itself is a DNA endonuclease and explain the in vivo effects caused by E2 in sensitive cells as well as the mechanism of immunity in E2-colicinogenic cells. Images PMID:1069283

  12. The Astro-E2 Mission

    NASA Technical Reports Server (NTRS)

    Kelley, Richard L.

    2004-01-01

    The Astro-E2 observatory is a rebuild of the original Astro-E observatory that was lost during launch in February 2000. It is scheduled for launch into low earth orbit on a Japanese M-V rocket in early 2005. The Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency, is developing the observatory with major contributions from the US. The three instruments on the observatory are the high-resolution x-ray spectrometer (the XRS) featuring a 30-pixel x-ray microcalorimeter array, a set of four CCD cameras (the XIS) and a combination photo-diode/scintillator detector system (the HXD) that will extend the band pass up to nearly 700 keV. A significant feature of Astro-E2 is that all of the instruments are coaligned and operated simultaneously. With its high spectral resolution and collecting area for spectroscopy above 1 keV, Astro-E2 should enable major discovery space and pioneer new technology for use in space. Prime areas for investigation are supernova remnants, active galaxies and the measurement of black hole properties via relativistically-broadened Fe-K emission galaxies. A number of enhancements have been made for the Astro-E2/XRS, including a higher resolution microcalorimeter array, ii mechanical cooler for longer cryogen life, and an improved in-flight calibration system. The Astro-E2/XIS has also been improved to include two back-side-illuminated CCDs to enhance the low energy response. Improvements have also been made to the x-ray mirrors used for both the XRS and XIS to sharpen the point spread function and reduce the effects of stray light. In this talk we will present the essential features of Astro-E2, paying particular attention to the enhancements, and describe the major scientific strengths of the observatory.

  13. Vortices in (e,2e) momentum distributions

    NASA Astrophysics Data System (ADS)

    Macek, J. H.; Ovchinnikov, S. Y.; Sternberg, J. B.

    2010-03-01

    Complete experiments measure all variables associated with atomic processes. Momentum distributions of ejected electrons in pure states, as for (e,2e) measurements, are examples of such complete experiments. All structures seen in such distributions are listed by Briggs and co-workersootnotetextJ. Berakdar and J. S. Briggs, J. Phys. B, 27, 4271 (1994). in 1994. Recently, we pointed out that there is a type of structure not included in the list. It has been shown that momentum distributions image time-dependent wave functions, and such wave functions may contain vortices owing to angular momentum transfer between species involved in the dynamical processes. The vortices are associated with exact zeros at single, isolated points. We have found such zeros in calculated momentum distributions for ion-atom collisions, photoionization, and (e,2e) distributions. By mapping distributions that image time-dependent wave functions we find velocity fields that circulate about exact zeros confirming their vortex structure. The vortices appear as unexpected holes in the (e,2e) momentum distributions. Our calculations suggest that one particular vortex has been observed.ootnotetextA. J. Murray and F. H. Read, Phys. Rev. A, 47, 3724 (1993).

  14. E2F-5, a new E2F family member that interacts with p130 in vivo.

    PubMed Central

    Hijmans, E M; Voorhoeve, P M; Beijersbergen, R L; van 't Veer, L J; Bernards, R

    1995-01-01

    E2F DNA binding sites are found in a number of genes whose expression is tightly regulated during the cell cycle. The activity of E2F transcription factors is regulated by association with specific repressor molecules that can bind and inhibit the E2F transactivation domain. For E2F-1, E2F-2, and E2F-3, the repressor is the product of the retinoblastoma gene, pRb. E2f-4 interacts with pRb-related p107 and not with pRb itself. Recently, a cDNA encoding a third member of the retinoblastoma gene family, p130, was isolated. p130 also interacts with E2F DNA binding activity, primarily in the G0 phase of the cell cycle. We report here the cloning of a fifth member of the E2F gene family. The human E2F-5 cDNA encodes a 346-amino-acid protein with a predicted molecular mass of 38 kDa. E2F-5 is more closely related to E2F-4 (78% similarity) than to E2F-1 (57% similarity). E2F-5 resembles the other E2Fs in that it binds to a consensus E2F site in a cooperative fashion with DP-1. By using a specific E2F-5 antiserum, we found that under physiological conditions, E2F-5 interacts preferentially with p130. PMID:7760804

  15. Anik-E2 recovery mission

    NASA Astrophysics Data System (ADS)

    Wang, D.; Martens, N.

    1993-09-01

    Anik-E2 was launched from Kourou on an Ariane 44P on 4 April 1991. Upon a successful completion of a series of apogee burns, the satellite reached the predicted drift orbit. Subsequently, two anomalies on deployment occurred. The first anomaly was the delay in deploying the Ku-band reflector. The full deployment did not occur until 2 days after the tie-down pyro firing. The second anomaly was noticed in connection with the deployment of the C-band reflector which did not deploy. An intensive investigation was immediately organized by Telesat and supported by Spar Aerospace and GE Astro to identify the possible cause(s) of the C-band antenna deployment failure. The investigations were focused on possible causes of failure which included the wiring of the pyro harness, freezing of the hinge damper, structural failure of the struts supporting the reflectors, alignment of the reflector tie-downs and interference of the thermal blankets surrounding the tie-down mechanisms. Due to some outstanding logistic planning effort and execution of a number of Anik-E2 flight manoeuvres and Anik-E1 ground testing, it was soon concluded that the most likely cause for the C-band reflector deployment failure was an interference resulting from the loosening of the thermal blankets surrounding the tie-down mechanisms. Based on the results of ground testing on Anik-E1, the required breakaway force for the snag was determined. This force requirement created a reflector deployment environment which was much more severe than the nominal design environment. In order to safely deploy the C-band reflector, another series of non-stop activities were performed by Telesat and its contractors to define a more dynamic satellite maneuver, e.g. high degree of nutation, coupled with a cold soak for the hinge dampers in order to provide a high breakaway force and efficient energy absorption during deployment to reduce the force at impact. A series of complex maneuvers with gradually increasing nutation was performed and the C-band reflector was eventually safely deployed on 3 July 1991 which was 3 months after the launch. Due to the constraints of the omni antenna coverage and the battery capacity, Telesat had conducted a large number of maneuvers which were never envisaged and rarely if ever carried out before. This paper will detail all the fault investigation events and mission maneuvers which led to the final recovery of the Anik-E2 satellite, and briefly describe the modification implemented on Anik-E1 which was subsequently launched and on which all deployments were executed flawlessly.

  16. Molecular mechanism of inhibition of estrogen-induced cathepsin D gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells

    SciTech Connect

    Krishnan, V.; Porter, W.; Santostefano, M.; Wang, Xiahong

    1995-12-01

    This report describes how 17{beta}-estradiol (E2) induces cathepsin D gene expression, but is inhibited by the aryl hydrocarbon receptor by disruption of the estrogen receptor/pBC12/S1/pac plasmid complex by interaction with an overlapping xenobiotic responsive element. It was also determined that 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) alone does not affect cathepsin D gene expression but can together with E2 to affect the rate of transcription and levels of immunoreactive protein. 85 refs., 6 figs., 2 tabs.

  17. 26 CFR 1.503(e)-2 - Requirements.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 7 2014-04-01 2013-04-01 true Requirements. 1.503(e)-2 Section 1.503(e)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Exempt Organizations § 1.503(e)-2 Requirements. (a) In general. The requirements which must be met under section 503(e)...

  18. E2f4 and E2f5 are essential for the development of the male reproductive system.

    PubMed

    Danielian, Paul S; Hess, Rex A; Lees, Jacqueline A

    2016-01-17

    The E2F transcription factors are primarily implicated in the regulation of entry and exit from the cell cycle. However, in vivo studies have established additional roles for E2Fs during organ development and homeostasis. With the goal of addressing the intestinal requirements of E2f4 and E2f5, we crossed mice carrying Vil-cre, E2f4 conditional and E2f5 germline alleles. E2f4 deletion had no detectable effect on intestinal development. However, E2f4f/f;E2f5+/-;Vil-cre males, but not E2f4f/f;Vil-cre littermates, were unexpectedly sterile. This defect was not due to defective spermatogenesis. Instead, the seminiferous tubules and rete testes showed significant dilation, and spermatozoa accumulated aberrantly in the rete testis and efferent ducts. Our data show that these problems result from defective efferent ducts, a tissue whose primary function is to concentrate sperm through fluid absorption. First, Vil-cre expression, and consequent E2F4 loss, was specific to the efferent ducts and not other reproductive tract tissues. Second, the E2f4f/f;E2f5+/-;Vil-cre efferent ducts had completely lost multiciliated cells and greatly reduced levels of critical absorptive cell proteins: aquaporin1, a water channel protein, and clusterin, an endocytic marker. Collectively, the observed testis phenotypes suggest a fluid flux defect. Remarkably, we observed rete testis dilation prior to the normal time of seminiferous fluid production, arguing that the efferent duct defects promote excessive secretory activity within the reproductive tract. Finally, we also detect key aspects of these testis defects in E2f5-/- mice. Thus, we conclude that E2f4 and E2f5 display overlapping roles in controlling the normal development of the male reproductive system. PMID:26825228

  19. E2 enzymes: more than just middle men

    PubMed Central

    Stewart, Mikaela D; Ritterhoff, Tobias; Klevit, Rachel E; Brzovic, Peter S

    2016-01-01

    Ubiquitin-conjugating enzymes (E2s) are the central players in the trio of enzymes responsible for the attachment of ubiquitin (Ub) to cellular proteins. Humans have ∼40 E2s that are involved in the transfer of Ub or Ub-like (Ubl) proteins (e.g., SUMO and NEDD8). Although the majority of E2s are only twice the size of Ub, this remarkable family of enzymes performs a variety of functional roles. In this review, we summarize common functional and structural features that define unifying themes among E2s and highlight emerging concepts in the mechanism and regulation of E2s. PMID:27002219

  20. Tax1BP1 interacts with papillomavirus E2 and regulates E2-dependent transcription and stability.

    PubMed

    Wang, Xiaoyu; Naidu, Samisubbu R; Sverdrup, Francis; Androphy, Elliot J

    2009-03-01

    The papillomavirus E2 proteins regulate viral replication, gene transcription, and genome maintenance by interacting with other viral and host proteins. From a yeast two-hybrid screen, we identified the cellular protein Tax1BP1 as a novel binding partner of human papillomavirus type 18 (HPV18) E2. Tax1BP1 also interacts with the HPV16 and bovine papillomavirus type 1 (BPV1) E2 proteins, with the C-terminal region of Tax1BP1 interacting with the N-terminal transactivation domain of BPV1 E2. Tax1BP1 complexes with p300 and acts synergistically as a coactivator with p300 to enhance E2-dependent transcription. Using chromatin immunoprecipitation assays, we show that Tax1BP1 and E2 localize to the long control region on the BPV1 genome. Tax1BP1 was recently reported to bind ubiquitin and to function as an essential component of an A20 ubiquitin-editing complex. We demonstrate that Tax1BP1 plays a role in the regulation of the steady-state level of E2 by preventing its proteasomal degradation. These studies provide new insights into the regulation of E2 functions. PMID:19109394

  1. Tax1BP1 Interacts with Papillomavirus E2 and Regulates E2-Dependent Transcription and Stability▿

    PubMed Central

    Wang, Xiaoyu; Naidu, Samisubbu R.; Sverdrup, Francis; Androphy, Elliot J.

    2009-01-01

    The papillomavirus E2 proteins regulate viral replication, gene transcription, and genome maintenance by interacting with other viral and host proteins. From a yeast two-hybrid screen, we identified the cellular protein Tax1BP1 as a novel binding partner of human papillomavirus type 18 (HPV18) E2. Tax1BP1 also interacts with the HPV16 and bovine papillomavirus type 1 (BPV1) E2 proteins, with the C-terminal region of Tax1BP1 interacting with the N-terminal transactivation domain of BPV1 E2. Tax1BP1 complexes with p300 and acts synergistically as a coactivator with p300 to enhance E2-dependent transcription. Using chromatin immunoprecipitation assays, we show that Tax1BP1 and E2 localize to the long control region on the BPV1 genome. Tax1BP1 was recently reported to bind ubiquitin and to function as an essential component of an A20 ubiquitin-editing complex. We demonstrate that Tax1BP1 plays a role in the regulation of the steady-state level of E2 by preventing its proteasomal degradation. These studies provide new insights into the regulation of E2 functions. PMID:19109394

  2. Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression

    PubMed Central

    Tarangelo, Amy; Lo, Nathanael; Teng, Rebecca; Kim, Eunsun; Le, Linh; Watson, Deborah; Furth, Emma E.; Raman, Pichai; Ehmer, Ursula; Viatour, Patrick

    2015-01-01

    Changes in gene expression during tumorigenesis are often considered the consequence of de novo mutations occurring in the tumour. An alternative possibility is that the transcriptional response of oncogenic transcription factors evolves during tumorigenesis. Here we show that aberrant E2f activity, following inactivation of the Rb gene family in a mouse model of liver cancer, initially activates a robust gene expression programme associated with the cell cycle. Slowly accumulating E2f1 progressively recruits a Pontin/Reptin complex to open the chromatin conformation at E2f target genes and amplifies the E2f transcriptional response. This mechanism enhances the E2f-mediated transactivation of cell cycle genes and initiates the activation of low binding affinity E2f target genes that regulate non-cell-cycle functions, such as the Warburg effect. These data indicate that both the physiological and the oncogenic activities of E2f result in distinct transcriptional responses, which could be exploited to target E2f oncogenic activity for therapy. PMID:26639898

  3. Gamma-ray spectrometer onboard Chang'E-2

    NASA Astrophysics Data System (ADS)

    Ma, T.; Chang, J.; Zhang, N.; Jian, W.; Cai, M. S.; Gong, Y. Z.; Tang, H. S.; Zhang, R. J.; Wang, N. S.; Yu, M.; Mao, J. P.; Hu, Y. M.; Xu, A. A.; Zhu, M. H.

    2013-10-01

    Chang'E-2 gamma-ray spectrometer (GRS) is included in the payload of Chinese second lunar mission Chang'E-2 that has been launched in October 2010. Specific objectives of the GRS are to map abundance of O, Si, Fe, Ti, U, Th, K, and, perhaps, Mg, Al, and Ca, to depth of about 20 cm. The energy resolution and detection efficiency were improved compared with Chang'E-1 GRS. We will describe the design of GRS, which used LaBr3 for its main detector, and present its performance in this paper. Moreover, the initial result of Chang'E-2 GRS is reported.

  4. Large Scale Genotype Comparison of Human Papillomavirus E2-Host Interaction Networks Provides New Insights for E2 Molecular Functions

    PubMed Central

    Muller, Mandy; Jacob, Yves; Jones, Louis; Weiss, Amlie; Brino, Laurent; Chantier, Thibault; Lotteau, Vincent; Favre, Michel; Demeret, Caroline

    2012-01-01

    Human Papillomaviruses (HPV) cause widespread infections in humans, resulting in latent infections or diseases ranging from benign hyperplasia to cancers. HPV-induced pathologies result from complex interplays between viral proteins and the host proteome. Given the major public health concern due to HPV-associated cancers, most studies have focused on the early proteins expressed by HPV genotypes with high oncogenic potential (designated high-risk HPV or HR-HPV). To advance the global understanding of HPV pathogenesis, we mapped the virus/host interaction networks of the E2 regulatory protein from 12 genotypes representative of the range of HPV pathogenicity. Large-scale identification of E2-interaction partners was performed by yeast two-hybrid screenings of a HaCaT cDNA library. Based on a high-confidence scoring scheme, a subset of these partners was then validated for pair-wise interaction in mammalian cells with the whole range of the 12 E2 proteins, allowing a comparative interaction analysis. Hierarchical clustering of E2-host interaction profiles mostly recapitulated HPV phylogeny and provides clues to the involvement of E2 in HPV infection. A set of cellular proteins could thus be identified discriminating, among the mucosal HPV, E2 proteins of HR-HPV 16 or 18 from the non-oncogenic genital HPV. The study of the interaction networks revealed a preferential hijacking of highly connected cellular proteins and the targeting of several functional families. These include transcription regulation, regulation of apoptosis, RNA processing, ubiquitination and intracellular trafficking. The present work provides an overview of E2 biological functions across multiple HPV genotypes. PMID:22761572

  5. Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

    SciTech Connect

    Kong, Leopold; Giang, Erick; Nieusma, Travis; Kadam, Rameshwar U.; Cogburn, Kristin E.; Hua, Yuanzi; Dai, Xiaoping; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Law, Mansun

    2014-08-26

    Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design.

  6. Evolutionary and biophysical relationships among the papillomavirus E2 proteins

    PubMed Central

    Blakaj, Dukagjin M.; Fernandez-Fuentes, Narcis; Chen, Zigui; Hegde, Rashmi; Fiser, Andras; Burk, Robert D.; Brenowitz, Michael

    2013-01-01

    Infection by a human papillomavirus (HPV) may result in a variety of clinical conditions ranging from benign warts to invasive cancer depending on the viral type. The HPV E2 protein represses transcription of the E6 and E7 genes in integrated papillomavirus genomes and together with the E1 protein is required for viral replication. E2 proteins bind with high affinity to palindromic DNA sequences consisting of two highly conserved four base pair sequences flanking a variable ‘spacer’ of identical length. The E2 proteins directly contact the conserved DNA but not the spacer DNA. However, variation in naturally occurring spacer sequences results in differential protein binding affinity. This discrimination in binding is dependent on their sensitivity to the unique conformational and/or dynamic properties of the spacer DNA in a process termed ‘indirect readout’. This article explores the structure of the E2 proteins and their interaction with DNA and other proteins, the effects of ions on affinity and specificity, and the phylogenetic and biophysical nature of this core viral protein. We have analyzed the sequence conservation and electrostatic features of three-dimensional models of the DNA binding domains of 146 papillomavirus types and variants with the goal of identifying characteristics that associated with risk of virally caused malignancy. The amino acid sequence, three-dimensional structure, and the electrostatic features of E2 protein DNA binding domain showed high conservation among all papillomavirus types. This indicates that the specific interactions between the E2 protein and its binding sites on DNA have been conserved throughout PV evolution. Analysis of the E2 protein’s transactivation domain showed that unlike the DNA binding domain, the transactivation domain does not have extensive surfaces of highly conserved residues. Rather, the regions of high conservation are localized to small surface patches. The invariance of the E2 DNA binding domain structure, electrostatics and sequence suggests that it may be a suitable target for the development of vaccines effective against a broad spectrum of HPV types. PMID:19273107

  7. A Structurally Unique E2-Binding Domain Activates Ubiquitination by the ERAD E2, Ubc7p, Through Multiple Mechanisms

    PubMed Central

    Metzger, Meredith B.; Liang, Yu-He; Das, Ranabir; Mariano, Jennifer; Li, Shengjian; Li, Jess; Kostova, Zlatka; Byrd, R. Andrew; Ji, Xinhua; Weissman, Allan M.

    2013-01-01

    SUMMARY Cue1p is an integral component of yeast endoplasmic reticulum (ER)-associated degradation (ERAD) ubiquitin ligase (E3) complexes. It tethers the ERAD ubiquitin-conjugating enzyme (E2), Ubc7p, to the ER and prevents its degradation, and also activates Ubc7p via unknown mechanisms. We have now determined the crystal structure of the Ubc7p-binding region (U7BR) of Cue1p with Ubc7p. The U7BR is a unique E2-binding domain that includes three α-helices that interact extensively with the ‘backside’ of Ubc7p. Residues essential for E2 binding are also required for activation of Ubc7p and for ERAD. We establish that the U7BR stimulates both RING-independent and dependent ubiquitin transfer from Ubc7p. Moreover, the U7BR enhances ubiquitin-activating enzyme (E1)-mediated charging of Ubc7p with ubiquitin. This is the first example where an essential component of E3 complexes both binds to E2 and enhances E2 loading with ubiquitin. These findings provide new insights into mechanisms of stimulating ubiquitination. PMID:23665230

  8. 26 CFR 1.503(e)-2 - Requirements.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... purchase by the employee trust. (b) For purposes of section 503(e), the price of the obligation prevailing... section 503(e) the offering price for the obligation at the time of the purchase means the price which... 26 Internal Revenue 7 2011-04-01 2009-04-01 true Requirements. 1.503(e)-2 Section...

  9. 26 CFR 1.503(e)-2 - Requirements.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... purchase by the employee trust. (b) For purposes of section 503(e), the price of the obligation prevailing... section 503(e) the offering price for the obligation at the time of the purchase means the price which... 26 Internal Revenue 7 2013-04-01 2013-04-01 false Requirements. 1.503(e)-2 Section...

  10. 26 CFR 1.503(e)-2 - Requirements.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... purchase by the employee trust. (b) For purposes of section 503(e), the price of the obligation prevailing... section 503(e) the offering price for the obligation at the time of the purchase means the price which... 26 Internal Revenue 7 2012-04-01 2012-04-01 false Requirements. 1.503(e)-2 Section...

  11. 26 CFR 1.503(e)-2 - Requirements.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... purchase by the employee trust. (b) For purposes of section 503(e), the price of the obligation prevailing... section 503(e) the offering price for the obligation at the time of the purchase means the price which... 26 Internal Revenue 7 2010-04-01 2010-04-01 true Requirements. 1.503(e)-2 Section...

  12. E-2C Loads Calibration in DFRC Flight Loads Lab

    NASA Technical Reports Server (NTRS)

    Schuster, Lawrence S.

    2008-01-01

    Objectives: a) Safely and efficiently perform structural load tests on NAVAIR E-2C aircraft to calibrate strain gage instrumentation installed by NAVAIR; b) Collect load test data and derive loads equations for use in NAVAIR flight tests; and c) Assist flight test team with use of loads equations measurements at PAX River.

  13. Astro-E2 education and public outreach program

    NASA Astrophysics Data System (ADS)

    Mukai, K.; Boyce, K.; Lochner, J.; Harrus, I.; Jacob, B.; Mitchell, S.

    Astro-E2 is a joint Japanese-US X-ray astronomy mission to be launched in early 2005, concentrating on high resolution X-ray spectroscopy. The Astro-E2 team at NASA's Goddard Space Flight Center is implementing an interconnected set of educational activities for this mission, aimed primarily at high school students. The focus of Astro-E2, X-ray spectroscopy, is a challenge and an opportunity: how do we communicate the power of spectroscopy, rather than that of images? We also feature two key technology components of Astro-E2, X-ray optics and cryogenics, as well as the processes of building a satellite, including the cultural aspects of this international collaboration. We have developed a web-based learning center, are producing an educational video, and will host a student competition. We will present the status of, and the plans for, these activities, as well as the lessons we have learned in developing them.

  14. Chang'e-2 spacecraft observations of asteroid 4179 Toutatis

    NASA Astrophysics Data System (ADS)

    Ji, Jianghui; Jiang, Yun; Zhao, Yuhui; Wang, Su; Yu, Liangliang

    2016-01-01

    On 13 December 2012, Chang'e-2 completed a successful flyby of the near-Earth asteroid 4179 Toutatis at a closest distance of 770 meters from the asteroid's surface. The observations show that Toutatis has an irregular surface and its shape resembles a ginger-root of a smaller lobe (head) and a larger lobe (body). Such bilobate shape is indicative of a contact binary origin for Toutatis. In addition, the high-resolution images better than 3 meters provide a number of new discoveries about this asteroid, such as an 800-meter depression at the end of the large lobe, a sharply perpendicular silhouette near the neck region, boulders, indicating that Toutatis is probably a rubble-pile asteroid. Chang'e-2 observations have significantly revealed new insights into the geological features and the formation and evolution of this asteroid. In final, we brief the future Chinese asteroid mission concept.

  15. Generalized seniority and E 2 transitions in the tin isotopes

    NASA Astrophysics Data System (ADS)

    Morales, Irving O.; Van Isacker, P.; Talmi, I.

    2011-09-01

    Recently, a shallow minimum was discovered in B(E2) values in even Sn isotopes around the middle of the neutron major shell. A peak in that region was expected according to calculations using generalized seniority. In a model calculation we show that the observed shape is consistent with generalized seniority. It seems to be due to the order of filling of j-orbits.

  16. Advanced Stirling Convertor (ASC-E2) Characterization Testing

    NASA Technical Reports Server (NTRS)

    Williams, Zachary D.; Oriti, Salvatore M.

    2012-01-01

    Testing has been conducted on Advanced Stirling Convertors (ASCs)-E2 at NASA Glenn Research Center in support of the Advanced Stirling Radioisotope Generator (ASRG) project. This testing has been conducted to understand sensitivities of convertor parameters due to environmental and operational changes during operation of the ASRG in missions to space. This paper summarizes test results and explains the operation of the ASRG during space missions

  17. Advanced Stirling Convertor (ASC-E2) Characterization Testing

    NASA Technical Reports Server (NTRS)

    Williams, Zachary D.; Oriti, Salvatore M.

    2012-01-01

    Testing has been conducted on Advanced Stirling Convertor (ASC-E2) convertors at NASA Glenn Research Center in support of the Advanced Stirling Radioisotope Generator (ASRG) Project. This testing has been conducted to understand sensitivities of convertor parameters due to environmental and operational changes during operation of the ASRG in missions to space. This paper summarizes test results and explains in terms of operation of the ASRG during space missions.

  18. Formulation of Ames 24E2 IR-black coating

    NASA Technical Reports Server (NTRS)

    Smith, Sheldon M.

    1991-01-01

    The formulation of Ames 24E2 IR-black coating and a rationale for the selection of its components are given. The objective was to make a very rough, very thick, and highly absorbing coating to attenuate the specular reflectance of telescope baffles at far-IR wavelengths. Application and curing instructions are also given. Outgassing measurements are quite low following a 24-hour radiative cure.

  19. Cadmium (e,2e) Experiments in the Autoionizing Region

    NASA Astrophysics Data System (ADS)

    Bauman, R. P.; Martin, N. L. S.; Martin, C. A.; Wilson, M.

    1998-05-01

    We recently completed a comprehensive set(N.L.S. Martin, R.P. Bauman and M. Wilson, submitted to Phys.Rev.A.) of (e,2e) measurements in the Cd 4d^95s^25p autoionizing region, with incident electron energy of 150 eV and scattering angles 2^circ→18^circ. The sum and difference of k_ej=±hat k pairs of (e,2e) energy spectra (N.L.S. Martin, D.B. Thompson, R.P. Bauman and M. Wilson, Phys. Rev. A 50), 3878 (1994). were compared with plane wave Born calculations. The calculated shape of the spectra agreed with experiment if phase corrections, independent of scattering angle, were made to ionization amplitudes, but spectral magnitudes differed by up to a factor of three. We have begun a series of experiments to monitor the phase correction as a function of incident electron energy for fixed momentum transfer, and we are also investigating the differences between (e,2e) spectra for equal angles either side of the momentum transfer axis. In a separate experiment we are measuring the cadmium beam profile in order to to improve beam management.

  20. The ancient function of RB-E2F Pathway: insights from its evolutionary history

    PubMed Central

    2010-01-01

    Background The RB-E2F pathway is conserved in most eukaryotic lineages, including animals and plants. E2F and RB family proteins perform crucial functions in cycle controlling, differentiation, development and apoptosis. However, there are two kinds of E2Fs (repressive E2Fs and active E2Fs) and three RB family members in human. Till now, the detail evolutionary history of these protein families and how RB-E2F pathway evolved in different organisms remain poorly explored. Results We performed a comprehensive evolutionary analysis of E2F, RB and DP (dimerization partners of E2Fs) protein family in representative eukaryotic organisms. Several interesting facts were revealed. First, orthologues of RB, E2F, and DP family are present in several representative unicellular organisms and all multicellular organisms we checked. Second, ancestral E2F, RB genes duplicated before placozoans and bilaterians diverged, thus E2F family was divided into E2F4/5 subgroup (including repressive E2Fs: E2F4 and E2F5) and E2F1/2/3 subgroup (including active E2Fs: E2F1, E2F2 and E2F3), RB family was divided into RB1 subgroup (including RB1) and RBL subgroup (including RBL1 and RBL2). Third, E2F4 and E2F5 share more sequence similarity with the predicted E2F ancestral sequence than E2F1, E2F2 and E2F3; E2F4 and E2F5 also possess lower evolutionary rates and higher purification selection pressures than E2F1, E2F2 and E2F3. Fourth, for RB family, the RBL subgroup proteins possess lower evolutionary rates and higher purification selection pressures compared with RB subgroup proteins in vertebrates, Conclusions Protein evolutionary rates and purification selection pressures are usually linked with protein functions. We speculated that function conducted by E2F4/5 subgroup and RBL subgroup proteins might mainly represent the ancient function of RB-E2F pathway, and the E2F1/2/3 subgroup proteins and RB1 protein might contribute more to functional diversification in RB-E2F pathway. Our results will enhance the current understanding of RB-E2F pathway and will also be useful to further functional studies in human and other model organisms. Reviewers This article was reviewed by Dr. Pierre Pontarotti, Dr. Arcady Mushegian and Dr. Zhenguo Lin (nominated by Dr. Neil Smalheiser). PMID:20849664

  1. An E2 accessory domain increases affinity for the anaphase-promoting complex and ensures E2 competition.

    PubMed

    Girard, Juliet R; Tenthorey, Jeanette L; Morgan, David O

    2015-10-01

    The anaphase-promoting complex/cyclosome (APC/C) is a member of the RING family of E3 ubiquitin ligases, which promote ubiquitin transfer from an E2 ubiquitin-conjugating enzyme to a substrate. In budding yeast, the APC/C collaborates with two E2s, Ubc4 and Ubc1, to promote the initiation and elongation, respectively, of polyubiquitin chains on the substrate. Ubc4 and Ubc1 are thought to compete for the same site on the APC/C, but it is not clear how their affinities are balanced. Here, we demonstrate that a C-terminal ubiquitin-associated (UBA) domain enhances the affinity of Ubc1 for the APC/C. Deletion of the UBA domain reduced apparent APC/C affinity for Ubc1 and decreased polyubiquitin chain length. Surprisingly, the positive effect of the UBA domain was not due to an interaction with the acceptor ubiquitin attached to the APC/C substrate or the donor ubiquitin attached to Ubc1 itself. Instead, our evidence suggests that the UBA domain binds to a site on the APC/C core, thereby increasing Ubc1 affinity and enhancing its ability to compete with Ubc4. The UBA domain is required for normal Ubc1 function and E2 competition in vivo. Thus, the UBA domain of Ubc1 ensures efficient polyubiquitination of substrate by balancing Ubc1 affinity with that of Ubc4. PMID:26306044

  2. Astro-E2 Magnesium Diboride High Current Leads

    NASA Technical Reports Server (NTRS)

    Panek, J. S.; Tuttle, J. G.; Riall, S.; Mustafi, S.; Gray, A.; Edmonds, R.; Marrero, V.

    2003-01-01

    The recent discovery of superconducting properties in MgB_2 and rapid development of small diameter steel-clad wires has opened up the possibility of enhancing the design of the baseline Astro-E2 high current lead assembly. Replacing YBCO filaments with MgB_2 wires and modifying the heat sink location can give much higher margins against quench from temperature oscillations of the 4 K heat sink, although wih some overall thermal penalty. The design and performance of a new lead assembly during flight qualification is discussed, with emphasis on thermal, structural, and electrical test results.

  3. E2F-1 binding affinity for pRb is not the only determinant of the E2F-1 activity

    PubMed Central

    Sahin, Fikret; Sladek, Todd L.

    2010-01-01

    E2F-1 is the major cellular target of pRB and is regulated by pRB during cell proliferation. Interaction between pRB and E2F-1 is dependent on the phosphorylation status of pRB. Despite the fact that E2F-1 and pRB have antagonistic activities when they are overexpressed, the role of the E2F-1-pRB interaction in cell growth largely remains unknown. Ideally, it would be better to study the properties of a pRB mutant that fails to bind to E2F, but retains all other activities. To date, no pRB mutation has been characterized in sufficient detail to show that it specifically eliminates E2F binding but leaves other interactions intact. An alternative approach to this issue is to ask whether mutations that change E2F proteins binding affinity to pRB are sufficient to change cell growth in aspect of cell cycle and tumor formation. Therefore, we used the E2F-1 mutants including E2F-1/S332-7A, E2F-1/S375A, E2F-1/S403A, E2F-1/Y411A and E2F-1/L132Q that have different binding affinities for pRB to better understand the roles of the E2F-1 phosphorylation and E2F-1-pRB interaction in the cell cycle, as well as in transformation and gene expression. Data presented in this study suggests that in vivo phosphorylation at amino acids 332-337, 375 and 403 is important for the E2F-1 and pRB interaction in vivo. However, although E2F-1 mutants 332-7, 375 and 403 showed similar binding affinity to pRB, they showed different characteristics in transformation efficiency, G0 accumulation, and target gene experiments. PMID:20616879

  4. Cadmium (e,2e) Energy Spectra in the Autoionizing Region

    NASA Astrophysics Data System (ADS)

    Martin, N. L. S.; Bauman, R. P.; Wilson, M.

    1997-04-01

    We will present an analysis of our comprehensive set of (e,2e) measurements in the Cd 4d^95s^25p autoionizing region, carried out for an incident electron-beam energy of 150 eV and scattering angles between 2^circ and 18^circ, corresponding to momentum transfer K=0.2 → 1 au. The results are presented as the sum and difference of k_ej=±hat k pairs of (e,2e) ejected-electron energy spectra(N.L.S. Martin, D.B. Thompson, R.P. Bauman and M. Wilson, Phys.Rev.Lett 72), 2163 (1994); Phys. Rev. A 50, 3878 (1994). for three special directions hat k, and compared with plane wave Born calculations that include ejected-electron partial waves l=0→7. It is found that the relative Born phases are incorrect for l=0,1,2 by amounts that are independent of scattering angle. The relative Born magnitudes are extremely good for hat k=hat K, but are extremely bad for the other two hat k directions. With increasing scattering angle we observe a reduction in the ^3P_1/^1P1 intensity ratio in the sum spectra, probably due to an increase in exchange scattering, and we also see a previously unobserved Cd 4d^95s^25p J=3 autoionizing level.

  5. (e,2e) Studies of Xenon Autoionizing Levels

    NASA Astrophysics Data System (ADS)

    Childers, J. G.; Martin, N. L. S.

    2001-05-01

    We have begun a series of (e,2e) experiments on Xe in the autoionizing region between the ^2P_3/2 and ^2P_1/2 ionic limits. These are analogous to the series of experiments that were carried out on autoionizing levels in Cd.(N.L.S. Martin, R.P. Bauman, M. Wilson, Phys. Rev. A. 59), 2764 (1999). Xe is of particular interest because the scattering kinematics are similar to Cd: the energy loss of these autoionizing levels and hence the momentum transferred is the same in both atoms. However, the ejected electron energies are very different -- much less than 1 eV in Xe compared with approximately 4 eV in Cd. Our preliminary experiments suggest that at 150 eV incident energy the binary peak in Xe can be much smaller than the recoil peak. This is in sharp contrast to Cd where the binary peak was always greater than the recoil peak. We will present data analyzed in terms of the sum and difference of (e,2e) energy spectra measured at ejected-electron angles 180^circ apart.

  6. An (e,2e) Spectrometer for Helium Autoionization Studies

    NASA Astrophysics Data System (ADS)

    Childers, J. G.; Martin, N. L. S.

    1998-05-01

    We are commissioning a new (e,2e) spectrometer which will be used to investigate the helium 2l2l' autoionizing region. The experimental technique to be used is similar to that of recent Cd experiments(N.L.S. Martin, D.B. Thompson, R.P. Bauman and M. Wilson, Phys. Rev. A 50), 3878 (1994). that measured pairs of (e,2e) energy spectra at ejected-electron directions 180^circ apart. At high incident electron energy the sum of these spectra is dominated by the dipole cross-section, while the difference isolates multipole interference cross-terms. A novel feature of the spectrometer is that in addition to an electron gun and scattered-electron detector, it has a pair of identical ejected-electron detectors located 180^circ apart on the same turntable; thus both spectra are taken simultaneously. The resistive anode of each of the two detectors is fed to a single position sensitive detection module; two non-overlapping images are formed which enables the separation of the spectra. Preliminary data will be presented.

  7. Triply differential (e,2e) studies of phenol

    NASA Astrophysics Data System (ADS)

    da Silva, G. B.; Neves, R. F. C.; Chiari, L.; Jones, D. B.; Ali, E.; Madison, D. H.; Ning, C. G.; Nixon, K. L.; Lopes, M. C. A.; Brunger, M. J.

    2014-09-01

    We have measured (e,2e) triple differential cross sections (TDCS) for the electron-impact ionisation of phenol with coplanar asymmetrical kinematics for an incident electron energy of 250 eV. Experimental measurements of the angular distribution of the slow outgoing electrons at 20 eV are obtained when the incident electron scatters through angles of -5°, -10°, and -15°, respectively. The TDCS data are compared with calculations performed within the molecular 3-body distorted wave model. In this case, a mixed level of agreement, that was dependent on the kinematical condition being probed, was observed between the theoretical and experimental results in the binary peak region. The experimental intensity of the recoil features under all kinematical conditions was relatively small, but was still largely underestimated by the theoretical calculations.

  8. Three body effects in low energy (e,2e) processes

    SciTech Connect

    Rasch, J.; Whelan, Colm T.

    1999-06-10

    Within the last two years a number of highly refined measurements have been performed on H targets which have yielded accurate absolute data for a range of energies and geometries and it would appear that the experimental situation for this, the simplest of atomic targets is now resolved. The theoretical situation is however far from satisfactory and in this paper we will analysis some of the main approaches and characterize their strengths and their weaknesses. We have developed a numerical method which allows us to evaluate triple differential cross sections (TDCS) using the most complex position dependent analytic ansatz wave function and we will present results, using this for low energy (e,2e) processes. We will see that this approach fails when incident channel effects, such as target polarization are likely to be strong.

  9. Dynamical (e,2e) studies of tetrahydrofurfuryl alcohol.

    PubMed

    Bellm, S M; Builth-Williams, J D; Jones, D B; Chaluvadi, Hari; Madison, D H; Ning, C G; Wang, F; Ma, X G; Lohmann, B; Brunger, M J

    2012-06-28

    Cross section data for electron scattering from DNA are important for modelling radiation damage in biological systems. Triply differential cross sections for the electron impact ionization of the highest occupied outer valence orbital of tetrahydrofurfuryl alcohol, which can be considered as an analogue to the deoxyribose backbone molecule in DNA, have been measured using the (e,2e) technique. The measurements have been performed with coplanar asymmetric kinematics at an incident electron energy of 250 eV, an ejected electron energy of 20 eV, and at scattered electron angles of -5°, -10°, and -15°. Experimental results are compared with corresponding theoretical calculations performed using the molecular 3-body distorted wave model. Some important differences are observed between the experiment and calculations. PMID:22755568

  10. The Astro-E2/XRS-2 helium insert system

    NASA Astrophysics Data System (ADS)

    Shirron, P. J.; DiPirro, M. J.; Panek, J.; Kelley, R.; Mitsuda, K.; Fujimoto, R.; Hirabayashi, M.; McCammon, D.

    2006-04-01

    The X-ray Spectrometer (XRS-2) instrument on the Japanese Space Agency (JAXA) Astro-E2 spacecraft will measure faint X-ray emissions in the energy range of 0.2-10 keV. A square array of 32 X-ray microcalorimeters used will be able to distinguish individual photons to better than 10 eV at 6 keV, with a quantum efficiency near 100%. The detectors are cooled to 60 mK by means of an adiabatic demagnetization refrigerator (ADR). The ADR rejects heat to a 1.3 K superfluid helium tank, which is surrounded by a 17 K solid neon tank. A Stirling cycle cryocooler precools an outer shield around the neon tank. This system will provide an estimated 3 years of on-orbit lifetime. This paper describes the helium insert, the ADR, the high temperature superconducting leads, and early on-orbit performance.

  11. Inflammatory mediator prostaglandin E2 in colorectal cancer

    PubMed Central

    Wang, Dingzhi; DuBois, Raymond N.

    2016-01-01

    It is widely accepted that dietary fats and chronic inflammation are risk factors for developing colorectal cancer. Arachidonic acid is a major component of animal fats and the bioactive lipids produced from this substrate play critical roles in a variety of biologic processes, including cancer. Cyclooxygenase-derived prostaglandin E2 (PGE2) is a known pro-inflammatory lipid mediator and promotes tumor progression. Metabolism of arachidonic acid by the cyclooxygenase pathway provides one mechanism for the contribution of dietary fats and chronic inflammation to carcinogenesis. In this review, we highlight recent advances in our understanding of how pro-inflammatory mediator PGE2 promotes colorectal cancer immune evasion. These findings may provide a rationale for the development of new therapeutic approaches to subvert tumor-induced immunosuppression. PMID:24270349

  12. Ionization excitation of helium by the (e,2e) reaction

    SciTech Connect

    Ren, X.G.; Ning, C.G.; Deng, J.K.; Su, G.L.; Zhang, S.F.; Huang, Y.R.; Li, G.Q.

    2005-10-15

    The (e,2e) cross sections for transitions to the n=1, 2, and 3 final state of He{sup +} have been measured at impact energies of 1000 and 1600 eV by using a newly developed energy and momentum dispersive spectrometer. Binding energy spectra in the range of 8 to 86 eV and momentum profiles for the transitions to the ground and excited ion states of He{sup +} are reported at different impact energies, and the experimental results are compared with plane wave impulse approximation calculations. The impact energy dependence of cross section ratios for the He{sup +} n=1, 2, and 3 final states are obtained. Some discrepancies are observed between experimental data and theoretical calculations.

  13. Triply differential (e,2e) studies of phenol

    SciTech Connect

    Silva, G. B. da; Neves, R. F. C.; Chiari, L.; Jones, D. B.; Ali, E.; Madison, D. H.; Ning, C. G.; Nixon, K. L.; Lopes, M. C. A.; Brunger, M. J.

    2014-09-28

    We have measured (e,2e) triple differential cross sections (TDCS) for the electron-impact ionisation of phenol with coplanar asymmetrical kinematics for an incident electron energy of 250 eV. Experimental measurements of the angular distribution of the slow outgoing electrons at 20 eV are obtained when the incident electron scatters through angles of −5°, −10°, and −15°, respectively. The TDCS data are compared with calculations performed within the molecular 3-body distorted wave model. In this case, a mixed level of agreement, that was dependent on the kinematical condition being probed, was observed between the theoretical and experimental results in the binary peak region. The experimental intensity of the recoil features under all kinematical conditions was relatively small, but was still largely underestimated by the theoretical calculations.

  14. E-2D Advanced Hawkeye: primary flight display

    NASA Astrophysics Data System (ADS)

    Paolillo, Paul W.; Saxena, Ragini; Garruba, Jonathan; Tripathi, Sanjay; Blanchard, Randy

    2006-05-01

    This paper is a response to the challenge of providing a large area avionics display for the E-2D AHE aircraft. The resulting display design provides a pilot with high-resolution visual information content covering an image area of almost three square feet (Active Area of Samsung display = 33.792cm x 27.0336 cm = 13.304" x 10.643" = 141.596 square inches = 0.983 sq. ft x 3 = 2.95 sq. ft). The avionics display application, design and performance being described is the Primary Flight Display for the E-2D Advanced Hawkeye aircraft. This cockpit display has a screen diagonal size of 17 inches. Three displays, with minimum bezel width, just fit within the available instrument panel area. The significant design constraints of supporting an upgrade installation have been addressed. These constraints include a display image size that is larger than the mounting opening in the instrument panel. This, therefore, requires that the Electromagnetic Interference (EMI) window, LCD panel and backlight all fit within the limited available bezel depth. High brightness and a wide dimming range are supported with a dual mode Cold Cathode Fluorescent Tube (CCFT) and LED backlight. Packaging constraints dictated the use of multiple U shaped fluorescent lamps in a direct view backlight design for a maximum display brightness of 300 foot-Lamberts. The low intensity backlight levels are provided by remote LEDs coupled through a fiber optic mesh. This architecture generates luminous uniformity within a minimum backlight depth. Cross-cockpit viewing is supported with ultra-wide field-of-view performance including contrast and the color stability of an advanced LCD cell design supports. Display system design tradeoffs directed a priority to high optical efficiency for minimum power and weight.

  15. The human papillomavirus type 16 L1 protein directly interacts with E2 and enhances E2-dependent replication and transcription activation.

    PubMed

    Siddiqa, Abida; Léon, Karen Campos; James, Claire D; Bhatti, Muhammad Faraz; Roberts, Sally; Parish, Joanna L

    2015-08-01

    The human papillomavirus (HPV) E2 protein is a multifunctional protein essential for the control of virus gene expression, genome replication and persistence. E2 is expressed throughout the differentiation-dependent virus life cycle and is functionally regulated by association with multiple viral and cellular proteins. Here, we show for the first time to our knowledge that HPV16 E2 directly associates with the major capsid protein L1, independently of other viral or cellular proteins. We have mapped the L1 binding region within E2 and show that the α-2 helices within the E2 DNA-binding domain mediate L1 interaction. Using cell-based assays, we show that co-expression of L1 and E2 results in enhanced transcription and virus origin-dependent DNA replication. Upon co-expression in keratinocytes, L1 reduces nucleolar association of E2 protein, and when co-expressed with E1 and E2, L1 is partially recruited to viral replication factories. Furthermore, co-distribution of E2 and L1 was detected in the nuclei of upper suprabasal cells in stratified epithelia of HPV16 genome-containing primary human keratinocytes. Taken together, our findings suggest that the interaction between E2 and L1 is important for the regulation of E2 function during the late events of the HPV life cycle. PMID:25911730

  16. The human papillomavirus type 16 L1 protein directly interacts with E2 and enhances E2-dependent replication and transcription activation

    PubMed Central

    Siddiqa, Abida; Léon, Karen Campos; James, Claire D.; Bhatti, Muhammad Faraz; Roberts, Sally

    2015-01-01

    The human papillomavirus (HPV) E2 protein is a multifunctional protein essential for the control of virus gene expression, genome replication and persistence. E2 is expressed throughout the differentiation-dependent virus life cycle and is functionally regulated by association with multiple viral and cellular proteins. Here, we show for the first time to our knowledge that HPV16 E2 directly associates with the major capsid protein L1, independently of other viral or cellular proteins. We have mapped the L1 binding region within E2 and show that the α-2 helices within the E2 DNA-binding domain mediate L1 interaction. Using cell-based assays, we show that co-expression of L1 and E2 results in enhanced transcription and virus origin-dependent DNA replication. Upon co-expression in keratinocytes, L1 reduces nucleolar association of E2 protein, and when co-expressed with E1 and E2, L1 is partially recruited to viral replication factories. Furthermore, co-distribution of E2 and L1 was detected in the nuclei of upper suprabasal cells in stratified epithelia of HPV16 genome-containing primary human keratinocytes. Taken together, our findings suggest that the interaction between E2 and L1 is important for the regulation of E2 function during the late events of the HPV life cycle. PMID:25911730

  17. Overexpression of E2F-1 in rat embryo fibroblasts leads to neoplastic transformation.

    PubMed Central

    Singh, P; Wong, S H; Hong, W

    1994-01-01

    The transcription factor E2F has been implicated in controlling the activation of multiple genes associated with cell proliferation. E2F-1, which is a component of E2F, can promote oncogenesis when transfected into REF cells. The transformation caused by E2F-1 correlates with constitutive overexpression of the transgene, increased transcription of E2F-dependent genes and the enhancement of two E2F DNA binding complexes containing the retinoblastoma susceptibility gene product (Rb) and E2F-1. The oncogenic potential of E2F-1 is dependent on functional DNA binding and transactivation domains but does not require the ability to interact directly with Rb. These findings provide the first direct evidence that sustained unregulated expression of E2F-1 can lead to the loss of cell proliferation control and that E2F-1 is a key component in cell cycle control. Images PMID:8045262

  18. Cesium vacancy ordering in phase-separated C sxF e2 -yS e2

    NASA Astrophysics Data System (ADS)

    Taddei, K. M.; Sturza, M.; Chung, D. Y.; Cao, H. B.; Claus, H.; Kanatzidis, M. G.; Osborn, R.; Rosenkranz, S.; Chmaissem, O.

    2015-09-01

    By simultaneously displaying magnetism and superconductivity in a single phase, the iron-based superconductors provide a model system for the study of magnetism's role in superconductivity. The class of intercalated iron selenide superconductors is unique among these in having the additional property of phase separation and coexistence of two distinct phases—one majority phase with iron vacancy ordering and strong antiferromagnetism, and the other a poorly understood minority microscopic phase with a contested structure. Adding to the intrigue, the majority phase has never been found to show superconductivity on its own while the minority phase has never been successfully synthesized separate from the majority phase. In order to better understand this minority phase, a series of high-quality C sxF e2 -yS e2 single crystals with (0.8 ≤x ≤1 ; 0 ≤y ≤0.3 ) were grown and studied. Neutron and x-ray powder diffraction performed on ground crystals show that the average I 4 /m m m structure of the minority phase is distinctly different from the high-temperature I 4 /m m m parent structure. Moreover, single-crystal diffraction reveals the presence of discrete superlattice reflections that remove the degeneracy of the Cs sites in both the majority and minority phases and reduce their structural symmetries from body centered to primitive. Group theoretical analysis in conjunction with structural modeling shows that the observed superlattice reflections originate from three-dimensional Cs vacancy ordering. This model predicts a 25 % vacancy of the Cs site in the minority phase which is consistent with the site's refined occupancy. Magnetization measurements performed in tandem with neutron single-crystal diffraction provide evidence that the minority phase is the host of superconductivity. Our results also reveal a superconducting dome in which the superconducting transition temperature varies as a function of the nominal valence of iron.

  19. NRIP, a Novel Calmodulin Binding Protein, Activates Calcineurin To Dephosphorylate Human Papillomavirus E2 Protein▿

    PubMed Central

    Chang, Szu-Wei; Tsao, Yeou-Ping; Lin, Chia-Yi; Chen, Show-Li

    2011-01-01

    Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interfering RNAs (siRNAs) decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression. PMID:21543494

  20. Developing a Biosensor for Estrogens in Water Samples: Study ofthe Real-time Response of Live Cells of the Estrogen-sensitive YeastStrain RMY/ER-ERE using Fluorescence Microscopy

    SciTech Connect

    Wozei, E.; Hermanowicz, S.W.; Holman, H-Y.N.

    2005-07-13

    Using a fluorescein di-{beta}-D-galactopyranoside (FDG) substrate we show that in live cells of an estrogen-sensitive yeast strain RMY/ER-ERE with human estrogen receptor (ER{alpha}) gene and the lacZ gene which encodes {beta}-galactosidase, the uptake of 17 {beta}-estradiol (E2) and the subsequent production of {beta}-galactosidase enzyme occur quite rapidly, with maximal enzyme-catalyzed product formation evident after about 30 minutes of exposure to E2. This finding which agrees with the well-known rates of enzyme-catalyzed reactions could have implications for shortening the duration of environmental sample screening and monitoring regimes using yeast-based estrogen assays, and the development of biosensors for environmental estrogens to complement quantification methods.

  1. Hormone replacement therapy and changes on pituitary function.

    PubMed

    Castelo-Branco, C; Fusté, P; Martínez de Osaba, M J; González-Merlo, J

    1992-01-01

    The aim of this study is to know how the pituitary function is affected by several delivery systems of estrogen replacement therapy. 116 menopausal women (54 natural and 62 surgical) were placed into three groups that received replacement therapy and in one control group. We determined prolactine (PRL), gonadotropins and 17 beta-estradiol (E2). We found a decrease in gonadotropin levels in treated patients with a natural menopause, and an increase in gonadotropin levels in the groups treated with conjugated estrogens in surgical menopausal women. 17 beta-E2 was found to be increased in all treated groups, mainly in the continuous therapy group (P less than 0.05). PRL was found to be decreased in patients treated with conjugated estrogens (oophorectomized and not oophorectomized) (P less than 0.05). Although these differences can not be attributed to the presence of the ovaries, we think that they may be the result of the treatment. PMID:1737610

  2. Electron emission from photo-excited testosterone in water-ethanol solution.

    PubMed

    Getoff, Nikola; Schittl, Heike; Hartmann, Johannes; Quint, Ruth Maria

    2009-03-01

    Testosterone (TES; 4-androstene-17beta-ol-3-on) is found for the first time to eject electrons from its singlet excited state in water-ethanol solvent mixture. This ability was very recently also observed for 17beta-estradiol (17betaE2) and progesterone (PRG)/1/. With increasing TES-concentration, the yield of solvated electrons (e(s)(-)) is decreasing, because of "associate" formation. At higher absorbed UV-doses (lambda=254 nm) the e(s)(-) yield is passing a sharp maximum by formation of TES-ethanol adducts, which are able likewise to emit electrons when excited. At prolonged irradiation the resulting photolytic products of TES-ethanol adducts are also able to emit electrons. The capability of the hormones: 17betaE2, PRG and TES to eject electrons and the resulting metabolites, some of which can induce cancer, is discussed. PMID:19124256

  3. Developing a Biosensor for Estrogens in Water Samples: Study ofthe Real-time Response of Live Cells of the Estrogen-sensitive YeastStrain RMY/ER-ERE using Fluorescence Microscopy

    SciTech Connect

    Wozei, E.; Hermanowicz, S.W.; Holman, H-Y.N.

    2006-01-01

    Using a fluorescein di-{beta}-d-galactopyranoside (FDG) substrate we show that in live cells of an estrogen-sensitive yeast strain RMY/ER-ERE with human estrogen receptor (ER{alpha}) gene and the lacZ gene which encodes {beta}-galactosidase, the uptake of 17{beta}-estradiol (E2) and the subsequent production of {beta}-galactosidase enzyme occur quite rapidly, with maximal enzyme-catalyzed product formation evident after about 30 min of exposure to E2. This finding which agrees with the well-known rates of enzyme-catalyzed reactions could have implications for shortening the duration of environmental sample screening and monitoring regimes using yeast-based estrogen assays, and the development of biosensors for environmental estrogens to complement quantification methods.

  4. Giant biquadratic interaction-induced magnetic anisotropy in the iron-based superconductor AxF e2 -yS e2

    NASA Astrophysics Data System (ADS)

    Zhu, Hai-Feng; Cao, Hai-Yuan; Xie, Yun; Hou, Yu-Sheng; Chen, Shiyou; Xiang, Hongjun; Gong, Xin-Gao

    2016-01-01

    The emergence of the electron-pocket-only iron-based superconductor AxF e2 -yS e2 (A =alkali metal ) challenges the Fermi-surface nesting picture established in iron pnictides. It was widely believed that magnetism is correlated with the superconductivity in AxF e2 -yS e2 . Unfortunately, the highly anisotropic exchange parameters and the disagreement between theoretical calculations and experimental results triggered a fierce debate on the nature of magnetism in AxF e2 -yS e2 . Here we find that the strong magnetic anisotropy is from the anisotropic biquadratic interaction. In order to accurately obtain the magnetic interaction parameters, we propose a universal method, which does not need to include other high-energy configurations as in the conventional energy-mapping method. We show that our model successfully captures the magnetic interactions in AxF e2 -yS e2 and correctly predicts the spin-wave spectrum, in quantitative agreement with the experimental observation. These results suggest that the local moment picture, including the biquadratic term, can describe accurately the magnetic properties and spin excitations in AxF e2 -yS e2 , which sheds new light on the future study of the high-Tc iron-based superconductors.

  5. High incidence of T-cell tumors in E2A-null mice and E2A/Id1 double-knockout mice.

    PubMed Central

    Yan, W; Young, A Z; Soares, V C; Kelley, R; Benezra, R; Zhuang, Y

    1997-01-01

    The basic-helix-loop-helix (bHLH) proteins encoded by the E2A gene are broadly expressed transcription regulators which function through binding to the E-box enhancer sequences. The DNA binding activities of E2A proteins are directly inhibited upon dimerization with the Id1 gene product. It has been shown that disruption of the E2A gene leads to a complete block in B-lymphocyte development and a high frequency of neonatal death. We report here that nearly half of the surviving E2A-null mice develop acute T-cell lymphoma between 3 to 10 months of age. We further show that disruption of the Id1 gene improves the chance of postnatal survival of E2A-null mice, indicating that Id1 is a canonical negative regulator of E2A and that the unbalanced ratio of E2A to Id1 may contribute to the postnatal death of the E2A-null mice. However, the E2A/Id1 double-knockout mice still develop T-cell tumors once they reach the age of 3 months. This result suggests that E2A may be essential for maintaining the homeostasis of T lymphocytes during their constant renewal in adult life. PMID:9372963

  6. Structure of the Rb C-Terminal Domain Bound to E2F1-DP1: A Mechanism for Phosphorylation-Induced E2F Release

    SciTech Connect

    Rubin,S.; Gall, A.; Zheng, N.; Pavletich, N.

    2005-01-01

    The retinoblastoma (Rb) protein negatively regulates the G1-S transition by binding to the E2F transcription factors, until cyclin-dependent kinases phosphorylate Rb, causing E2F release. The Rb pocket domain is necessary for E2F binding, but the Rb C-terminal domain (RbC) is also required for growth suppression. Here we demonstrate a high-affinity interaction between RbC and E2F-DP heterodimers shared by all Rb and E2F family members. The crystal structure of an RbC-E2F1-DP1 complex reveals an intertwined heterodimer in which the marked box domains of both E2F1 and DP1 contact RbC. We also demonstrate that phosphorylation of RbC at serines 788 and 795 destabilizes one set of RbC-E2F-DP interactions directly, while phosphorylation at threonines 821 and 826 induces an intramolecular interaction between RbC and the Rb pocket that destabilizes the remaining interactions indirectly. Our findings explain the requirement of RbC for high-affinity E2F binding and growth suppression and establish a mechanism for the regulation of Rb-E2F association by phosphorylation.

  7. Differential Stem and Progenitor Cell Trafficking by Prostaglandin E2

    PubMed Central

    Hoggatt, Jonathan; Mohammad, Khalid S.; Singh, Pratibha; Hoggatt, Amber F.; Chitteti, Brahmananda Reddy; Speth, Jennifer M.; Hu, Peirong; Poteat, Bradley A.; Stilger, Kayla N.; Ferraro, Francesca; Silberstein, Lev; Wong, Frankie K.; Farag, Sherif S.; Czader, Magdalena; Milne, Ginger L.; Breyer, Richard M.; Serezani, Carlos H.; Scadden, David T.; Guise, Theresa; Srour, Edward F.; Pelus, Louis M.

    2013-01-01

    SUMMARY To maintain lifelong production of blood cells, hematopoietic stem cells (HSC) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSC (LT-HSC) reside in several, perhaps overlapping, niches that produce regulatory molecules/signals necessary for homeostasis and increased output following stress/injury 1–5. Despite significant advances in specific cellular or molecular mechanisms governing HSC/niche interactions, little is understood about regulatory function within the intact mammalian hematopoietic niche. Recently, we and others described a positive regulatory role for Prostaglandin E2 (PGE2) on HSC function ex vivo 6,7. While exploring the role of endogenous PGE2 we unexpectedly observed hematopoietic egress after nonsteroidal anti-inflammatory drug (NSAID) treatment. Surprisingly, this was independent of the SDF-1/CXCR4 axis. Stem and progenitor cells were found to have differing mechanisms of egress, with HSC transit to the periphery dependent on niche attenuation and reduction in the retentive molecule osteopontin (OPN). Hematopoietic grafts mobilized with NSAIDs had superior repopulating ability and long-term engraftment. Treatment of non-human primates and healthy human volunteers confirmed NSAID-mediated egress in higher species. PGE2 receptor knockout mice demonstrated that progenitor expansion and stem/progenitor egress resulted from reduced EP4 receptor signaling. These results not only uncover unique regulatory roles for EP4 signaling in HSC retention in the niche but also define a rapidly translatable strategy to therapeutically enhance transplantation. PMID:23485965

  8. Influence of prostaglandin E2 on parturition in cattle.

    PubMed

    Hirsbrunner, G; Zanolari, P; Althaus, H; Hüsler, J; Steiner, A

    2007-09-22

    A double-blinded, randomised, placebo-controlled field study of the influence of prostaglandin E2 (PGE2) on cattle at parturition was carried out. The extent of cervical opening and the intensity of labour were scored before administration of the compound and 10 minutes later; routine birth assistance was then continued by the veterinarian. Successful birth occurred more quickly in the cows treated with PGE2. The extent of cervical opening before the administration of the drug had a significant effect on the time to delivery, but the intensity of labour and a concomitant infusion of calcium did not have significant effects on this period. The less open the cervix before administration of the drug, the more the duration of parturition differed between the two groups, with the placebo group taking longer. A telephone follow-up inquiry found no significant differences between the cows postpartum; there were cases of mastitis and hypocalcaemia in both groups. The incidence of retained fetal membranes and the mortality of the calves were higher in the placebo group, but in neither case was the difference significant. PMID:17890770

  9. STS-70 Launch - Nikon E-2 Digital Image

    NASA Technical Reports Server (NTRS)

    1995-01-01

    This test image was taken with a Nikon E-2 Digital Imaging System camera and are provided courtesy of Nikon (GIF 450x450 JPEG 1280x1000): The second Shuttle launch in 16 days hurtles off the pad into a sweltering summer sky. The unstable weather typical to Florida in the summertime didn't have a chance to coalesce and impact this morning's launch window, and the Space Shuttle Discovery began its planned seven-day, 22-hour flight on Mission STS-70 from Launch Pad 39B at 9:41:55.078 a.m. EDT, just seconds off schedule. On board for Discovery's 21st spaceflight are a crew of five: Commander Terence 'Tom' Henricks; Pilot Kevin R. Kregel; and Mission Specialists Nancy Jane Currie, Donald A. Thomas and Mary Ellen Weber. The crew's primary objective during the 70th Shuttle flight is to deploy the Tracking and Data Relay Satellite (TDRS-G), which will join a constellation of other TDRS spacecraft already on orbit. TDRS-G is destined to become an on- orbit, fully operational 'ready reserve' satellite, available along with one other ready reserve TDRS spacecraft to back up the two primary TDRS satellites positions, TDRS East over the Atlantic Ocean and TDRS West over the Pacific. Assured capability of the TDRS communications network is essential for linking Earth-orbiting spacecraft such as the Shuttle and the Hubble Space Telescope with the ground.

  10. STS-70 Launch - Nikon E-2 Digital Image

    NASA Technical Reports Server (NTRS)

    1995-01-01

    This test images was taken with a Nikon E-2 Digital Imaging System camera and are provided courtesy of Nikon (GIF 450x450 JPEG 1280x1000): The second Shuttle launch in 16 days hurtles off the pad into a sweltering summer sky. The unstable weather typical to Florida in the summertime didn't have a chance to coalesce and impact this morning's launch window, and the Space Shuttle Discovery began its planned seven-day, 22-hour flight on Mission STS-70 from Launch Pad 39B at 9:41:55.078 a.m. EDT, just seconds off schedule. On board for Discovery's 21st spaceflight are a crew of five: Commander Terence 'Tom' Henricks; Pilot Kevin R. Kregel; and Mission Specialists Nancy Jane Currie, Donald A. Thomas and Mary Ellen Weber. The crew's primary objective during the 70th Shuttle flight is to deploy the Tracking and Data Relay Satellite (TDRS-G), which will join a constellation of other TDRS spacecraft already on orbit. TDRS-G is destined to become an on- orbit, fully operational 'ready reserve' satellite, available along with one other ready reserve TDRS spacecraft to back up the two primary TDRS satellites positions, TDRS East over the Atlantic Ocean and TDRS West over the Pacific. Assured capability of the TDRS communications network is essential for linking Earth-orbiting spacecraft such as the Shuttle and the Hubble Space Telescope with the ground.

  11. Prostaglandin E2 Prevents Disuse-Induced Cortical Bone Loss

    NASA Technical Reports Server (NTRS)

    Jee, Webster S. S.; Akamine, T.; Ke, Hua Zhu; Li, Xiao Jian; Tang, L. Y.; Zeng, Q. Q.

    1992-01-01

    The object of this study was to determine whether prostaglandin E2 (PGE2) can prevent disuse (underloaded)-induced cortical bone loss as well as add extra bone to underloaded bones. Thirteen-month-old retired female Sprague-Dawley breeders served as controls or were subjected to simultaneous right hindlimb immobilization by bandaging and daily subcutaneous doses of 0, 1, 3, or 6 mg PGE2/kg/d for two and six weeks. Histomorphometric analyses were performed on double-fluorescent labeled undecalcified tibial shaft sections (proximal to the tibiofibular junction). Disuse-induced cortical bone loss occurred by enlarging the marrow cavity and increasing intracortical porosity. PGE2 treatment of disuse shafts further increased intracortical porosity above that in disuse alone controls. This bone loss was counteracted by enhancement of periosteal and corticoendosteal bone formation. Stimulation of periosteal and corticoendosteal bone formation slightly enlarged the total tissue (cross-sectional) area and inhibited marrow cavity enlargement. These PGE2-induced activities netted the same percentage of cortical bone with a different distribution than the beginning and age related controls. These findings indicate the PGE2-induced increase in bone formation compensated for the disuse and PGE2-induced bone loss, and thus prevented immobilization induced bone loss.

  12. (e,2e) Angular Distributions and Energy Spectra in Cadmium

    NASA Astrophysics Data System (ADS)

    Martin, N. L. S.; Bauman, R. P.; Ross, K. J.; Wilson, M.

    1996-05-01

    Early angular distribution measurements on the Cd 4d^95s^25p ^3P1 autoionizing level( N.L.S. Martin and K.J. Ross, J. Phys. B 17), 4033 (1984). did not correspond with those expected from a single level of mixed ^3P+^1P character. An analysis indicated that the results were consistent with the combined angular distributions of the ^3P1 level and a previously unknown ^1D2 even parity autoionizing level at a slightly displaced ejected-electron energy. Recent (e,2e) energy spectra measurements( N.L.S. Martin, D.B. Thompson, R.P. Bauman, M. Wilson, Phys.Rev.A 50), 3878 (1994). that spanned the 4d^95s^25p energy region were interpreted with the help of ab initio structure and plane wave Born amplitude calculations. It was found that the experimental data could be modeled satisfactorily without including a ^1D2 level close to the ^3P1 level. We will present new calculations which reconcile these apparent contradictions between the angular distributions and energy spectra.

  13. Parametrization of a spin-polarized (e,2e) experiment

    NASA Astrophysics Data System (ADS)

    Mazevet, S.; McCarthy, I. E.; Weigold, E.

    1998-03-01

    We use the density-matrix formalism to parametrize a spin-polarized (e,2e) experiment where a p electron is ejected from a closed-shell system and the fine structure of the ion is resolved experimentally. This formulation allows a definition from general principles of the recently observed spin up-down asymmetry [X. Guo and co-workers, Phys. Rev. Lett. 76, 1228 (1996); J. Phys. B 30, 4097 (1997)] due to the fine-structure effect [S. Jones et al., Phys. Rev. Lett. 72, 2554 (1994); G. F. Hanne, in Abstracts of Contributed Papers, XVIIth International Conference on the Physics of Electronic and Atomic Collisions, edited by W. R. MacGillivray, I. E. McCarthy, and M. C. Standage (Hilger, Bristol, 1992), p. 199] and the complete definition of potentially observable quantities such as the polarization of the fast- and slow-emitted-electron beams and the orientation of the ion beam after the collision. These parameters are defined for the general case where the incoming-electron beam has an arbitrary polarization P=(Px,Py,Pz) and calculated in an asymmetric kinematic for the case where the initial-electron beam is, respectively, unpolarized and polarized perpendicular to the scattering plane.

  14. Feedback regulation between atypical E2Fs and APC/CCdh1 coordinates cell cycle progression.

    PubMed

    Boekhout, Michiel; Yuan, Ruixue; Wondergem, Annelotte P; Segeren, Hendrika A; van Liere, Elsbeth A; Awol, Nesibu; Jansen, Imke; Wolthuis, Rob Mf; de Bruin, Alain; Westendorp, Bart

    2016-03-01

    E2F transcription factors control the oscillating expression pattern of multiple target genes during the cell cycle. Activator E2Fs, E2F1-3, induce an upswing of E2F targets, which is essential for the G1-to-S phase transition, whereas atypical E2Fs, E2F7 and E2F8, mediate a downswing of the same targets during late S, G2, and M phases. Expression of atypical E2Fs is induced by E2F1-3, but it is unknown how atypical E2Fs are inactivated in a timely manner. Here, we demonstrate that E2F7 and E2F8 are substrates of the anaphase-promoting complex/cyclosome (APC/C). Removal of CDH1, or mutating the CDH1-interacting KEN boxes, stabilized E2F7/8 from anaphase onwards and during G1. Expressing KEN mutant E2F7 during G1 impairs S phase entry and eventually results in cell death. Furthermore, we show that E2F8, but not E2F7, interacts also with APC/C(C) (dc20). Importantly, atypical E2Fs can activate APC/C(C) (dh1) by repressing its inhibitors cyclin A, cyclin E, and Emi1. In conclusion, we discovered a feedback loop between atypical E2Fs and APC/C(C) (dh1), which ensures balanced expression of cell cycle genes and normal cell cycle progression. PMID:26882548

  15. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    SciTech Connect

    Wang, Jimin Li, Yue; Modis, Yorgo

    2014-04-15

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed.

  16. DNA-binding and trans-activation properties of Drosophila E2F and DP proteins.

    PubMed Central

    Dynlacht, B D; Brook, A; Dembski, M; Yenush, L; Dyson, N

    1994-01-01

    The temporal activation of E2F transcriptional activity appears to be an important component of the mechanisms that prepare mammalian cells for DNA replication. Regulation of E2F activity appears to be a highly complex process, and the dissection of the E2F pathway will be greatly facilitated by the ability to use genetic approaches. We report the isolation of two Drosophila genes that can stimulate E2F-dependent transcription in Drosophila cells. One of these genes, dE2F, contains three domains that are highly conserved in the human homologs E2F-1, E2F-2, and E2F-3. Interestingly, one of these domains is highly homologous to the retinoblastoma protein (RB)-binding sequences of human E2F genes. The other gene, dDP, is closely related to the human DP-1 and DP-2 genes. We demonstrate that dDP and dE2F interact and cooperate to give sequence-specific DNA binding and optimal trans-activation. These features suggest that endogenous Drosophila E2F, like human E2F, may be composed of heterodimers and may be regulated by RB-like proteins. The isolation of these genes will provide important reagents for the genetic analysis of the E2F pathway. Images PMID:8022787

  17. E2 protein cage as a multifunctional nanoplatform

    NASA Astrophysics Data System (ADS)

    Dalmau Mallorqui, Merce

    Caged protein systems such as viral capsids, heat shock proteins, and ferritin are spherical structures that occur naturally in living organisms and are a growing class of biomimetic templates used to create new materials in nanotechnology. Such systems have been proposed as general drug carriers since they form highly symmetric nanoscale architectures that offer the potential to be tailored according to the desired application. Within this framework, this dissertation focuses on the design and development of a new drug delivery nanoplatform based on the E2 subunit of the pyruvate dehydrogenase protein from Bacillus stearothermophilus. This scaffold forms a 25-nm nanocapsule structure with a hollow cavity. We produced a variant of this protein consisting only of the structural core, and found the thermostability of this self-assembled scaffold to be unusually high, with an onset unfolding temperature of 81.1 +/- 0.9°C and an apparent midpoint unfolding temperature of 91.4 +/- 1.4°C. To evaluate the potential of this scaffold for encapsulation of guest molecules in the internal cavity, we made variants which altered the physicochemical properties of the hollow internal surface. These mutants, yielding up to 240 mutations within this cavity, assembled into correct architectures and exhibited high thermostability that was also comparable to the wild-type scaffold. To show the applicability of this scaffold we coupled two drug-like small molecules to the internal cavity. We also developed a new strategy for encapsulation of small hydrophobic drug molecules. This method is based on hydrophobic differences between the interior cavity and the external buffer to nucleate drug-like agents inside the protein cage. We demonstrate that internal mutations can introduce non-native functionality and enable molecular encapsulation within the cavity while still retaining the dodecahedral structure. Another surface amenable to modifications is the interface between subunits. Such a region was modified to introduce pH-dependent scaffold disassembly ability to assist drug release upon endocytosis inside the cells. Moreover, we demonstrated that modulation of the pH at which disassembly occurs can be achieved by modulation of electrostatic interactions through mutagenesis or changing ionic strength. Together, these results demonstrate the potential of our scaffold as a robust nanoscale platform for biomedical applications.

  18. MPN patients harbor recurrent truncating mutations in transcription factor NF-E2

    PubMed Central

    Jutzi, Jonas S.; Bogeska, Ruzhica; Nikoloski, Gorica; Schmid, Corina A.; Seeger, Thalia S.; Stegelmann, Frank; Schwemmers, Sven; Grnder, Albert; Peeken, Jan C.; Gothwal, Monika; Wehrle, Julius; Aumann, Konrad; Hamdi, Kamar; Dierks, Christine; Wang, Wei; Dhner, Konstanze; Jansen, Joop H.

    2013-01-01

    The molecular etiology of myeloproliferative neoplasms (MPNs) remains incompletely understood, despite recent advances incurred through the discovery of several different mutations in MPN patients. We have recently described overexpression of the transcription factor NF-E2 in MPN patients and shown that elevated NF-E2 levels in vivo cause an MPN phenotype and predispose to leukemic transformation in transgenic mice. We report the presence of acquired insertion and deletion mutations in the NF-E2 gene in MPN patients. These result in truncated NF-E2 proteins that enhance wild-type (WT) NF-E2 function and cause erythrocytosis and thrombocytosis in a murine model. NF-E2 mutant cells acquire a proliferative advantage, witnessed by clonal dominance over WT NF-E2 cells in MPN patients. Our data underscore the role of increased NF-E2 activity in the pathophysiology of MPNs. PMID:23589569

  19. Curcumin blocks prostaglandin E2 biosynthesis through direct inhibition of the microsomal prostaglandin E2 synthase-1.

    PubMed

    Koeberle, Andreas; Northoff, Hinnak; Werz, Oliver

    2009-08-01

    Prostaglandin E(2) (PGE(2)) plays a crucial role in the apparent link between tumor growth and chronic inflammation. Cyclooxygenase (COX)-2 and microsomal PGE(2) synthase-1, which are overexpressed in many cancers, are functionally coupled and thus produce massive PGE(2) in various tumors. Curcumin, a polyphenolic beta-diketone from tumeric with anti-carcinogenic and anti-inflammatory activities, was shown to suppress PGE(2) formation and to block the expression of COX-2 and of microsomal PGE(2) synthase-1. Here, we identified microsomal PGE(2) synthase-1 as a molecular target of curcumin and we show that inhibition of microsomal PGE(2) synthase-1 activity is the predominant mechanism of curcumin to suppress PGE(2) biosynthesis. Curcumin reversibly inhibited the conversion of PGH(2) to PGE(2) by microsomal PGE(2) synthase-1 in microsomes of interleukin-1beta-stimulated A549 lung carcinoma cells with an IC(50) of 0.2 to 0.3 micromol/L. Closely related polyphenols (e.g., resveratrol, coniferyl alcohol, eugenol, rosmarinic acid) failed in this respect, and isolated ovine COX-1 and human recombinant COX-2 were not inhibited by curcumin up to 30 micromol/L. In lipopolysaccharide-stimulated human whole blood, curcumin inhibited COX-2-derived PGE(2) formation from endogenous or from exogenous arachidonic acid, whereas the concomitant formation of COX-2-mediated 6-keto PGF(1)alpha and COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid was suppressed only at significant higher concentrations. Based on the key function of PGE(2) in inflammation and carcinogenesis, inhibition of microsomal PGE(2) synthase-1 by curcumin provides a molecular basis for its anticarcinogenic and anti-inflammatory activities. PMID:19671757

  20. Modulation of the E2F1-driven cancer cell fate by the DNA damage response machinery and potential novel E2F1 targets in osteosarcomas.

    PubMed

    Liontos, Michalis; Niforou, Katerina; Velimezi, Georgia; Vougas, Konstantinos; Evangelou, Konstantinos; Apostolopoulou, Kalliopi; Vrtel, Radek; Damalas, Alexandros; Kontovazenitis, Panayiotis; Kotsinas, Athanassios; Zoumpourlis, Vassilis; Tsangaris, George Th; Kittas, Christos; Ginsberg, Doron; Halazonetis, Thanos D; Bartek, Jiri; Gorgoulis, Vassilis G

    2009-07-01

    Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either "oncogenic" or "oncosuppressor" properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network. Surprisingly, induction of p73, an established E2F1 target, was also DNA damage response-dependent. Furthermore, a global proteome analysis associated with bioinformatics revealed novel E2F1-regulated genes and potential E2F1-driven signaling networks that could provide useful targets in challenging this aggressive neoplasm by innovative therapies. PMID:19541929

  1. Ability of the bed bug (Hemiptera: Cimicidae) defensive secretions (E)-2-hexenal and (E)-2-octenal to attract adult bed bugs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accurate and timely surveillance of bed bug infestations is critical for development of effective control strategies. While the bed bug produced volatiles (E)-2-hexenal and (E)-2-octenal are considered defensive secretions, through use of EthoVision® video-tracking software we demonstrate that low ...

  2. Inhibition of the entomopathogenic fungus Metarhizium anisopliae in vitro by the bed bug defensive secretions (E)-2-hexenal and (E)-2-octenal

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The two major aldehydes (E)-2-hexenal and (E)-2-octenal emitted as defensive secretions by bed bugs Cimex lectularius L. (Hemiptera: Cimicidae), inhibit the in vitro growth of Metarhizium anisopliae (Metsch.) Sokorin (Hypocreales: Clavicipitaceae). These chemicals inhibit fungal growth by direct con...

  3. 26 CFR 48.4216(e)-2 - Limitation on aggregate of exclusions and price readjustments.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... readjustments. 48.4216(e)-2 Section 48.4216(e)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... Applicable to Manufacturers Taxes § 48.4216(e)-2 Limitation on aggregate of exclusions and price... advertising, as provided in section 4216(e)(1) and § 48.4216(e)-1, plus the amount of the readjustments...

  4. 26 CFR 48.4216(e)-2 - Limitation on aggregate of exclusions and price readjustments.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... readjustments. 48.4216(e)-2 Section 48.4216(e)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... Applicable to Manufacturers Taxes § 48.4216(e)-2 Limitation on aggregate of exclusions and price... advertising, as provided in section 4216(e)(1) and § 48.4216(e)-1, plus the amount of the readjustments...

  5. 26 CFR 48.4216(e)-2 - Limitation on aggregate of exclusions and price readjustments.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... readjustments. 48.4216(e)-2 Section 48.4216(e)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... Applicable to Manufacturers Taxes § 48.4216(e)-2 Limitation on aggregate of exclusions and price... advertising, as provided in section 4216(e)(1) and § 48.4216(e)-1, plus the amount of the readjustments...

  6. 26 CFR 48.4216(e)-2 - Limitation on aggregate of exclusions and price readjustments.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... readjustments. 48.4216(e)-2 Section 48.4216(e)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... Applicable to Manufacturers Taxes § 48.4216(e)-2 Limitation on aggregate of exclusions and price... advertising, as provided in section 4216(e)(1) and § 48.4216(e)-1, plus the amount of the readjustments...

  7. 17 CFR 270.30e-2 - Reports to shareholders of unit investment trusts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... unit investment trusts. 270.30e-2 Section 270.30e-2 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT OF 1940 § 270.30e-2 Reports to shareholders of unit investment trusts. (a) At least semiannually every registered unit...

  8. Structural insights into the DNA-binding specificity of E2F family transcription factors

    PubMed Central

    Morgunova, Ekaterina; Yin, Yimeng; Jolma, Arttu; Dave, Kashyap; Schmierer, Bernhard; Popov, Alexander; Eremina, Nadejda; Nilsson, Lennart; Taipale, Jussi

    2015-01-01

    The mammalian cell cycle is controlled by the E2F family of transcription factors. Typical E2Fs bind to DNA as heterodimers with the related dimerization partner (DP) proteins, whereas the atypical E2Fs, E2F7 and E2F8 contain two DNA-binding domains (DBDs) and act as repressors. To understand the mechanism of repression, we have resolved the structure of E2F8 in complex with DNA at atomic resolution. We find that the first and second DBDs of E2F8 resemble the DBDs of typical E2F and DP proteins, respectively. Using molecular dynamics simulations, biochemical affinity measurements and chromatin immunoprecipitation, we further show that both atypical and typical E2Fs bind to similar DNA sequences in vitro and in vivo. Our results represent the first crystal structure of an E2F protein with two DBDs, and reveal the mechanism by which atypical E2Fs can repress canonical E2F target genes and exert their negative influence on cell cycle progression. PMID:26632596

  9. Suppression of newborn natural killer cell activity by prostaglandin E2

    SciTech Connect

    Milch, P.O.; Salvatore, W.; Luft, B.; Baker, D.A.

    1988-10-01

    The effect of prostaglandin E2 on natural killer cell activity of cord blood was examined. Natural killer cell activity, determined by chromium 51 release, was significantly reduced after prostaglandin E2 (1 microgram/ml) treatment. Prostaglandin E2 has been found to enhance the cellular spread of herpesvirus. Thus prostaglandins may enhance viral infections indirectly by suppressing natural killer cell activity.

  10. Identification of novel target genes specifically activated by deregulated E2F in human normal fibroblasts.

    PubMed

    Kitamura, Hodaka; Ozono, Eiko; Iwanaga, Ritsuko; Bradford, Andrew P; Okuno, Junko; Shimizu, Emi; Kurayoshi, Kenta; Kugawa, Kazuyuki; Toh, Hiroyuki; Ohtani, Kiyoshi

    2015-09-01

    The transcription factor E2F is the principal target of the tumor suppressor pRB. E2F plays crucial roles not only in cell proliferation by activating growth-related genes but also in tumor suppression by activating pro-apoptotic and growth-suppressive genes. We previously reported that, in human normal fibroblasts, the tumor suppressor genes ARF, p27(Kip1) and TAp73 are activated by deregulated E2F activity induced by forced inactivation of pRB, but not by physiological E2F activity induced by growth stimulation. In contrast, growth-related E2F targets are activated by both E2F activities, underscoring the roles of deregulated E2F in tumor suppression in the context of dysfunctional pRB. In this study, to further understand the roles of deregulated E2F, we explored new targets that are specifically activated by deregulated E2F using DNA microarray. The analysis identified nine novel targets (BIM, RASSF1, PPP1R13B, JMY, MOAP1, RBM38, ABTB1, RBBP4 and RBBP7), many of which are involved in the p53 and RB tumor suppressor pathways. Among these genes, the BIM gene was shown to be activated via atypical E2F-responsive promoter elements and to contribute to E2F1-mediated apoptosis. Our results underscore crucial roles of deregulated E2F in growth suppression to counteract loss of pRB function. PMID:26201719

  11. 29 CFR 2584.8477(e)-2 - Allocation of fiduciary duties.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 9 2011-07-01 2011-07-01 false Allocation of fiduciary duties. 2584.8477(e)-2 Section 2584.8477(e)-2 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION... AND REGULATIONS FOR THE ALLOCATION OF FIDUCIARY RESPONSIBILITY § 2584.8477(e)-2 Allocation...

  12. Compensation and specificity of function within the E2F family.

    PubMed

    Kong, L-J; Chang, J T; Bild, A H; Nevins, J R

    2007-01-18

    Functions encoded by single genes in lower organisms are often represented by multiple related genes in the mammalian genome. An example is the retinoblastoma and E2F families of proteins that regulate transcription during the cell cycle. Analysis of gene function using germline mutations is often confounded by overlapping function resulting in compensation. Indeed, in cells deleted of the E2F1 or E2F3 genes, there is an increase in the expression of the other family member. To avoid complications of compensatory effects, we have used small-interfering RNAs that target individual E2F proteins to generate a temporary loss of E2F function. We find that both E2F1 and E2F3 are required for cells to enter the S phase from a quiescent state, whereas only E2F3 is necessary for the S phase in growing cells. We also find that the acute loss of E2F3 activity affects the expression of genes encoding DNA replication and mitotic activities, whereas loss of E2F1 affects a limited number of genes that are distinct from those regulated by E2F3. We conclude that the long-term loss of E2F activity does lead to compensation by other family members and that the analysis of acute loss of function reveals specific and distinct roles for these proteins. PMID:16909124

  13. 26 CFR 1.669(e)-2A - Illustration of the provisions of section 669.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 8 2011-04-01 2011-04-01 false Illustration of the provisions of section 669. 1.669(e)-2A Section 1.669(e)-2A Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... Applicable to Taxable Years Beginning Before January 1, 1969 § 1.669(e)-2A Illustration of the provisions...

  14. 26 CFR 1.669(e)-2A - Illustration of the provisions of section 669.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 8 2010-04-01 2010-04-01 false Illustration of the provisions of section 669. 1.669(e)-2A Section 1.669(e)-2A Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... Taxable Years Beginning Before January 1, 1969 § 1.669(e)-2A Illustration of the provisions of section...

  15. E2F Activators Signal and Maintain Centrosome Amplification in Breast Cancer Cells

    PubMed Central

    Lee, Mi-Young; Moreno, Carlos S.

    2014-01-01

    Centrosomes ensure accurate chromosome segregation by directing spindle bipolarity. Loss of centrosome regulation results in centrosome amplification, multipolar mitosis and aneuploidy. Since centrosome amplification is common in premalignant lesions and breast tumors, it is proposed to play a central role in breast tumorigenesis, a hypothesis that remains to be tested. The coordination between the cell and centrosome cycles is of paramount importance to maintain normal centrosome numbers, and the E2Fs may be responsible for regulating these cycles. However, the role of E2F activators in centrosome amplification is unclear. Because E2Fs are deregulated in Her2+ cells displaying centrosome amplification, we addressed whether they signal this abnormal process. Knockdown of E2F1 or E2F3 in Her2+ cells decreased centrosome amplification without significantly affecting cell cycle progression, whereas the overexpression of E2F1, E2F2, or E2F3 increased centrosome amplification in MCF10A mammary epithelial cells. Our results revealed that E2Fs affect the expression of proteins, including Nek2 and Plk4, known to influence the cell/centrosome cycles and mitosis. Downregulation of E2F3 resulted in cell death and delays/blocks in cytokinesis, which was reversed by Nek2 overexpression. Nek2 overexpression enhanced centrosome amplification in Her2+ breast cancer cells silenced for E2F3, revealing a role for the E2F activators in maintaining centrosome amplification in part through Nek2. PMID:24797070

  16. Estrogens in streams associated with a concentrated animal feeding operation in upstate New York, USA.

    PubMed

    Zhao, Sherry; Zhang, Pengfei; Melcer, Michael E; Molina, John F

    2010-04-01

    Estrogens (estrone, 17 alpha-estradiol, 17beta-estradiol, and estriol) in three headwater streams within a concentrated animal feed operation (CAFO) site were monitored on a monthly base for a year (November 2006-October 2007). This CAFO is certified as organic (no growth promoters are administrated) and uses many Whole Farm Planning practices (e.g., 12-month-capacity waste storage lagoons). In general, estrogen concentrations in the streams are low (<1 ng L(-1)), and appeared to increase in spring, likely due to the mobilization of estrogens from soils upon snow melting/precipitation. Estrogens were detected in the streams during dry periods, indicating the contribution of estrogens from groundwater. The low concentrations of estrogens in stream water were probably the result of the long residence time (approximately 8 months) of the manure in the lagoons where most of the estrogens were degraded during storage. An analysis of liquid manure at the beginning of manure application season (after approximately 8 months storage) showed that over 99.8% of the estrogens potentially excreted by the cows were degraded. Moreover, about 90% of the estrogens in the liquid manure were associated with particulates larger than 0.7 microm. Batch experiments with spiked deuterium-labeled 17beta-estradiol-16,16,17-d(3) (d(3)-E2 beta) in the liquid manure demonstrated sorption of d(3)-E2 beta onto particulates in the liquid manure, and rapid degradation of d(3)-E2 beta in the aqueous phase and on particulates of the liquid manure under aerobic conditions. PMID:20172589

  17. Overexpression of aprE2, a fibrinolytic enzyme gene from Bacillus subtilis CH3-5, in Escherichia coli and the properties of AprE2.

    PubMed

    Jeong, Seon-Ju; Cho, Kye Man; Lee, Chang Kwon; Kim, Gyoung Min; Shin, Jung-Hye; Kim, Jong Sang; Kim, Jeong Hwan

    2014-07-01

    The aprE2 gene with its prosequence from Bacillus subtilis CH3-5 was overexpressed in Escherichia coli BL21(DE3) by using plasmid pET26b(+). After IPTG induction, active and mature AprE2 was produced when cells were grown at 20°C, whereas inactive and insoluble enzyme was produced in a large amount when cells were grown at 37°C. The insoluble fraction was resuspended with 6 M guanidine-HCl and dialyzed against 2 M Tris-HCl (pH 7.0) or 0.5 M sodium acetate (pH 7.0) buffer. Then active AprE2 was regenerated and purified by a Ni-NTA column. Purified AprE2 from the soluble fraction had a specific activity of 1,069.4 ± 42.4 U/mg protein, higher than that from the renatured insoluble fraction. However, more active AprE2 was obtained by renaturation of the insoluble fraction. AprE2 was most stable at pH 7 and 40°C, respectively. The fibrinolytic activity of AprE2 was inhibited by PMSF, but not by EDTA and metal ions. AprE2 degraded Aα and Bβ chains of fibrinogen quickly, but not the γ-chain. AprE2 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-pNA. The Km and kcat/Km of AprE2 was 0.56 mM and 3.10 × 10(4) S(-1) M(-1), respectively. PMID:24743573

  18. Subunit affinities and stoichiometries of the human papillomavirus type 11 E1:E2:DNA complex.

    PubMed

    Chao, S F; Rocque, W J; Daniel, S; Czyzyk, L E; Phelps, W C; Alexander, K A

    1999-04-01

    The association between the papillomavirus E1 and E2 proteins is an important regulatory interaction, imparting coordinated control of viral transcription and replication. Using fluorescence polarization, we have characterized the interactions between HPV-11 E1, HPV-11 E2, and DNA in solution at equilibrium. For these studies, two double-stranded fluorescein-labeled oligonucleotides were prepared. The first fluorescent oligonucleotide, designated Fl-E2BS and containing a single E2 binding-site palindrome (ACCGN6CGGT), was used to determine the affinity of E2 for its DNA binding site. HPV-11 E2 bound Fl-E2BS with an apparent Kd of 0.84 nM. Binding was saturable and consistent with a single class of noninteracting sites. The second oligonucleotide, designated Fl-E1E2BS, contained both E1 and E2 sites in sequence derived directly from the HPV-11 origin of replication. Under titration conditions identical to those used for Fl-E2BS, the E2 protein exhibited reduced affinity for Fl-E1E2BS (Kd > 100 nM). E1 binding to Fl-E1E2BS was of very low affinity. Addition of excess HPV-11 E1 to Fl-E1E2BS lowered the dissociation constant for the E2:Fl-E1E2BS interaction to 2 nM. This effect was not dependent upon ATP or magnesium ion. Fluorescence polarization and other data suggest formation of a complex containing six E1 molecules and a single dimer of E2 bound to a single Fl-E1E2BS oligonucleotide; E2 dissociation from the final complex did not occur. In summary, physical interaction between E1 and E2 increases the DNA binding affinity of each. The role of this energy coupling may be to promote origin-specific binding of both E1 and E2 to DNA. PMID:10194380

  19. In vivo delivery of bovine viral diahorrea virus, E2 protein using hollow mesoporous silica nanoparticles.

    PubMed

    Mahony, D; Cavallaro, A S; Mody, K T; Xiong, L; Mahony, T J; Qiao, S Z; Mitter, N

    2014-06-21

    Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral diarrhoea virus (BVDV). BVDV infection occurs in the target species of cattle and sheep herds worldwide and is therefore of economic importance. E2 is a major immunogenic determinant of BVDV and is an ideal candidate for the development of a subunit based nanovaccine using mesoporous silica nanoparticles. Hollow type mesoporous silica nanoparticles with surface amino functionalisation (termed HMSA) were characterised and assessed for adsorption and desorption of E2. A codon-optimised version of the E2 protein (termed Opti-E2) was produced in Escherichia coli. HMSA (120 nm) had an adsorption capacity of 80 μg Opti-E2 per mg HMSA and once bound E2 did not dissociate from the HMSA. Immunisation studies in mice with a 20 μg dose of E2 adsorbed to 250 μg HMSA was compared to immunisation with Opti-E2 (50 μg) together with the traditional adjuvant Quillaja saponaria Molina tree saponins (QuilA, 10 μg). The humoral responses with the Opti-E2/HMSA nanovaccine although slightly lower than those obtained for the Opti-E2 + QuilA group demonstrated that HMSA particles are an effective adjuvant that stimulated E2-specific antibody responses. Importantly the cell-mediated immune responses were consistently high in all mice immunised with Opti-E2/HMSA nanovaccine formulation. Therefore we have shown the Opti-E2/HMSA nanoformulation acts as an excellent adjuvant that gives both T-helper 1 and T-helper 2 mediated responses in a small animal model. This study has provided proof-of-concept towards the development of an E2 subunit nanoparticle based vaccine. PMID:24811899

  20. [The effect of E2 and progesterone on the receptor status in endometrial carcinoma].

    PubMed

    Gorchev, G; Milkov, V; Tanchev, S; Popov, I

    1992-01-01

    The authors study the plasma levels of E2 and progesterone in 62 women with EC. Parallelly the ER and PR in the same number of cases have been studied. The values of E2 in the plasma of ER+ are much lower than those of E2 in ER-. The quantity of E2 and progesterone influence the values of ER and PR in cancers of women in pre and postmenopausal age. In positive ER and PR patients the level of E2 and progesterone is lower in comparison of ER- and PR-. PMID:1342552

  1. E2F7 overexpression leads to tamoxifen resistance in breast cancer cells by competing with E2F1 at miR-15a/16 promoter

    PubMed Central

    Chu, Junjun; Zhu, Yinghua; Liu, Yujie; Sun, Lijuan; Lv, Xiaobin; Wu, Yanqin; Hu, Pengnan; Su, Fengxi; Gong, Chang; Song, Erwei; Liu, Bodu; Liu, Qiang

    2015-01-01

    About 50–70% of breast cancers are estrogen receptor α (ERα) positive and most of them are sensitive to endocrine therapy including tamoxifen. However, one third of these patients will eventually develop resistance and relapse. We found that the expression of miR-15a and miR-16 were significantly decreased in tamoxifen resistant ER positive breast cancer cell lines. Exogenous expression of miR-15a/16 mimics re-sensitized resistant cells to tamoxifen by inhibiting Cyclin E1 and B cell lymphoma-2 (Bcl-2) to induce cell growth arrest and apoptosis respectively. Further, we identified that a repressive member of E2F family, E2F7, was responsible for the suppression of miR-15a/16 cluster by competing with E2F1 for E2F binding site at the promoter of their host gene DLEU2. Moreover, high expression of E2F7 is correlated with high risk of relapse and poor prognosis in breast cancer patients receiving tamoxifen treatment. Together, our results suggest that overexpression of E2F7 represses miR-15a/16 and then increases Cyclin E1 and Bcl-2 that result in tamoxifen resistance. E2F7 may be a valuable prognostic marker and a therapeutic target of tamoxifen resistance in breast cancer. PMID:26397135

  2. A genetic, non-transcriptional assay for nuclear receptor ligand binding in yeast.

    PubMed

    Köhler, Fabian; Zimmermann, André; Hager, Martin; Sippel, Albrecht E

    2004-08-01

    We describe the development of a genetic, non-transcriptional assay for the detection of ligand binding to nuclear receptors based on the ligand-dependent reconstitution of the defective Ras/cAMP viability pathway of the Saccharomyces cerevisiae strain CDC25-2. We have characterized the assay using the estrogen receptor (ER) alpha as an example and found it to be extremely sensitive, stringent, rapid and selective. We applied this assay to different ligands and ligand-binding domains (LBDs) and analyzed co-stimulation with 17beta-estradiol (E2) and the synthetic ligand 4-hydroxytamoxifen (4-OHT) in vivo. This simple and inexpensive assay may be useful for the study of steroid hormone receptor (SHR) actions at the plasma membrane and for the analysis of ligand binding in vivo. Furthermore, it may allow for the selection of novel ligands and ligand-binding domains and has significant potential for application in compound screening. PMID:15276207

  3. The banana E2 gene family: Genomic identification, characterization, expression profiling analysis.

    PubMed

    Dong, Chen; Hu, Huigang; Jue, Dengwei; Zhao, Qiufang; Chen, Hongliang; Xie, Jianghui; Jia, Liqiang

    2016-04-01

    The E2 is at the center of a cascade of Ub1 transfers, and it links activation of the Ub1 by E1 to its eventual E3-catalyzed attachment to substrate. Although the genome-wide analysis of this family has been performed in some species, little is known about analysis of E2 genes in banana. In this study, 74 E2 genes of banana were identified and phylogenetically clustered into thirteen subgroups. The predicted banana E2 genes were distributed across all 11 chromosomes at different densities. Additionally, the E2 domain, gene structure and motif compositions were analyzed. The expression of all of the banana E2 genes was analyzed in the root, stem, leaf, flower organs, five stages of fruit development and under abiotic stresses. All of the banana E2 genes, with the exception of few genes in each group, were expressed in at least one of the organs and fruit developments, which indicated that the E2 genes might involve in various aspects of the physiological and developmental processes of the banana. Quantitative RT-PCR (qRT-PCR) analysis identified that 45 E2s under drought and 33 E2s under salt were induced. To the best of our knowledge, this report describes the first genome-wide analysis of the banana E2 gene family, and the results should provide valuable information for understanding the classification, cloning and putative functions of this family. PMID:26940488

  4. E2F-1 has dual roles depending on the cell cycle

    PubMed Central

    Sahin, Fikret; Sladek, Todd L.

    2010-01-01

    The E2F family of transcription factors play a critical role in the control of cell proliferation. E2F-1 is the major cellular target of pRB and is regulated by pRB during cell proliferation. E2F-1-mediated activation and repression of target genes occurs in different settings. The role of E2F-1 and E2F-1/pRB complexes in regulation of different target genes, and in cycling versus quiescent cells, is unclear. In this study, effects of free E2F-1 (doesn't complex with pRb) and E2F-1/pRb complex, on E2F-1 target gene expression were compared in different cell growth conditions. Findings suggest that E2F-1 acts in different ways, not only depending on the target gene but also depending on different stages of the cell cycle. For example, E2F-1 acts as part of the repression complex with pRB in the expression of DHFR, b-myb, TK and cdc2 in asynchronously growing cells; on the other hand, E2F-1 acts as an activator in the expression of the same genes in cells that are re-entering the cycle. PMID:20224733

  5. POH1 deubiquitylates and stabilizes E2F1 to promote tumour formation

    PubMed Central

    Wang, Boshi; Ma, Aihui; Zhang, Li; Jin, Wei-Lin; Qian, Yu; Xu, Guiqin; Qiu, Bijun; Yang, Zhaojuan; Liu, Yun; Xia, Qiang; Liu, Yongzhong

    2015-01-01

    Hyperactivation of the transcriptional factor E2F1 occurs frequently in human cancers and contributes to malignant progression. E2F1 activity is regulated by proteolysis mediated by the ubiquitin–proteasome system. However, the deubiquitylase that controls E2F1 ubiquitylation and stability remains undefined. Here we demonstrate that the deubiquitylase POH1 stabilizes E2F1 protein through binding to and deubiquitylating E2F1. Conditional knockout of Poh1 alleles results in reduced E2F1 expression in primary mouse liver cells. The POH1-mediated regulation of E2F1 expression strengthens E2F1-downstream prosurvival signals, including upregulation of Survivin and FOXM1 protein levels, and efficiently facilitates tumour growth of liver cancer cells in nude mice. Importantly, human hepatocellular carcinomas (HCCs) recapitulate POH1 regulation of E2F1 expression, as nuclear abundance of POH1 is increased in HCCs and correlates with E2F1 overexpression and tumour growth. Thus, our study suggests that the hyperactivated POH1–E2F1 regulation may contribute to the development of liver cancer. PMID:26510456

  6. Immunogenicity of recombinant BCGs expressing predicted antigenic epitopes of bovine viral diarrhea virus E2 gene.

    PubMed

    Liu, Dongxu; Lu, Huijun; Shi, Kun; Su, Fengyan; Li, Jianming; Du, Rui

    2014-10-01

    To develop a vaccine to prevent diseases caused by Mycobacterium tuberculosis and bovine viral diarrhea virus (BVDV) simultaneously, recombinant Bacillus Calmette-Guerin (rBCG) vaccines expressing different regions of the BVDV E2 gene were constructed. Using DNASTAR 6.0 software, potential antigenic epitopes were predicted, and six regions were chosen to generate recombinant plasmids with the pMV361 vector (pMV361-E2-1, pMV361-E2-2, pMV361-E2-3, pMV361-E2-4, pMV361-E2-5 and pMV361-E2-6, respectively). The recombinant plasmids were transformed into BCG, and protein expression was thermally induced at 45 °C. Mice were immunized with 5 × 10(6) CFU/200 µL of each rBCG strain. Compared with other groups, BVDV E2 specific antibody titers were higher in mice immunized with rBCG-E2-6. Ratios and numbers of CD4+, CD8+ and IL-12 expressing spleen lymphocytes of the rBCG-E2-6 group also were higher than those of other groups. Thus, the rBCG-E2-6 vaccine showed the highest immunogenicity of all groups based on the humoral and cellular responses to vaccination. PMID:25135492

  7. E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis

    PubMed Central

    Müller, Heiko; Bracken, Adrian P.; Vernell, Richard; Moroni, M. Cristina; Christians, Fred; Grassilli, Emanuela; Prosperini, Elena; Vigo, Elena; Oliner, Jonathan D.; Helin, Kristian

    2001-01-01

    The retinoblastoma protein (pRB) and its two relatives, p107 and p130, regulate development and cell proliferation in part by inhibiting the activity of E2F-regulated promoters. We have used high-density oligonucleotide arrays to identify genes in which expression changed in response to activation of E2F1, E2F2, and E2F3. We show that the E2Fs control the expression of several genes that are involved in cell proliferation. We also show that the E2Fs regulate a number of genes involved in apoptosis, differentiation, and development. These results provide possible genetic explanations to the variety of phenotypes observed as a consequence of a deregulated pRB/E2F pathway. PMID:11159908

  8. Pumilio facilitates miRNA regulation of the E2F3 oncogene.

    PubMed

    Miles, Wayne O; Tschöp, Katrin; Herr, Anabel; Ji, Jun-Yuan; Dyson, Nicholas J

    2012-02-15

    E2F transcription factors are important regulators of cell proliferation and are frequently dysregulated in human malignancies. To identify novel regulators of E2F function, we used Drosophila as a model system to screen for mutations that modify phenotypes caused by reduced levels of dE2F1. This screen identified components of the Pumilio translational repressor complex (Pumilio, Nanos, and Brain tumor) as suppressors of dE2F1-RNAi phenotypes. Subsequent experiments provided evidence that Pumilio complexes repress dE2F1 levels and that this mechanism of post-transcriptional regulation is conserved in human cells. The human Pumilio homologs Pum 1 and Pum 2 repress the translation of E2F3 by binding to the E2F3 3' untranslated region (UTR) and also enhance the activity of multiple E2F3 targeting microRNAs (miRNAs). E2F3 is an oncogene with strong proliferative potential and is regularly dysregulated or overexpressed in cancer. Interestingly, Pumilio/miRNA-mediated regulation of E2F3 is circumvented in cancer cells in several different ways. Bladder carcinomas selectively down-regulate miRNAs that cooperate with Pumilio to target E2F3, and multiple tumor cell lines shorten the 3' end of the E2F3 mRNA, removing the Pumilio regulatory elements. These studies suggest that Pumilio-miRNA repression of E2F3 translation provides an important level of E2F regulation that is frequently abrogated in cancer cells. PMID:22345517

  9. Pumilio facilitates miRNA regulation of the E2F3 oncogene

    PubMed Central

    Miles, Wayne O.; Tschöp, Katrin; Herr, Anabel; Ji, Jun-Yuan; Dyson, Nicholas J.

    2012-01-01

    E2F transcription factors are important regulators of cell proliferation and are frequently dysregulated in human malignancies. To identify novel regulators of E2F function, we used Drosophila as a model system to screen for mutations that modify phenotypes caused by reduced levels of dE2F1. This screen identified components of the Pumilio translational repressor complex (Pumilio, Nanos, and Brain tumor) as suppressors of dE2F1-RNAi phenotypes. Subsequent experiments provided evidence that Pumilio complexes repress dE2F1 levels and that this mechanism of post-transcriptional regulation is conserved in human cells. The human Pumilio homologs Pum 1 and Pum 2 repress the translation of E2F3 by binding to the E2F3 3′ untranslated region (UTR) and also enhance the activity of multiple E2F3 targeting microRNAs (miRNAs). E2F3 is an oncogene with strong proliferative potential and is regularly dysregulated or overexpressed in cancer. Interestingly, Pumilio/miRNA-mediated regulation of E2F3 is circumvented in cancer cells in several different ways. Bladder carcinomas selectively down-regulate miRNAs that cooperate with Pumilio to target E2F3, and multiple tumor cell lines shorten the 3′ end of the E2F3 mRNA, removing the Pumilio regulatory elements. These studies suggest that Pumilio–miRNA repression of E2F3 translation provides an important level of E2F regulation that is frequently abrogated in cancer cells. PMID:22345517

  10. Regulation of E2F1-induced apoptosis by poly(ADP-ribosyl)ation

    PubMed Central

    Kumari, A; Iwasaki, T; Pyndiah, S; Cassimere, E K; Palani, C D; Sakamuro, D

    2015-01-01

    The transcription factor adenovirus E2 promoter-binding factor (E2F)-1 normally enhances cell-cycle progression, but it also induces apoptosis under certain conditions, including DNA damage and serum deprivation. Although DNA damage facilitates the phosphorylation and stabilization of E2F1 to trigger apoptosis, how serum starvation renders cells vulnerable to E2F1-induced apoptosis remains unclear. Because poly(ADP-ribose) polymerase 1 (PARP1), a nuclear enzyme essential for genomic stability and chromatin remodeling, interacts directly with E2F1, we investigated the effects of PARP1 on E2F1-mediated functions in the presence and absence of serum. PARP1 attenuation, which increased E2F1 transactivation, induced G2/M cell-cycle arrest under normal growth conditions, but enhanced E2F1-induced apoptosis in serum-starved cells. Interestingly, basal PARP1 activity was sufficient to modify E2F1 by poly(ADP-ribosyl)ation, which stabilized the interaction between E2F1 and the BIN1 tumor suppressor in the nucleus. Accordingly, BIN1 acted as an RB1-independent E2F1 corepressor. Because E2F1 directly activates the BIN1 gene promoter, BIN1 curbed E2F1 activity through a negative-feedback mechanism. Conversely, when the BIN1–E2F1 interaction was abolished by PARP1 suppression, E2F1 continuously increased BIN1 levels. This is functionally germane, as PARP1-depletion-associated G2/M arrest was reversed by the transfection of BIN1 siRNA. Moreover, PARP-inhibitor-associated anti-transformation activity was compromised by the coexpression of dominant-negative BIN1. Because serum starvation massively reduced the E2F1 poly(ADP-ribosyl)ation, we conclude that the release of BIN1 from hypo-poly(ADP-ribosyl)ated E2F1 is a mechanism by which serum starvation promotes E2F1-induced apoptosis. PMID:25257171

  11. [Aromatase inhibitor letrozole induces sex inversion in the protogynous red spotted grouper (Epinephelus akaara).].

    PubMed

    Li, Guang-Li; Liu, Xiao-Chun; Lin, Hao-Ran

    2005-08-25

    The objective of this study was to investigate the effects of the aromatase inhibitor (AI) letrozole on gonadal development, serum steroids and aromatase activities in 2-year-old female red spotted grouper (Epinephelus akaara) during reproductive season. Groupers were divided into two groups, one implanted with aromatase inhibitor (AI, 5 mg/kg body weight) and the other elastomer without AI into peritoneal cavity once every four weeks for 8 weeks. Spermiation was checked through gentle abdominal pressure every 2 weeks. Blood samples were obtained from 6 fish of each group every 4 weeks for later analysis of sex steroids. After blood samples were collected, forebrain, midbrain, hindbrain, and gonads were collected and stored at -70 degrees C for later aromatase activity measurement and gonadal histological study. Significantly lower gondadosomatic index (GSI) was observed in AI-implanted group. Fish implanted with AI once showed complete degradation of oocytes and sex inversion with developing testicular tissues in the 4th week. AI induced females to develop into functional males with authentic males testes similar in structure to those in normal males. Spermiating rate of AI-treated males were 14.3%, 35.3%, and 48.4%in the 4th, 6th, and 8th week, respectively, while all fish in the control group were still female with developing ovaries. Aromatase activities in gonads decreased significantly after implantation with aromatase inhibitor, but showed no significant difference between control and AI-implanted group. No difference in serum testosterone (T) levels was observed in control and AI-treated group, while serum levels of 17beta-estradiol (E(2)) decreased but 11-ketotestosterone (11-KT) concentration increased significantly. The present results suggest that the decrease in serum 17beta-estradiol (E(2)) and increase in 11-KT levels may be important for sex inversion induced by aromatase inhibitor in red spotted grouper. PMID:16094495

  12. Transcriptional regulation of human RANK ligand gene expression by E2F1

    SciTech Connect

    Hu Yan; Sun Meng; Nadiminty, Nagalakshmi; Lou Wei; Pinder, Elaine; Gao, Allen C.

    2008-06-06

    Receptor activator of nuclear factor kappa B ligand (RANKL) is a critical osteoclastogenic factor involved in the regulation of bone resorption, immune function, the development of mammary gland and cardiovascular system. To understand the transcriptional regulation of RANKL, we amplified and characterized a 1890 bp 5'-flanking sequence of human RANKL gene (-1782 bp to +108 bp relative to the transcription start site). Using a series of deletion mutations of the 1890 bp RANKL promoter, we identified a 72 bp region (-172 to -100 bp) mediating RANKL basal transcriptional activity. Sequence analysis revealed a putative E2F binding site within this 72 bp region in the human RANKL promoter. Overexpression of E2F1 increased RANKL promoter activity, while down-regulation of E2F1 expression by small interfering RNA decreased RANKL promoter activity. RT-PCR and enzyme linked immunosorbent assays (ELISA) further demonstrated that E2F1 induced the expression of RANKL. Electrophoretic gel mobility shift assays (EMSA) and antibody competition assays confirmed that E2F1 proteins bind to the consensus E2F binding site in the RANKL promoter. Mutation of the E2F consensus binding site in the RANKL promoter profoundly reduced the basal promoter activity and abolished the transcriptional modulation of RANKL by E2F1. These results suggest that E2F1 plays an important role in regulating RANKL transcription through binding to the E2F consensus binding site.

  13. The evolutionary history of the E2F and DEL genes in Viridiplantae.

    PubMed

    Rauber, Rafael; Cabreira, Caroline; de Freitas, Loreta Brandão; Turchetto-Zolet, Andreia Carina; Margis-Pinheiro, Marcia

    2016-06-01

    The E2 promoter binding factor (E2F) proteins are present in almost all eukaryotic organisms and are essential to control several processes, such as the cell cycle progression, cell division, DNA replication, and apoptosis. The E2F family comprises two different types of proteins: the typical E2Fs and atypical E2Fs, which differ structurally and have specific functions. The E2F gene family was described for the first time in plants in 1999, and since then several studies have focused on the functional aspects, but the evolutionary history of this gene family is still unknown. Here, we investigated the evolutionary history of the E2F gene family in plants. Our findings suggest that E2F proteins arose early after the emergence of the eukaryotic species, while DEL proteins appear to have arisen before the metazoan and plants origin probably through a partial duplication of an ancient E2F protein. Our data also suggest that E2Fs activators and repressors appeared twice during evolution, once in the metazoan lineage and again in the embryophyte lineage. PMID:27033948

  14. Regulation of VEGF receptors by Rb and E2F1: Role of acetylation

    PubMed Central

    Pillai, Smitha; Kovacs, Michelle; Chellappan, Srikumar

    2010-01-01

    E2F transcription factors regulate a variety of cellular processes but their role in angiogenesis is not clear. We find that many genes involved in angiogenesis like FLT-1, KDR and angiopoietin 2 have potential E2F1 binding sites in their promoter. ChIP assays showed that E2F1 can associate with these promoters and the recruitment of E2F1 was enhanced upon VEGF stimulation with concomitant dissociation of Rb leading to the transcriptional activation of these promoters. Transient transfection experiments showed that these promoters were induced by E2F1 and repressed by Rb while depletion of E2F1 decreased their expression. The increased binding of E2F1 to these promoters upon VEGF stimulation correlated with acetylation of histones and E2F1; this required VEGFR function, as seen in ChIP-re-ChIP experiments. This suggests the existence of a positive feedback loop regulating E2F1 acetylation and VEGFR expression. Acetylation associated with VEGF signaling appears to be predominantly mediated by PCAF and depletion of histone acetyl transferases disrupted the formation of angiogenic tubules. These results suggest a novel role for E2F1 and acetylation in the angiogenic process. PMID:20516113

  15. Amplification of the E2F1 transcription factor gene in the HEL erythroleukemia cell line

    SciTech Connect

    Saito, M.; Valentine, M.B.; Look, A.T.

    1995-01-01

    The E2F transcription factor plays an important regulatory role in cell proliferation, mediating the expression of genes whose products are essential for inducing resting cells to enter the cell cycle and synthesize DNA. To investigate the possible involvement of E2F in hematopoietic malignancies, we isolated genomic clones encompassing the human E2F1 gene. We then used fluorescence in situ hybridization to localize E2F1 to human chromosome 20q11, telomeric to the p107 locus, a gene whose product is related to the retinoblastoma gene product (pRb). This finding contrasts with the 1p36 and 6q22 chromosomal locations previously assigned E2F2 and E2F3, two additional members of the E2F family. Although deletions or structural rearrangements of E2F1 were not detected in 14 primary acute leukemia or myelodysplasia samples with structural abnormalities of chromosome 20q11, the gene was amplified and overexpressed in HEL erythroleukemia cells and translocated to other chromosomes in several established human leukemia cell lines. This study provides the first evidence of gene amplification involving a member of the E2F family of transcription factors. We propose that E2F1 overexpression in erythroid progenitors may stimulate abnormal cell proliferation by overriding negative regulatory signals mediated by tumor suppressor proteins such as pRb. 76 refs., 5 figs., 2 tabs.

  16. E2F function in muscle growth is necessary and sufficient for viability in Drosophila

    PubMed Central

    Zappia, Maria Paula; Frolov, Maxim V.

    2016-01-01

    The E2F transcription factor is a key cell cycle regulator. However, the inactivation of the entire E2F family in Drosophila is permissive throughout most of animal development until pupation when lethality occurs. Here we show that E2F function in the adult skeletal muscle is essential for animal viability since providing E2F function in muscles rescues the lethality of the whole-body E2F-deficient animals. Muscle-specific loss of E2F results in a significant reduction in muscle mass and thinner myofibrils. We demonstrate that E2F is dispensable for proliferation of muscle progenitor cells, but is required during late myogenesis to directly control the expression of a set of muscle-specific genes. Interestingly, E2f1 provides a major contribution to the regulation of myogenic function, while E2f2 appears to be less important. These findings identify a key function of E2F in skeletal muscle required for animal viability, and illustrate how the cell cycle regulator is repurposed in post-mitotic cells. PMID:26823289

  17. The transcription factor E2F: a crucial switch in the control of homeostasis and tumorigenesis.

    PubMed

    Fang, Z H; Han, Z C

    2006-04-01

    The transcription factor E2F plays a crucial role in governing cell proliferation through manipulation of the expression of many genes required for cell cycle progression. As studies are exploring in depth, E2F has grown into a multimember family and has been required for the regulation of a large number of genes involved in various cellular processes. The expanding E2F membership and biological function provide us some new insights relating to the evolution of E2F. One of them is to understand the exact mechanisms by which E2F executes in these different cellular processes during ontogenesis. This review summarizes recent advances in this field, with an emphasis on a notion that E2F acts as a molecular switch in the control of both normal cell and tumor development. PMID:16437386

  18. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis

    PubMed Central

    Plechanovová, Anna; Jaffray, Ellis; Tatham, Michael H.; Naismith, James H.; Hay, Ronald T.

    2012-01-01

    SUMMARY Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING of RNF4 in complex with E2 (UbcH5a) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The C-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilise the consequent tetrahedral transition state intermediate. PMID:22842904

  19. Use of Chromatin Immunoprecipitation To Clone Novel E2F Target Promoters

    PubMed Central

    Weinmann, Amy S.; Bartley, Stephanie M.; Zhang, Theresa; Zhang, Michael Q.; Farnham, Peggy J.

    2001-01-01

    We have taken a new approach to the identification of E2F-regulated promoters. After modification of a chromatin immunoprecipitation assay, we cloned nine chromatin fragments which represent both strong and weak in vivo E2F binding sites. Further characterization of three of the cloned fragments revealed that they are bound in vivo not only by E2Fs but also by members of the retinoblastoma tumor suppressor protein family and by RNA polymerase II, suggesting that these fragments represent promoters regulated by E2F transcription complexes. In fact, database analysis indicates that all three fragments correspond to genomic DNA located just upstream of start sites for previously identified mRNAs. One clone, ChET 4, corresponds to the promoter region for beclin 1, a candidate tumor suppressor protein. We demonstrate that another of the clones, ChET 8, is strongly bound by E2F family members in vivo but does not contain a consensus E2F binding site. However, this fragment functions as a promoter whose activity can be repressed by E2F1. Finally, we demonstrate that the ChET 9 promoter contains a consensus E2F binding site, can be activated by E2F1, and drives expression of an mRNA that is upregulated in colon and liver tumors. Interestingly, the characterized ChET promoters do not display regulation patterns typical of known E2F target genes in a U937 cell differentiation system. In summary, we have provided evidence that chromatin immunoprecipitation can be used to identify E2F-regulated promoters which contain both consensus and nonconsensus binding sites and have shown that not all E2F-regulated promoters show identical expression profiles. PMID:11564866

  20. RNAi-mediated knockdown of E2F2 inhibits tumorigenicity of human glioblastoma cells

    PubMed Central

    NAKAHATA, ADRIANA M.; SUZUKI, DANIELA E.; RODINI, CAROLINA O.; FIUZA, MAYARA L.; OKAMOTO, OSWALDO K.

    2014-01-01

    In a previous genome-wide expression profiling study, we identified E2F2 as a hyperexpressed gene in stem-like cells of distinct glioblastoma multiforme (GBM) specimens. Since the encoded E2F2 transcription factor has been implicated in both tumor suppression and tumor development, we conducted a functional study to investigate the pertinence of E2F2 to human gliomagenesis. E2F2 expression was knocked down by transfecting U87MG cells with plasmids carrying a specific silencing shRNA. Upon E2F2 silencing, in vitro cell proliferation was significantly reduced, as indicated by a time-course analysis of viable tumor cells. Anchorage-independent cell growth was also significantly inhibited after E2F2 silencing, based on cell colony formation in soft agar. Subcutaneous and orthotopic xenograft models of GBM in nude mice also indicated inhibition of tumor development in vivo, following E2F2 silencing. As expression of the E2F2 gene is associated with glioblastoma stem cells and is involved in the transformation of human astrocytes, the present findings suggest that E2F2 is involved in gliomagenesis and could be explored as a potential therapeutic target in malignant gliomas. PMID:25202354

  1. AMF-1/Gps2 binds p300 and enhances its interaction with papillomavirus E2 proteins.

    PubMed

    Peng, Y C; Breiding, D E; Sverdrup, F; Richard, J; Androphy, E J

    2000-07-01

    The cellular protein AMF-1 (Gps2) positively modulates gene expression by the papillomavirus E2 protein (D. E. Breiding et al., Mol. Cell. Biol. 17:7208-7219, 1997). We show here that AMF-1 also binds the transcriptional coactivator p300 in vitro and in vivo. E2 interacted weakly with p300. These observations led to a model in which AMF-1 recruits p300 into a complex with E2. Cotransfection of AMF-1 or p300 stimulated levels of E2-dependent transcription, while cotransfection of both AMF-1 and p300 showed an additive effect. The functional significance of p300 recruitment for E2 transactivation was evidenced by repression of E2-activated transcription by adenovirus E1A, which inhibits both coactivator and acetylase activities of p300. Antibodies to AMF-1 or E2 immunoprecipitated histone acetylase activity from cell lysates. Western blotting using antibody against acetyl-lysine failed to detect acetylation of AMF-1 or E2 in complex with p300. These results suggest that AMF-1 facilitates the recruitment of p300 and its histone acetylase activity into complexes with E2 and represents a novel mechanism of transcriptional activation. PMID:10846067

  2. Characterization of the nuclear localization signal of high risk HPV16 E2 protein

    SciTech Connect

    Klucevsek, Kristin; Wertz, Mary; Lucchi, John; Leszczynski, Anna; Moroianu, Junona . E-mail: moroianu@bc.edu

    2007-03-30

    The E2 protein of high risk human papillomavirus type 16 (HPV16) contains an amino-terminal (N) domain, a hinge (H) region and a carboxyl-terminal (C) DNA-binding domain. Using enhanced green fluorescent protein (EGFP) fusions with full length E2 and E2 domains in transfection assays in HeLa cells, we found that the C domain is responsible for the nuclear localization of E2 in vivo, whereas the N and H domains do not contain additional nuclear localization signals (NLSs). Deletion analysis of EGFP-E2 and EGFP-cE2 determined that the C domain contains an {alpha} helix cNLS that overlaps with the DNA-binding region. Mutational analysis revealed that the arginine and lysine residues in this cNLS are essential for nuclear localization of HPV16 E2. Interestingly, these basic amino acid residues are well conserved among the E2 proteins of BPV-1 and some high risk HPV types but not in the low risk HPV types, suggesting that there are differences between the NLSs and corresponding nuclear import pathways between these E2 proteins.

  3. Downregulation of E2F1 during ER stress is required to induce apoptosis.

    PubMed

    Pagliarini, Vittoria; Giglio, Paola; Bernardoni, Paolo; De Zio, Daniela; Fimia, Gian Maria; Piacentini, Mauro; Corazzari, Marco

    2015-03-15

    The endoplasmic reticulum (ER) has recently emerged as an alternative target to induce cell death in tumours, because prolonged ER stress results in the induction of apoptosis even in chemoresistant transformed cells. Here, we show that the DNA-damage-responsive pro-apoptotic factor E2F1 is unexpectedly downregulated during the ER stress-mediated apoptotic programme. E2F1 decline is a late event during the ER response and is mediated by the two unfolded protein response (UPR) sensors ATF6 and IRE1 (also known as ERN1). Whereas ATF6 directly interacts with the E2F1 promoter, IRE1 requires the involvement of the known E2F1 modulator E2F7, through the activation of its main target Xbp-1. Importantly, inhibition of the E2F1 decrease prevents ER-stress-induced apoptosis, whereas E2F1 knockdown efficiently sensitises cells to ER stress-dependent apoptosis, leading to the upregulation of two main factors in the UPR pro-apoptotic execution phase, Puma and Noxa (also known as BBC3 and PMAIP1, respectively). Our results point to a novel key role of E2F1 in the cell survival/death decision under ER stress, and unveil E2F1 inactivation as a valuable novel potential therapeutic strategy to increase the response of tumour cells to ER stress-based anticancer treatments. PMID:25616897

  4. The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity

    PubMed Central

    Hunt, Ann R.; Frederickson, Shana; Maruyama, Toshiaki; Roehrig, John T.; Blair, Carol D.

    2010-01-01

    Background Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2c,d,e,f,g,h) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182–207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE. Methods We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants. Findings Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115–119. Using a 9 Å resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope. Conclusions The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo. PMID:20644615

  5. The E2F2 Transcription Factor Sustains Hepatic Glycerophospholipid Homeostasis in Mice

    PubMed Central

    Maldonado, Eduardo N.; Delgado, Igotz; Furland, Natalia E.; Buqué, Xabier; Iglesias, Ainhoa; Aveldaño, Marta I.; Zubiaga, Ana; Fresnedo, Olatz; Ochoa, Begoña

    2014-01-01

    Increasing evidence links metabolic signals to cell proliferation, but the molecular wiring that connects the two core machineries remains largely unknown. E2Fs are master regulators of cellular proliferation. We have recently shown that E2F2 activity facilitates the completion of liver regeneration after partial hepatectomy (PH) by regulating the expression of genes required for S-phase entry. Our study also revealed that E2F2 determines the duration of hepatectomy-induced hepatic steatosis. A transcriptomic analysis of normal adult liver identified “lipid metabolism regulation” as a major E2F2 functional target, suggesting that E2F2 has a role in lipid homeostasis. Here we use wild-type (E2F2+/+) and E2F2 deficient (E2F2−/−) mice to investigate the in vivo role of E2F2 in the composition of liver lipids and fatty acids in two metabolically different contexts: quiescence and 48-h post-PH, when cellular proliferation and anabolic demands are maximal. We show that liver regeneration is accompanied by large triglyceride and protein increases without changes in total phospholipids both in E2F2+/+ and E2F2−/− mice. Remarkably, we found that the phenotype of quiescent liver tissue from E2F2−/− mice resembles the phenotype of proliferating E2F2+/+ liver tissue, characterized by a decreased phosphatidylcholine to phosphatidylethanolamine ratio and a reprogramming of genes involved in generation of choline and ethanolamine derivatives. The diversity of fatty acids in total lipid, triglycerides and phospholipids was essentially preserved on E2F2 loss both in proliferating and non-proliferating liver tissue, although notable exceptions in inflammation-related fatty acids of defined phospholipid classes were detected. Overall, our results indicate that E2F2 activity sustains the hepatic homeostasis of major membrane glycerolipid components while it is dispensable for storage glycerolipid balance. PMID:25396754

  6. Prostaglandin E2 levels and platelet function are different in cord blood compared to adults.

    PubMed

    Schlagenhauf, Axel; Haidl, Harald; Leschnik, Bettina; Leis, Hans-Joerg; Heinemann, Akos; Muntean, Wolfgang

    2015-01-01

    Neonatal platelets support primary haemostasis and thrombin generation as well as adult platelets, despite observable hypoaggregability in vitro. High prostaglandin E2 levels at accouchement could account for inhibited platelet function via the EP4 receptor. We set out to determine prostaglandin E2 plasma levels in cord blood of healthy neonates and evaluate the impact of prostaglandin E2 on platelet function in adult and cord blood samples. Prostaglandin E2 plasma levels were measured in cord blood and venous adult blood using GC-MS. Impact of prostaglandin E2 on platelet aggregation was measured by spiking cord blood and adult samples. Contributions of EP3 and EP4 receptors were evaluated using respective antagonists. Intracellular cAMP concentrations were measured using a commercial ELISA-kit. Prostaglandin E2 plasma levels were substantially higher in cord blood than in adult samples. Spiking with prostaglandin E2 resulted in a slight but consistent reduction of platelet aggregation in adult blood, but response to PGE2 was blunted in cord blood samples. Aggregation response of spiked adult samples was still higher than with non-spiked cord blood samples. Blockage of EP4 receptors resulted in improved platelet aggregation in adult platelets upon prostaglandin E2 spiking, while aggregation in cord blood samples remained unaltered. Intracellular cAMP concentrations after preincubation with prostaglandin E2 were only increased in adult samples. In conclusion, very high prostaglandin E2 concentrations in cord blood affect platelet function. This effect may partially explain neonatal platelet hypoaggregability. Peak levels of prostaglandin E2 can potentially protect against birth stress-induced platelet activation. PMID:25118631

  7. E2 conjugating enzyme selectivity and requirements for function of the E3 ubiquitin ligase CHIP.

    PubMed

    Soss, Sarah E; Yue, Yuanyuan; Dhe-Paganon, Sirano; Chazin, Walter J

    2011-06-17

    The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2∼Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2∼Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2∼Ub conjugate is derived from other molecular determinants. PMID:21518764

  8. Transcription factor E2F3 overexpressed in prostate cancer independently predicts clinical outcome.

    PubMed

    Foster, Christopher S; Falconer, Alison; Dodson, Andrew R; Norman, Andrew R; Dennis, Nening; Fletcher, Anne; Southgate, Christine; Dowe, Anna; Dearnaley, David; Jhavar, Sameer; Eeles, Rosalind; Feber, Andrew; Cooper, Colin S

    2004-08-01

    E2F transcription factors, including E2F3, directly modulate expression of EZH2. Recently, overexpression of the EZH2 gene has been implicated in the development of human prostate cancer. In tissue microrarray studies we now show that expression of high levels of nuclear E2F3 occurs in a high proportion (98/147, 67%) of human prostate cancers, but is a rare event in non-neoplastic prostatic epithelium suggesting a role for E2F3 overexpression in prostate carcinogenesis. Patients with prostate cancer exhibiting immunohistochemically detectable nuclear E2F3 expression have poorer overall survival (P=0.0022) and cause-specific survival (P=0.0047) than patients without detectable E2F3 expression. When patients are stratified according to the maximum percentage of E2F3-positive nuclei identified within their prostate cancers (up to 20, 21-40%, etc.), there is an increasingly significant association between E2F3 staining and risk of death both for overall survival (P=0.0014) and for cause-specific survival (P=0.0004). Multivariate analyses select E2F3 expression as an independent factor predicting overall survival (unstratified P=0.0103, stratified P=0.0086) and cause-specific survival (unstratified P=0.0288, stratified P=0.0072). When these results are considered together with published data on EZH2 and on the E2F3 control protein pRB, we conclude that the pRB-E2F3-EZH2 control axis may have a critical role in modulating aggressiveness of individual human prostate cancer. PMID:15184867

  9. E2F-1:DP-1 induces p53 and overrides survival factors to trigger apoptosis.

    PubMed Central

    Hiebert, S W; Packham, G; Strom, D K; Haffner, R; Oren, M; Zambetti, G; Cleveland, J L

    1995-01-01

    The E2F DNA binding activity consists of a heterodimer between E2F and DP family proteins, and these interactions are required for association of E2F proteins with pRb and the pRb-related proteins p107 and p130, which modulate E2F transcriptional activities. E2F-1 expression is sufficient to release fibroblasts from G0 and induce entry into S phase, yet it also initiates apoptosis. To investigate the mechanisms of E2F-induced apoptosis, we utilized interleukin-3 (IL-3)-dependent 32D.3 myeloid cells, a model of hematopoietic progenitor programmed cell death. In the absence of IL-3, E2F-1 alone was sufficient to induce apoptosis, and p53 levels were diminished. DP-1 alone was not sufficient to induce cell cycle progression or alter rates of death following IL-3 withdrawal. However, overexpression of both E2F-1 and DP-1 led to the rapid death of cells even in the presence of survival factors. In the presence of IL-3, levels of endogenous wild-type p53 increased in response to E2F-1, and coexpression of DP-1 further augmented p53 levels. These results provide evidence that E2F is a functional link between the tumor suppressors p53 and pRb. However, induction of p53 alone was not sufficient to trigger apoptosis, suggesting that the ability of E2F to override survival factors involves additional effectors. PMID:8524253

  10. Clinical significance of E2F1 protein expression in non-small cell lung cancer

    PubMed Central

    2012-01-01

    Background The transcription factor E2F1 has been implicated in cell cycle control and DNA damage response. Paradoxically, E2F1 can promote apoptosis and function as tumor suppressor. In non-small cell lung cancer (NSCLC), there are conflicting data for clinical significance of E2F1 expression. In this study, we investigated the protein expression of E2F1 in patients with stage I-III NSCLC, and its correlation with clinical outcome. Results 56 paired adjacent non-tumor/tumor matched samples were prospectively obtained from patients undergoing surgery for stage I-III NSCLC at Taipei Veterans General Hospital. The protein expression of E2F1 was determined by Western blot analysis. The levels of E2F1 protein were significantly higher in tumor samples than in non-tumor lung specimens (P = 0.008). Overexpression of E2F1 was defined as a more than 2-fold expression in the tumorous sample compared with the corresponding nontumorous one, and was noted in 21 patients (37.5%). There was no significant difference in overall survival (P = 0.44) or probability of freedom from recurrence (P = 0.378) between patients with E2F1 overexpression vs. non-overexpressors. Additionally, there was no significant association between E2F1 overexpression and any clinicopathologic parameter such as histological type, stage, or angiolymphatic invasion of tumor. Conclusion E2F1 protein is frequently overexpressed in NSCLC. There is no correlation between E2F1 protein expression and clinical outcome such as survival and freedom from progression. PMID:23210897

  11. A human ubiquitin conjugating enzyme (E2)-HECT E3 ligase structure-function screen.

    PubMed

    Sheng, Yi; Hong, Jenny H; Doherty, Ryan; Srikumar, Tharan; Shloush, Jonathan; Avvakumov, George V; Walker, John R; Xue, Sheng; Neculai, Dante; Wan, Janet W; Kim, Sung K; Arrowsmith, Cheryl H; Raught, Brian; Dhe-Paganon, Sirano

    2012-08-01

    Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an "acidic trough" located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7). Mass spectrometry was used to characterize the autoubiquitylation products of a number of functional E2-HECT pairs, and demonstrated that HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. Our data set represents the first comprehensive analysis of E2-HECT E3 interactions, and thus provides a framework for better understanding the molecular mechanisms of ubiquitylation. PMID:22496338

  12. NRIP enhances HPV gene expression via interaction with either GR or E2

    SciTech Connect

    Chang, Szu-Wei; Lu, Pei-Yu; Guo, Jih-Huong; Tsai, Tzung-Chieh; Tsao, Yeou-Ping; Chen, Show-Li

    2012-02-05

    We previously identified a gene, nuclear receptor-interaction protein (NRIP), which functions as a transcription cofactor in glucocorticoid receptor (GR) and human papillomavirus E2 (HPV E2)-driven gene expression. Here, we comprehensively evaluated the role of NRIP in HPV-16 gene expression. NRIP acts as a transcription cofactor to enhance GR-regulated HPV-16 gene expression in the presence of hormone. NRIP also can form complex with E2 that caused NRIP-induced HPV gene expression via E2-binding sites in a hormone-independent manner. Furthermore, NRIP can associate with GR and E2 to form tri-protein complex to activate HPV gene expression via GRE, not the E2-binding site, in a hormone-dependent manner. These results indicate that NRIP and GR are viral E2-binding proteins and that NRIP regulates HPV gene expression via GRE and/or E2 binding site in the HPV promoter in a hormone-dependent or independent manner, respectively.

  13. Cell cycle-related transformation of the E2F4-p130 repressor complex

    SciTech Connect

    Popov, Boris . E-mail: popov_478@hotmail.com; Chang, L.-S.; Serikov, Vladimir

    2005-10-28

    During G0 phase the p130, member of the pRb tumor suppressor protein family, forms a repressor complex with E2F4 which is inactivated in G1/S by hyperphosphorylation of the p130. The role of p130 after G1/S remains poorly investigated. We found that in nuclear extracts of T98G cells, the p130-E2F4-DNA (pp-E2F4) complex does not dissociate at G1/S transition, but instead reverts to the p130-E2F4-cyclin E/A-cdk2 (cyc/cdk-pp-E2F4) complex, which is detected in S and G2/M phases of the cell cycle. Hyperphosphorylation of the p130 at G1/S transition is associated with a decrease of its total amount; however, this protein is still detected during the rest of the cell cycle, and it is increasingly hyperphosphorylated in the cytosol, but continuously dephosphorylated in the nucleus. Both nuclear and cytosol cell fractions in T98G cells contain a hyperphosphorylated form of p130 in complex with E2F4 at S and G2/M cell cycle phases. In contrast to T98G cells, transformation of the p130 containing cyc/cdk-pp-E2F4 complex into the p130-pp-E2F4 repressor does not occur in HeLa cells under growth restriction conditions.

  14. Insights into the mechanism of human papillomavirus E2-induced procaspase-8 activation and cell death

    PubMed Central

    Singh, Nitu; Senapati, Sanjib; Bose, Kakoli

    2016-01-01

    High-risk human papillomavirus (HR-HPV) E2 protein, the master regulator of viral life cycle, induces apoptosis of host cell that is independent of its virus-associated regulatory functions. E2 protein of HR-HPV18 has been found to be involved in novel FADD-independent activation of caspase-8, however, the molecular basis of this unique non-death-fold E2-mediated apoptosis is poorly understood. Here, with an interdisciplinary approach that involves in silico, mutational, biochemical and biophysical probes, we dissected and characterized the E2-procasapse-8 binding interface. Our data demonstrate direct non-homotypic interaction of HPV18 E2 transactivation domain (TAD) with α2/α5 helices of procaspase-8 death effector domain-B (DED-B). The observed interaction mimics the homotypic DED-DED complexes, wherein the conserved hydrophobic motif of procaspase-8 DED-B (F122/L123) occupies a groove between α2/α3 helices of E2 TAD. This interaction possibly drives DED oligomerization leading to caspase-8 activation and subsequent cell death. Furthermore, our data establish a model for E2-induced apoptosis in HR-HPV types and provide important clues for designing E2 analogs that might modulate procaspase-8 activation and hence apoptosis. PMID:26906543

  15. 29 CFR 2584.8477(e)-2 - Allocation of fiduciary duties.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 9 2010-07-01 2010-07-01 false Allocation of fiduciary duties. 2584.8477(e)-2 Section 2584..., DEPARTMENT OF LABOR FIDUCIARY RESPONSIBILITY UNDER THE FEDERAL EMPLOYEES' RETIREMENT SYSTEM ACT OF 1986 RULES AND REGULATIONS FOR THE ALLOCATION OF FIDUCIARY RESPONSIBILITY § 2584.8477(e)-2 Allocation...

  16. E2F Inhibition Synergizes with Paclitaxel in Lung Cancer Cell Lines

    PubMed Central

    Kurtyka, Courtney A.; Chen, Lu; Cress, W. Douglas

    2014-01-01

    The CDK/Rb/E2F pathway is commonly disrupted in lung cancer, and thus, it is predicted that blocking the E2F pathway would have therapeutic potential. To test this hypothesis, we have examined the activity of HLM006474 (a small molecule pan-E2F inhibitor) in lung cancer cell lines as a single agent and in combination with other compounds. HLM006474 reduces the viability of both SCLC and NSCLC lines with a biological IC50 that varies between 15 and 75 µM, but with no significant difference between the groups. Combination of HLM006474 with cisplatin and gemcitabine demonstrate little synergy; however, HLM006474 synergizes with paclitaxel. Surprisingly, we discovered that brief treatment of cells with HLM006474 led to an increase of E2F3 protein levels (due to de-repression of these promoter sites). Since paclitaxel sensitivity has been shown to correlate with E2F3 levels, we hypothesized that HLM006474 synergy with paclitaxel may be mediated by transient induction of E2F3. To test this, H1299 cells were depleted of E2F3a and E2F3b with siRNA and treated with paclitaxel. Assays of proliferation showed that both siRNAs significantly reduced paclitaxel sensitivity, as expected. Taken together, these results suggest that HLM006474 may have efficacy in lung cancer and may be useful in combination with taxanes. PMID:24831239

  17. The Spectrum of E2F in Liver Disease-Mediated Regulation in Biology and Cancer.

    PubMed

    Huntington, Justin T; Tang, Xing; Kent, Lindsey N; Schmidt, Carl R; Leone, Gustavo

    2016-07-01

    Uncoordinated cell growth is one of the fundamental concepts in carcinogenesis and occurs secondary to dysregulation of the cell cycle. The E2Fs are a large family of transcription factors and are key regulators of the cell cycle. The activation of E2Fs is intimately regulated by retinoblastoma 1 (RB1). The RB pathway has been implicated in almost every human malignancy. Recently there have been exciting developments in the E2F field using animal models to better understand the role of E2Fs in vivo. Genetic mouse models have proven essential in implicating E2Fs in hepatocellular carcinoma (HCC) and liver disease. In this review, the general structure and function of E2Fs as well as the role for E2Fs in the development of HCC and liver disease is evaluated. Specifically, what is known about E2Fs in human disease is explored in depth, and future directions are discussed. J. Cell. Physiol. 231: 1438-1449, 2016. © 2015 Wiley Periodicals, Inc. PMID:26566968

  18. Retinoblastoma protein (RB) interacts with E2F3 to control terminal differentiation of Sertoli cells

    PubMed Central

    Rotgers, E; Rivero-Müller, A; Nurmio, M; Parvinen, M; Guillou, F; Huhtaniemi, I; Kotaja, N; Bourguiba-Hachemi, S; Toppari, J

    2014-01-01

    The retinoblastoma protein (RB) is essential for normal cell cycle control. RB function depends, at least in part, on interactions with the E2F family of DNA-binding transcription factors (E2Fs). To study the role of RB in the adult testis, a Sertoli cell (SC)-specific Rb knockout mouse line (SC-RbKO) was generated using the Cre/loxP recombination system. SC-RbKO mice exhibited an age-dependent testicular atrophy, impaired fertility, severe SC dysfunction, and spermatogenic defects. Removal of Rb in SC induced aberrant SC cycling, dedifferentiation, and apoptosis. Here we show that E2F3 is the only E2F expressed in mouse SCs and that RB interacts with E2F3 during mouse testicular development. In the absence of RB, the other retinoblastoma family members p107 and p130 began interacting with E2F3 in the adult testes. In vivo silencing of E2F3 partially restored the SC maturation and survival as well as spermatogenesis in the SC-RbKO mice. These results point to RB as a key regulator of SC function in adult mice and that the RB/E2F3 pathway directs SC maturation, cell cycle quiescence, and RB protects SC from apoptosis. PMID:24901045

  19. 26 CFR 1.1402(e)(2)-1 - Time limitation for filing waiver certificate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 12 2010-04-01 2010-04-01 false Time limitation for filing waiver certificate. 1.1402(e)(2)-1 Section 1.1402(e)(2)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Tax on Self-Employment Income §...

  20. Insights into the mechanism of human papillomavirus E2-induced procaspase-8 activation and cell death.

    PubMed

    Singh, Nitu; Senapati, Sanjib; Bose, Kakoli

    2016-01-01

    High-risk human papillomavirus (HR-HPV) E2 protein, the master regulator of viral life cycle, induces apoptosis of host cell that is independent of its virus-associated regulatory functions. E2 protein of HR-HPV18 has been found to be involved in novel FADD-independent activation of caspase-8, however, the molecular basis of this unique non-death-fold E2-mediated apoptosis is poorly understood. Here, with an interdisciplinary approach that involves in silico, mutational, biochemical and biophysical probes, we dissected and characterized the E2-procasapse-8 binding interface. Our data demonstrate direct non-homotypic interaction of HPV18 E2 transactivation domain (TAD) with α2/α5 helices of procaspase-8 death effector domain-B (DED-B). The observed interaction mimics the homotypic DED-DED complexes, wherein the conserved hydrophobic motif of procaspase-8 DED-B (F122/L123) occupies a groove between α2/α3 helices of E2 TAD. This interaction possibly drives DED oligomerization leading to caspase-8 activation and subsequent cell death. Furthermore, our data establish a model for E2-induced apoptosis in HR-HPV types and provide important clues for designing E2 analogs that might modulate procaspase-8 activation and hence apoptosis. PMID:26906543

  1. A Human Ubiquitin Conjugating Enzyme (E2)-HECT E3 Ligase Structure-function Screen*

    PubMed Central

    Sheng, Yi; Hong, Jenny H.; Doherty, Ryan; Srikumar, Tharan; Shloush, Jonathan; Avvakumov, George V.; Walker, John R.; Xue, Sheng; Neculai, Dante; Wan, Janet W.; Kim, Sung K.; Arrowsmith, Cheryl H.; Raught, Brian; Dhe-Paganon, Sirano

    2012-01-01

    Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an “acidic trough” located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7). Mass spectrometry was used to characterize the autoubiquitylation products of a number of functional E2-HECT pairs, and demonstrated that HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. Our data set represents the first comprehensive analysis of E2-HECT E3 interactions, and thus provides a framework for better understanding the molecular mechanisms of ubiquitylation. PMID:22496338

  2. Transcription factor E2-2 inhibits the proliferation of endothelial progenitor cells by suppressing autophagy

    PubMed Central

    ZHANG, LI; YU, YANG; XIA, XI; MA, YANG; CHEN, XIE-WAN; NI, ZHEN-HONG; WANG, HONG

    2016-01-01

    Endothelial progenitor cells (EPCs) play a key role in repairing the injured vascular endothelium by differentiating into mature endothelial cells (ECs) or secreting cytokines in a paracrine manner to promote proliferation of existing ECs. However, the mechanisms underlying the proliferation of EPCs were not fully understood. In order to investigate the mechanisms of EPC proliferation, we isolated EPCs from mononuclear cells of mouse spleens. By manipulating E2-2 expression in vitro, we observed that E2-2 negatively regulated the proliferation of EPCs. Moreover, we noted that E2-2 negatively regulated the autophagy of EPCs by studying the expression of LC3II and p62. We also demonstrated that an autophagy inhibitor chloroquine (CQ) decreased the proliferation of EPCs in a concentration-dependent manner. Interestingly, CQ reversed the increase in cell proliferation and autophagy in the E2-2 knockdown group. Furthermore, we detected the expression of autophagy-related protein ATG7 in EPCs which had been transfected with small interfering (siRNA)-E2-2 and siRNA-autophagy related 7 (ATG7) or were untransfected. Our study revealed that E2-2 regulated EPC autophagy via mediating ATG7 expression. We conclude that E2-2 inhibited EPC proliferation via suppressing their autophagy, and E2-2 regulated EPC autophagy by mediating the expression of ATG7. PMID:26986900

  3. Increased prostaglandin F2 alpha and E2 production in late passage WI38 diploid fibroblasts.

    PubMed

    Mets, T; Korteweg, M; Verdonk, G

    1979-11-01

    The prostaglandin (PG) F2alpha and E2 concentrations in the culture medium of WI38 cells were determined at various passage levels. It was found that late passage cells produce significantly higher amounts of both PG F2alpha and PG E2. PMID:509542

  4. Functional characterization of the Sindbis virus E2 glycoprotein by transposon linker-insertion mutagenesis

    SciTech Connect

    Navaratnarajah, Chanakha K.; Kuhn, Richard J. . E-mail: kuhnr@purdue.edu

    2007-06-20

    The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1-E2 contacts, and receptor binding.

  5. B (E 2 ) values in neutron-excess nuclei near A =16

    NASA Astrophysics Data System (ADS)

    Fortune, H. T.

    2016-04-01

    A simple model is used to compute B (E 2 ) 's in several nuclei that have one or two s d -shell neutrons and no s d -shell protons. The model works well for all six nuclei if I use later experimental values for 16C for which the measured B (E 2 ) is about four to eight times earlier values.

  6. [Transcriptional repressive activity of mutated E2 protein of human papillomavirus 2 (HPV-2) variant].

    PubMed

    Lei, Yan-jun; Gao, Chen; Jiang, Hui-ying; Han, Jun; Chen, Jian-ming; Shi, Qi; Zhou, Wei; Yuan, Yu-kang; Dong, Xiao-ping

    2008-07-01

    Common warts are close associated with HPVs infection. In this study, we amplified and sequenced the LCR fragment and E2 gene of HPV-2 that infected the patient of extensive common wart with cutaneous horns, and we constructed the recombinant CAT-reporter plasmids pBLCAT-LCR containing HPV-2 prototype or variant LCR and mammalian expression plasmids pcDNA3. 1-E2 containing prototype or variant E2 ORF individually. The promoter activities of HPV-2 variant and the transcriptional repression activities of the mutated E2 protein were evaluated by transient transfection into HeLa cells. The results showed that there were several mutations in LCR and E2 gene of HPV-2 variant. Compared with the prototype, the viral early promoter activity of variant was significantly increased uder the control of LCR. Compared with the wild type E2 protein, the transcriptional repression activities of the mutated E2 protein was abolished partially. We speculate herein that increased promoter activities and decreased repression effect of the mutated E2 protein are linked, at least partially, with the clinical phenotypes of the uncommon huge common wart. PMID:18780628

  7. E2F1 mediates sustained lipogenesis and contributes to hepatic steatosis.

    PubMed

    Denechaud, Pierre-Damien; Lopez-Mejia, Isabel C; Giralt, Albert; Lai, Qiuwen; Blanchet, Emilie; Delacuisine, Brigitte; Nicolay, Brandon N; Dyson, Nicholas J; Bonner, Caroline; Pattou, Franois; Annicotte, Jean-Sbastien; Fajas, Lluis

    2016-01-01

    E2F transcription factors are known regulators of the cell cycle, proliferation, apoptosis, and differentiation. Here, we reveal that E2F1 plays an essential role in liver physiopathology through the regulation of glycolysis and lipogenesis. We demonstrate that E2F1 deficiency leads to a decrease in glycolysis and de novo synthesis of fatty acids in hepatocytes. We further demonstrate that E2F1 directly binds to the promoters of key lipogenic genes, including Fasn, but does not bind directly to genes encoding glycolysis pathway components, suggesting an indirect effect. In murine models, E2F1 expression and activity increased in response to feeding and upon insulin stimulation through canonical activation of the CDK4/pRB pathway. Moreover, E2F1 expression was increased in liver biopsies from obese, glucose-intolerant humans compared with biopsies from lean subjects. Finally, E2f1 deletion completely abrogated hepatic steatosis in different murine models of nonalcoholic fatty liver disease (NAFLD). In conclusion, our data demonstrate that E2F1 regulates lipid synthesis and glycolysis and thus contributes to the development of liver pathology. PMID:26619117

  8. Identical Structural and Receptor Binding Defects in Apolipoprotein E2 in Hypo-, Normo-, and Hypercholesterolemic Dysbetalipoproteinemia

    PubMed Central

    Rall, Stanley C.; Weisgraber, Karl H.; Innerarity, Thomas L.; Mahley, Robert W.; Assmann, Gerd

    1983-01-01

    Apolipoprotein E (apoprotein E or apo-E) from type III hyperlipoproteinemic subjects with the E2/2 homozygous phenotype displays both structural and receptor binding heterogeneity. The apo-E from all subjects thus far studied, however, has been functionally defective, though to different degrees. Although nearly every type III hyperlipoproteinemic subject has the E2/2 phenotype, 95-99% of the people with this same phenotype do not display type III hyperlipoproteinemia, nor do they have elevated plasma cholesterol levels. Consequently, it became important to determine whether the apo-E2 from hypo- and normocholesterolemic individuals with the E2/2 phenotype is also functionally abnormal. To do this, apo-E2 was isolated from two hypo-, two normo- and two hypercholesterolemic homozygous E2/2 subjects. The apo-E2 was recombined with vesicles and tested for its ability to displace 125I-low density lipoproteins (LDL) from apo-B,E (LDL) receptors on human fibroblasts. The apo-E2 from all six subjects was found to be severely defective in receptor binding (<2% of the binding activity of normal apo-E3). In all cases, the binding activity of the apo-E2 was increased 10- to 20-fold by treating the apoproteins with cysteamine, a reagent that converts cysteine residues to positively charged lysine analogues. The cysteine content of each apo-E was determined by monitoring the change in the isoelectric focusing position of the cysteamine-treated apo-E2. Using this method, it was found that the apo-E2 from each subject contained two cysteine residues per mole. A partial sequence analysis of the cysteine-containing regions of the apo-E from three of the six subjects indicated that the two cysteine residues were at residues 112 and 158 in the amino acid sequence. The cysteine at residue 158 has previously been implicated in the severe binding defect of the apo-E2 from a type III hyperlipoproteinemic subject. Since the apo-E2 of the hypo-, normo-, and hypercholesterolemic subjects in this study all displayed a severe functional abnormality, it is apparent that factors in addition to the defective receptor binding activity of the apo-E2 are necessary for the manifestation of type III hyperlipoproteinemia. Images PMID:6300187

  9. Inhibition of HPV-16 E7 oncogenic activity by HPV-16 E2.

    PubMed

    Gammoh, N; Isaacson, E; Tomaić, V; Jackson, D J; Doorbar, J; Banks, L

    2009-06-11

    Human papillomavirus (HPV) E7 is essential in inducing S-phase progression in differentiating epithelial cells. We have previously shown that HPV-16 E7 activity can be controlled by a direct interaction with the viral transcriptional activator E2, thereby inhibiting transforming potential of E7. We have extended these analyses to show that E2 induces a generalized re-localization of E7 within the cell nucleus, one potential consequence of which is the inhibition of E7-induced degradation of pRb. Most importantly, we show that E2 can also inhibit the ability of E7 to induce centrosome abnormalities, thus preventing aberrant mitoses. Taken together, these studies highlight the central importance of E2 in controlling the functions of E7, independently of the ability of E2 to regulate transcription. PMID:19421149

  10. Tables of E2 transition probabilities from the first 2+ states in even-even nuclei

    NASA Astrophysics Data System (ADS)

    Pritychenko, B.; Birch, M.; Singh, B.; Horoi, M.

    2016-01-01

    Experimental results of E2 transition probabilities or B(E2) values for the known first 2+ states in 447 even-even nuclei have been compiled and evaluated. The evaluation policies for the analysis of experimental data have been described and new results are discussed. The recommended B(E2) values have been compared with comprehensive shell model calculations for a selected set of nuclei, where such theoretical procedures are amenable. The present work was motivated by a rapid increase in the number of new B(E2) measurements for the first 2+ states since the previous evaluation of such data by S. Raman et al. published in 2001. Future plans to investigate the systematics of B(E2) ↑ values, and intercomparison of different experimental techniques to obtain these data are outlined.

  11. In vivo delivery of bovine viral diahorrea virus, E2 protein using hollow mesoporous silica nanoparticles

    NASA Astrophysics Data System (ADS)

    Mahony, D.; Cavallaro, A. S.; Mody, K. T.; Xiong, L.; Mahony, T. J.; Qiao, S. Z.; Mitter, N.

    2014-05-01

    Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral diarrhoea virus (BVDV). BVDV infection occurs in the target species of cattle and sheep herds worldwide and is therefore of economic importance. E2 is a major immunogenic determinant of BVDV and is an ideal candidate for the development of a subunit based nanovaccine using mesoporous silica nanoparticles. Hollow type mesoporous silica nanoparticles with surface amino functionalisation (termed HMSA) were characterised and assessed for adsorption and desorption of E2. A codon-optimised version of the E2 protein (termed Opti-E2) was produced in Escherichia coli. HMSA (120 nm) had an adsorption capacity of 80 μg Opti-E2 per mg HMSA and once bound E2 did not dissociate from the HMSA. Immunisation studies in mice with a 20 μg dose of E2 adsorbed to 250 μg HMSA was compared to immunisation with Opti-E2 (50 μg) together with the traditional adjuvant Quillaja saponaria Molina tree saponins (QuilA, 10 μg). The humoral responses with the Opti-E2/HMSA nanovaccine although slightly lower than those obtained for the Opti-E2 + QuilA group demonstrated that HMSA particles are an effective adjuvant that stimulated E2-specific antibody responses. Importantly the cell-mediated immune responses were consistently high in all mice immunised with Opti-E2/HMSA nanovaccine formulation. Therefore we have shown the Opti-E2/HMSA nanoformulation acts as an excellent adjuvant that gives both T-helper 1 and T-helper 2 mediated responses in a small animal model. This study has provided proof-of-concept towards the development of an E2 subunit nanoparticle based vaccine.Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral diarrhoea virus (BVDV). BVDV infection occurs in the target species of cattle and sheep herds worldwide and is therefore of economic importance. E2 is a major immunogenic determinant of BVDV and is an ideal candidate for the development of a subunit based nanovaccine using mesoporous silica nanoparticles. Hollow type mesoporous silica nanoparticles with surface amino functionalisation (termed HMSA) were characterised and assessed for adsorption and desorption of E2. A codon-optimised version of the E2 protein (termed Opti-E2) was produced in Escherichia coli. HMSA (120 nm) had an adsorption capacity of 80 μg Opti-E2 per mg HMSA and once bound E2 did not dissociate from the HMSA. Immunisation studies in mice with a 20 μg dose of E2 adsorbed to 250 μg HMSA was compared to immunisation with Opti-E2 (50 μg) together with the traditional adjuvant Quillaja saponaria Molina tree saponins (QuilA, 10 μg). The humoral responses with the Opti-E2/HMSA nanovaccine although slightly lower than those obtained for the Opti-E2 + QuilA group demonstrated that HMSA particles are an effective adjuvant that stimulated E2-specific antibody responses. Importantly the cell-mediated immune responses were consistently high in all mice immunised with Opti-E2/HMSA nanovaccine formulation. Therefore we have shown the Opti-E2/HMSA nanoformulation acts as an excellent adjuvant that gives both T-helper 1 and T-helper 2 mediated responses in a small animal model. This study has provided proof-of-concept towards the development of an E2 subunit nanoparticle based vaccine. Electronic supplementary information (ESI) available: Desorption studies of Opti-E2 bound HMSA. See DOI: 10.1039/c4nr01202j

  12. Materials characterization activities for %E2%80%9CTake Our Sons&Daughters to Work Day%E2%80%9D 2013.

    SciTech Connect

    Mowry, Curtis Dale; Pimentel, Adam S.; Sparks, Elizabeth Schares; Hanlon, Brittany Paula

    2013-09-01

    We created interactive demonstration activities for Take Our Daughters&Sons to Work Day (TODSTWD) 2013 in order to promote general interest in chemistry and also generate awareness of the type of work our laboratories can perform. %E2%80%9CCurious about Mars Rover Curiosity?%E2%80%9D performed an elemental analysis on rocks brought to our lab using the same technique utilized on the planet Mars by the NASA robotic explorer Curiosity. %E2%80%9CFood is Chemistry?%E2%80%9D utilized a mass spectrometer to measure, in seconds, each participant's breath in order to identify the food item consumed for the activity. A total of over 130 children participated in these activities over a 3 hour block, and feedback was positive. This document reports the materials (including handouts), experimental procedures, and lessons learned so that future demonstrations can benefit from the baseline work performed. We also present example results used to prepare the Food activity and example results collected during the Curiosity demo.

  13. Oncogenic ETS fusions deregulate E2F3 target genes in Ewing sarcoma and prostate cancer

    PubMed Central

    Bilke, Sven; Schwentner, Raphaela; Yang, Fan; Kauer, Maximilian; Jug, Gunhild; Walker, Robert L.; Davis, Sean; Zhu, Yuelin J.; Pineda, Marbin; Meltzer, Paul S.; Kovar, Heinrich

    2013-01-01

    Deregulated E2F transcription factor activity occurs in the vast majority of human tumors and has been solidly implicated in disturbances of cell cycle control, proliferation, and apoptosis. Aberrant E2F regulatory activity is often caused by impairment of control through pRB function, but little is known about the interplay of other oncoproteins with E2F. Here we show that ETS transcription factor fusions resulting from disease driving rearrangements in Ewing sarcoma (ES) and prostate cancer (PC) are one such class of oncoproteins. We performed an integrative study of genome-wide DNA-binding and transcription data in EWSR1/FLI1 expressing ES and TMPRSS2/ERG containing PC cells. Supported by promoter activity and mutation analyses, we demonstrate that a large fraction of E2F3 target genes are synergistically coregulated by these aberrant ETS proteins. We propose that the oncogenic effect of ETS fusion oncoproteins is in part mediated by the disruptive effect of the E2F–ETS interaction on cell cycle control. Additionally, a detailed analysis of the regulatory targets of the characteristic EWSR1/FLI1 fusion in ES identifies two functionally distinct gene sets. While synergistic regulation in concert with E2F in the promoter of target genes has a generally activating effect, EWSR1/FLI1 binding independent of E2F3 is predominantly associated with repressed differentiation genes. Thus, EWSR1/FLI1 appears to promote oncogenesis by simultaneously promoting cell proliferation and perturbing differentiation. PMID:23940108

  14. Functional interaction of a novel cellular protein with the papillomavirus E2 transactivation domain.

    PubMed

    Breiding, D E; Sverdrup, F; Grossel, M J; Moscufo, N; Boonchai, W; Androphy, E J

    1997-12-01

    The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells. PMID:9372953

  15. The Sclerotinia sclerotiorum FoxE2 Gene Is Required for Apothecial Development.

    PubMed

    Wang, Lu; Liu, Yanzhi; Liu, Jinliang; Zhang, Yanhua; Zhang, Xianghui; Pan, Hongyu

    2016-05-01

    Sclerotinia sclerotiorum is a widely dispersed plant pathogenic fungus causing many diseases such as white mold, Sclerotinia stem rot, stalk rot, and Sclerotinia head rot on many varieties of broadleaf crops worldwide. Previous studies have shown that the Forkhead-box transcription factors (FOX TFs) play key regulatory roles in the sexual reproduction of some fungi. Ss-FoxE2 is one of four FOX TF family member genes in S. sclerotiorum. Based on ortholog function in other fungi it is hypothesized to function in S. sclerotiorum sexual reproduction. In this study, the role of Ss-FoxE2 in S. sclerotiorum was identified with a gene knock-out strategy. Following transformation and screening, strains having undergone homologous recombination in which the hygromycin resistance gene replaced the gene Ss-FoxE2 from the genomic DNA were identified. No difference in hyphae growth, number, and weight of sclerotia and no obvious change in virulence was observed among the wild type Ss-FoxE2 knock-out mutant and genetically complemented mutant; however, following induction of sclerotia for sexual development, apothecia were not formed in Ss-FoxE2 knock-out mutant. The Ss-FoxE2 gene expressed significantly higher in the apothecial stages than in other developmental stages. These results indicate that Ss-FoxE2 appears to be necessary for the regulation of sexual reproduction, but may not affect the pathogenicity and vegetative development of S. sclerotiorum significantly. PMID:26756829

  16. Functional interaction of a novel cellular protein with the papillomavirus E2 transactivation domain.

    PubMed Central

    Breiding, D E; Sverdrup, F; Grossel, M J; Moscufo, N; Boonchai, W; Androphy, E J

    1997-01-01

    The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells. PMID:9372953

  17. OTUB1 Co-opts Lys48-Linked Ubiquitin Recognition to Suppress E2 Enzyme Function

    SciTech Connect

    Juang, Yu-Chi; Landry, Marie-Claude; Sanches, Mario; Vittal, Vinayak; Leung, Charles C.Y.; Ceccarelli, Derek F.; Mateo, Abigail-Rachele F.; Pruneda, Jonathan N.; Mao, Daniel Y.L.; Szilard, Rachel K.; Orlicky, Stephen; Munro, Meagan; Brzovic, Peter S.; Klevit, Rachel E.; Sicheri, Frank; Durocher, Daniel

    2012-03-26

    Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.

  18. The Arabidopsis her1 mutant implicates GABA in E-2-hexenal responsiveness.

    PubMed

    Mirabella, Rossana; Rauwerda, Han; Struys, Eduard A; Jakobs, Cornelis; Triantaphylidès, Christian; Haring, Michel A; Schuurink, Robert C

    2008-01-01

    When wounded or attacked by herbivores or pathogens, plants produce a blend of six-carbon alcohols, aldehydes and esters, known as C6-volatiles. Undamaged plants, when exposed to C6-volatiles, respond by inducing defense-related genes and secondary metabolites, suggesting that C6-volatiles can act as signaling molecules regulating plant defense responses. However, to date, the molecular mechanisms by which plants perceive and respond to these volatiles are unknown. To elucidate such mechanisms, we decided to isolate Arabidopsis thaliana mutants in which responses to C6-volatiles were altered. We observed that treatment of Arabidopsis seedlings with the C6-volatile E-2-hexenal inhibits root elongation. Among C6-volatiles this response is specific to E-2-hexenal, and is not dependent on ethylene, jasmonic and salicylic acid. Using this bioassay, we isolated 18 E-2-hexenal-response (her) mutants that showed sustained root growth after E-2-hexenal treatment. Here, we focused on the molecular characterization of one of these mutants, her1. Microarray and map-based cloning revealed that her1 encodes a gamma-amino butyric acid transaminase (GABA-TP), an enzyme that degrades GABA. As a consequence of the mutation, her1 plants accumulate high GABA levels in all their organs. Based on the observation that E-2-hexenal treatment induces GABA accumulation, and that high GABA levels confer resistance to E-2-hexenal, we propose a role for GABA in mediating E-2-hexenal responses. PMID:17971036

  19. The transcription factor E2F-1 mediates the autoregulation of RB gene expression.

    PubMed Central

    Shan, B; Chang, C Y; Jones, D; Lee, W H

    1994-01-01

    The retinoblastoma (RB) gene is the prototype tumor suppressor gene. Mutations in this gene are often associated with the occurrence of various tumors. Several mutations have been found in the promoter region of the gene, suggesting that inappropriate transcriptional regulation of the RB gene contributes to tumorigenesis. Sequence analysis of the RB promoter has revealed a potential E2F recognition site within a region critical for RB gene transcription. By using the cloned E2F-1 gene, here we report that (i) RB expression is negatively regulated by its own gene product, (ii) E2F-1 binds specifically to an E2F recognition sequence in the RB promoter and transactivates the RB promoter, (iii) overexpression of RB suppresses E2F-1-mediated stimulation of RB promoter activity, and (iv) the expression of the RB gene is paralleled by the expression of the E2F-1 gene during cell cycle progression. These results demonstrate that expression of RB is negatively autoregulated through E2F-1. Images PMID:8264596

  20. 17β-estradiol (E2) in membranes: Orientation and dynamic properties.

    PubMed

    Guo, Jason J; Yang, De-Ping; Tian, Xiaoyu; Vemuri, V Kiran; Yin, Dali; Li, Chen; Duclos, Richard I; Shen, Lingling; Ma, Xiaoyu; Janero, David R; Makriyannis, Alexandros

    2016-02-01

    Non-genomic membrane effects of estrogens are of great interest because of the diverse biological activities they may elicit. To further our understanding of the molecular features of the interaction between estrogenic hormones and membrane bilayers, we have determined the preferred orientation, location, and dynamic properties of 17β-estradiol (E2) in two different phospholipid membrane environments using (2)H-NMR and 2D (1)H-(13)C HSQC in conjunction with molecular dynamics simulations. Unequivocal spectral assignments to specific (2)H labels were made possible by synthesizing six selectively deuterated E2 molecules. The data allow us to conclude that the E2 molecule adopts a nearly "horizontal" orientation in the membrane bilayer with its long axis essentially perpendicular to the lipid acyl-chains. All four rings of the E2 molecule are located near the membrane interface, allowing both the E2 3-OH and the 17β-OH groups to engage in hydrogen bonding and electrostatic interactions with polar phospholipid groups. The findings augment our knowledge of the molecular interactions between E2 and membrane bilayer and highlight the asymmetric nature of the dynamic motions of the rigid E2 molecule in a membrane environment. PMID:26607010

  1. Citrullination-acetylation interplay guides E2F-1 activity during the inflammatory response

    PubMed Central

    Ghari, Fatemeh; Quirke, Anne-Marie; Munro, Shonagh; Kawalkowska, Joanna; Picaud, Sarah; McGouran, Joanna; Subramanian, Venkataraman; Muth, Aaron; Williams, Richard; Kessler, Benedikt; Thompson, Paul R.; Fillipakopoulos, Panagis; Knapp, Stefan; Venables, Patrick J.; La Thangue, Nicholas B.

    2016-01-01

    Peptidyl arginine deiminase 4 (PAD4) is a nuclear enzyme that converts arginine residues to citrulline. Although increasingly implicated in inflammatory disease and cancer, the mechanism of action of PAD4 and its functionally relevant pathways remains unclear. E2F transcription factors are a family of master regulators that coordinate gene expression during cellular proliferation and diverse cell fates. We show that E2F-1 is citrullinated by PAD4 in inflammatory cells. Citrullination of E2F-1 assists its chromatin association, specifically to cytokine genes in granulocyte cells. Mechanistically, citrullination augments binding of the BET (bromodomain and extra-terminal domain) family bromodomain reader BRD4 (bromodomain-containing protein 4) to an acetylated domain in E2F-1, and PAD4 and BRD4 coexist with E2F-1 on cytokine gene promoters. Accordingly, the combined inhibition of PAD4 and BRD4 disrupts the chromatin-bound complex and suppresses cytokine gene expression. In the murine collagen-induced arthritis model, chromatin-bound E2F-1 in inflammatory cells and consequent cytokine expression are diminished upon small-molecule inhibition of PAD4 and BRD4, and the combined treatment is clinically efficacious in preventing disease progression. Our results shed light on a new transcription-based mechanism that mediates the inflammatory effect of PAD4 and establish the interplay between citrullination and acetylation in the control of E2F-1 as a regulatory interface for driving inflammatory gene expression. PMID:26989780

  2. In vivo association of E2F and DP family proteins.

    PubMed Central

    Wu, C L; Zukerberg, L R; Ngwu, C; Harlow, E; Lees, J A

    1995-01-01

    The mammalian transcription factor E2F plays an important role in regulating the expression of genes that are required for passage through the cell cycle. This transcriptional activity is inhibited by association with the retinoblastoma tumor suppressor protein (pRB) or its relatives p107 and p103. The first cDNA from the E2F family to be cloned was designated E2F-1, and multiple E2F family members have now been identified. They bind to DNA as heterodimers, interacting with proteins known as DP. Here we demonstrate that DP is also a family of polypeptides with at least two members (hDP-1 and hDP-2). Both hDP-1 and hDP-2 bind to all E2F family members in vivo, and each complex is capable of activating transcription. However, the various E2F/DP complexes display strong differences in the ability to bind to either pRB or p107 in vivo, and the specificity of pRB or p107 binding is mediated by the E2F subunit. PMID:7739537

  3. Atypical E2F activity coordinates PHR1 photolyase gene transcription with endoreduplication onset

    PubMed Central

    Radziejwoski, Amandine; Vlieghe, Kobe; Lammens, Tim; Berckmans, Barbara; Maes, Sara; Jansen, Marcel A K; Knappe, Claudia; Albert, Andreas; Seidlitz, Harald K; Bahnweg, Günther; Inzé, Dirk; De Veylder, Lieven

    2011-01-01

    Because of their sessile life style, plants have evolved the ability to adjust to environmentally harsh conditions. An important aspect of stress adaptation involves the reprogramming of the cell cycle to ensure optimal growth. The atypical E2F transcription factor DP-E2F-like 1 (E2Fe/DEL1) had been found previously to be an important regulator of the endocycle onset. Here, a novel role for E2Fe/DEL1 was identified as a transcriptional repressor of the type-II cyclobutane pyrimidine dimer-photolyase DNA repair gene PHR1. Upon ultraviolet-B (UV-B) treatment, plants knocked out for E2Fe/DEL1 had improved DNA repair abilities when compared with control plants, whereas those overexpressing it performed less well. Better DNA repair allowed E2Fe/DEL1 knockout plants to resume endoreduplication faster than control plants, contributing in this manner to UV-B radiation resistance by compensating the stress-induced reduction in cell number by ploidy-dependent cell growth. As E2Fe/DEL1 levels decreased upon UV-B treatment, we hypothesize that the coordinated transcriptional induction of PHR1 with the endoreduplication onset contributes to the adaptation of plants exposed to UV-B stress. PMID:21131907

  4. The action of colicin E2 on supercoiled lambda DNA. I. Experiments in vivo.

    PubMed

    Saxe, L S

    1975-05-20

    A lambda DNA supercoil system has been developed to study the effects of colicin E2 on DNA in vivo. Colicin E2, a protein antibiotic synthesized by strains of coliform bacteria that carry the Col E2 plasmid, had as its most conspicious effect damage to the DNA of sensitive strains. Colicine E2 attacks the supercoiled molecul formed by labeled lambda DNA in superinfected cells as well as it attacks the bacterial DNA. The rate and extent of acid solubilization of the lambda supercoils and of host bacterial DNA induced by E2 treatment are nearly identical. Treatment of superinfected cells with colicin E2 results in the progressive conversion of lambda DNA supercoils to open circles and/or linear full lenght molecules, and subsequently to fragments less than full lambda in size. The first endonucleolytic reactions are single-strand and or double-strand breaks. The rate of supercoil breakdown as well as the final percent supercoils remaining unconverted, the size of the final lambda fragments, and the extent of solubilization are dependent on the multiplicity of colicin used. Additions of trypsin to E2-treated superinfected cells results in a cessation of further breakdown of the lambda molecules, presumably as a result of digestion of accessible colicin molecules. Energy is essential for an early event in colicin E2 action. The host enzymes, endonuclease I and Rec BC, may be instrumental in the nucleolytic process caused by colicin E2: endonuclease I in reaction preceding cell killing and Rec BC in a secondary degradation of the bacterial DNA. PMID:1148158

  5. Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network.

    PubMed

    Markson, Gabriel; Kiel, Christina; Hyde, Russell; Brown, Stephanie; Charalabous, Panagoula; Bremm, Anja; Semple, Jennifer; Woodsmith, Jonathan; Duley, Simon; Salehi-Ashtiani, Kourosh; Vidal, Marc; Komander, David; Serrano, Luis; Lehner, Paul; Sanderson, Christopher M

    2009-10-01

    In eukaryotic cells the stability and function of many proteins are regulated by the addition of ubiquitin or ubiquitin-like peptides. This process is dependent upon the sequential action of an E1-activating enzyme, an E2-conjugating enzyme, and an E3 ligase. Different combinations of these proteins confer substrate specificity and the form of protein modification. However, combinatorial preferences within ubiquitination networks remain unclear. In this study, yeast two-hybrid (Y2H) screens were combined with true homology modeling methods to generate a high-density map of human E2/E3-RING interactions. These data include 535 experimentally defined novel E2/E3-RING interactions and >1300 E2/E3-RING pairs with more favorable predicted free-energy values than the canonical UBE2L3-CBL complex. The significance of Y2H predictions was assessed by both mutagenesis and functional assays. Significantly, 74/80 (>92%) of Y2H predicted complexes were disrupted by point mutations that inhibit verified E2/E3-RING interactions, and a approximately 93% correlation was observed between Y2H data and the functional activity of E2/E3-RING complexes in vitro. Analysis of the high-density human E2/E3-RING network reveals complex combinatorial interactions and a strong potential for functional redundancy, especially within E2 families that have undergone evolutionary expansion. Finally, a one-step extended human E2/E3-RING network, containing 2644 proteins and 5087 edges, was assembled to provide a resource for future functional investigations. PMID:19549727

  6. Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network

    PubMed Central

    Markson, Gabriel; Kiel, Christina; Hyde, Russell; Brown, Stephanie; Charalabous, Panagoula; Bremm, Anja; Semple, Jennifer; Woodsmith, Jonathan; Duley, Simon; Salehi-Ashtiani, Kourosh; Vidal, Marc; Komander, David; Serrano, Luis; Lehner, Paul; Sanderson, Christopher M.

    2009-01-01

    In eukaryotic cells the stability and function of many proteins are regulated by the addition of ubiquitin or ubiquitin-like peptides. This process is dependent upon the sequential action of an E1-activating enzyme, an E2-conjugating enzyme, and an E3 ligase. Different combinations of these proteins confer substrate specificity and the form of protein modification. However, combinatorial preferences within ubiquitination networks remain unclear. In this study, yeast two-hybrid (Y2H) screens were combined with true homology modeling methods to generate a high-density map of human E2/E3-RING interactions. These data include 535 experimentally defined novel E2/E3-RING interactions and >1300 E2/E3-RING pairs with more favorable predicted free-energy values than the canonical UBE2L3–CBL complex. The significance of Y2H predictions was assessed by both mutagenesis and functional assays. Significantly, 74/80 (>92%) of Y2H predicted complexes were disrupted by point mutations that inhibit verified E2/E3-RING interactions, and a ∼93% correlation was observed between Y2H data and the functional activity of E2/E3-RING complexes in vitro. Analysis of the high-density human E2/E3-RING network reveals complex combinatorial interactions and a strong potential for functional redundancy, especially within E2 families that have undergone evolutionary expansion. Finally, a one-step extended human E2/E3-RING network, containing 2644 proteins and 5087 edges, was assembled to provide a resource for future functional investigations. PMID:19549727

  7. Rendezvous with Toutatis from the Moon: The Chang'e-2 mission

    NASA Astrophysics Data System (ADS)

    Huang, J.; Tang, X.; Meng, L.

    2014-07-01

    Chang'e-2 probe was the second lunar probe of China, with the main objectives to demonstrate some key features of the new lunar soft landing technology, and its applications to future exploration missions. After completing the planned mission successfully, Chang'e-2 flew away from the Moon and entered into the interplanetary space. Later, at a distance of 7 million km from the Earth, Chang'e-2 encountered asteroid (4179) Toutatis with a very close fly-by distance and obtained colorful images with a 3-m resolution. Given some surplus velocity increment as well as the promotion of autonomous flight ability and improvement of control, propulsion, and thermal systems in the initial design, Chang'e-2 had the capabilities necessary for escaping from the Moon. By taking advantage of the unique features of the Lagrangian point, the first close fly-by of asteroid Toutatis was realized despite the tight constraints of propellant allocation, spacecraft-Earth communication, and coordination of execution sequences. Chang'e-2 realized the Toutatis flyby with a km-level distance at closest approach. In the absence of direct measurement method, based on the principle of relative navigation and through the use of the sequence of target images, we calculated the rendezvous parameters such as relative distance and image resolution. With the help of these parameters, some fine and new scientific discoveries about the asteroid were obtained by techniques of optical measurements and image processing. Starting with an innovative design, followed by high-fidelity testing and demonstration, elaborative implementation, and optimal usage of residual propellant, Chang'e-2 has for the first time successfully explored the Moon, L2 point and an asteroid, while achieving the purpose of 'faster, better, cheaper'. What Chang'e-2 has accomplished was far beyond our expectations. *J. Huang is the chief designer (PI) of Chang'e-2 probe, planned Chang'e-2's multi-objective and multitasking exploration mission.

  8. Silencing of E2F3 suppresses tumor growth of Her2+ breast cancer cells by restricting mitosis

    PubMed Central

    Lee, Miyoung; Oprea-Ilies, Gabriela; Saavedra, Harold I.

    2015-01-01

    The E2F transcriptional activators E2F1, E2F2 and E2F3a regulate many important cellular processes, including DNA replication, apoptosis and centrosome duplication. Previously, we demonstrated that silencing E2F1 or E2F3 suppresses centrosome amplification (CA) and chromosome instability (CIN) in Her2+ breast cancer cells without markedly altering proliferation. However, it is unknown whether and how silencing a single E2F activator, E2F3, affects malignancy of human breast cancer cells. Thus, we injected HCC1954 Her2+ breast cancer cells silenced for E2F3 into mammary fat pads of immunodeficient mice and demonstrated that loss of E2F3 retards tumor growth. Surprisingly, silencing of E2F3 led to significant reductions in mitotic indices relative to vector controls, while the percentage of cells undergoing S phase were not affected. Nek2 is a mitotic kinase commonly upregulated in breast cancers and a critical regulator of Cdk4- or E2F- mediated CA. In this report, we found that Nek2 overexpression rescued back the CA caused by silencing of shE2F3. However, the effects of Nek2 overexpression in affecting tumor growth rates of shE2F3 and shE2F3; GFP cells were inconclusive. Taken together, our results indicate that E2F3 silencing decreases mammary tumor growth by reducing percentage of cells undergoing mitosis. PMID:26512919

  9. 26 CFR 301.6223(e)-2 - Elections if Internal Revenue Service fails to provide timely notice.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... years beginning prior to October 4, 2001, see § 301.6223(e)-2T contained in 26 CFR part 1, revised April... provide timely notice. 301.6223(e)-2 Section 301.6223(e)-2 Internal Revenue INTERNAL REVENUE SERVICE... In General § 301.6223(e)-2 Elections if Internal Revenue Service fails to provide timely notice....

  10. The RB/E2F pathway and regulation of RNA processing

    SciTech Connect

    Ahlander, Joseph; Bosco, Giovanni

    2009-07-03

    The retinoblastoma tumor suppressor protein (RB) is inactivated in a majority of cancers. RB restricts cell proliferation by inhibiting the E2F family of transcription factors. The current model for RB/E2F function describes its role in regulating transcription at gene promoters. Whether the RB or E2F proteins might play a role in gene expression beyond transcription initiation is not well known. This review describes evidence that points to a novel role for the RB/E2F network in the regulation of RNA processing, and we propose a model as a framework for future research. The elucidation of a novel role of RB in RNA processing will have a profound impact on our understanding of the role of this tumor suppressor family in cell and developmental biology.

  11. Identification of New Functional Regions in Hepatitis C Virus Envelope Glycoprotein E2?

    PubMed Central

    Albecka, Anna; Montserret, Roland; Krey, Thomas; Tarr, Alexander W.; Diesis, Eric; Ball, Jonathan K.; Descamps, Vronique; Duverlie, Gilles; Rey, Felix; Penin, Franois; Dubuisson, Jean

    2011-01-01

    Little is known about the structure of the envelope glycoproteins of hepatitis C virus (HCV). To identify new regions essential for the function of these glycoproteins, we generated HCV pseudoparticles (HCVpp) containing HCV envelope glycoproteins, E1 and E2, from different genotypes in order to detect intergenotypic incompatibilities between these two proteins. Several genotype combinations were nonfunctional for HCV entry. Of interest, a combination of E1 from genotype 2a and E2 from genotype 1a was nonfunctional in the HCVpp system. We therefore used this nonfunctional complex and the recently described structural model of E2 to identify new functional regions in E2 by exchanging protein regions between these two genotypes. The functionality of these chimeric envelope proteins in the HCVpp system and/or the cell-cultured infectious virus (HCVcc) was analyzed. We showed that the intergenotypic variable region (IgVR), hypervariable region 2 (HVR2), and another segment in domain II play a role in E1E2 assembly. We also demonstrated intradomain interactions within domain I. Importantly, we also identified a segment (amino acids [aa] 705 to 715 [segment 705-715]) in the stem region of E2, which is essential for HCVcc entry. Circular dichroism and nuclear magnetic resonance structural analyses of the synthetic peptide E2-SC containing this segment revealed the presence of a central amphipathic helix, which likely folds upon membrane binding. Due to its location in the stem region, segment 705-715 is likely involved in the reorganization of the glycoprotein complexes taking place during the fusion process. In conclusion, our study highlights new functional and structural regions in HCV envelope glycoprotein E2. PMID:21147916

  12. Regression of Human Papillomavirus Intraepithelial Lesions Is Induced by MVA E2 Therapeutic Vaccine

    PubMed Central

    López-Contreras, Mario; Rosales, Carlos; Magallanes-Molina, Jose-Roberto; Gonzalez-Vergara, Roberto; Arroyo-Cazarez, Jose Martin; Ricardez-Arenas, Antonio; del Follo-Valencia, Armando; Padilla-Arriaga, Santiago; Guerrero, Miriam Veronica; Pirez, Miguel Angel; Arellano-Fiore, Claudia; Villarreal, Freddy

    2014-01-01

    Abstract Human papilloma viruses can induce warts, condylomas, and other intraepithelial cervical lesions that can progress to cancer. Cervical cancer is a serious problem in developing countries because early detection is difficult, and thus proper early treatment is many times missing. In this phase III clinical trial, we evaluated the potential use of MVA E2 recombinant vaccinia virus to treat intraepithelial lesions associated with papillomavirus infection. A total of 1176 female and 180 male patients with intraepithelial lesions were studied. They were injected with 107 MVA E2 virus particles directly into their uterus, urethra, vulva, or anus. Patients were monitored by colposcopy and cytology. Immune response was determined by measuring the antibody titer against MVA E2 virus and by analyzing the cytotoxic activity against cancer cells bearing papillomavirus DNA. Papillomavirus was determined by the Hybrid Capture method or by polymerase chain reaction analysis. By histology, 1051 (89.3%) female patients showed complete elimination of lesions after treatment with MVA E2. In 28 (2.4%) female patients, the lesion was reduced to CIN 1. Another 97 (8.3%) female patients presented isolated koilocytes after treatment. In men, all lesions were completely eliminated. All MVA E2–treated patients developed antibodies against the MVA E2 vaccine and generated a specific cytotoxic response against papilloma-transformed cells. Papillomavirus DNA was not detected after treatment in 83% of total patients treated. MVA E2 did not generate any apparent side effects. These data suggest that therapeutic vaccination with MVA E2 vaccine is an excellent candidate to stimulate the immune system and generate regression in intraepithelial lesions when applied locally. PMID:25275724

  13. Increased metastasis with loss of E2F2 in Myc-driven tumors

    PubMed Central

    Yuwanita, Inez; Barnes, Danielle; Monterey, Michael D.; O'Reilly, Sandra; Andrechek, Eran R.

    2015-01-01

    In human breast cancer, mortality is associated with metastasis to distant sites. Therefore, it is critical to elucidate the biological mechanisms that underlie tumor progression and metastasis. Using signaling pathway signatures we previously predicted a role for E2F transcription factors in Myc induced tumors. To test this role we interbred MMTV-Myc transgenic mice with E2F knockouts. Surprisingly, we observed that the loss of E2F2 sharply increased the percentage of lung metastasis in MMTV-Myc transgenic mice. Examining the gene expression profile from these tumors, we identified genetic components that were potentially involved in mediating metastasis. These genes were filtered to uncover the genes involved in metastasis that also impacted distant metastasis free survival in human breast cancer. In order to elucidate the mechanism by which E2F2 loss enhanced metastasis we generated knockdowns of E2F2 in MDA-MB-231 cells and observed increased migration in vitro and increased lung colonization in vivo. We then examined genes that were differentially regulated between tumors from MMTV-Myc, MMTV-Myc E2F2−/−, and lung metastases samples and identified PTPRD. To test the role of PTPRD in E2F2-mediated breast cancer metastasis, we generated a knockdown of PTPRD in MDA-MB-231 cells. We noted that decreased levels of PTPRD resulted in decreased migration in vitro and decreased lung colonization in vivo. Taken together, these data indicate that E2F2 loss results in increased metastasis in breast cancer, potentially functioning through a PTPRD dependent mechanism. PMID:26474282

  14. Transcriptional control of stem cell fate by E2Fs and pocket proteins

    PubMed Central

    Julian, Lisa M.; Blais, Alexandre

    2015-01-01

    E2F transcription factors and their regulatory partners, the pocket proteins (PPs), have emerged as essential regulators of stem cell fate control in a number of lineages. In mammals, this role extends from both pluripotent stem cells to those encompassing all embryonic germ layers, as well as extra-embryonic lineages. E2F/PP-mediated regulation of stem cell decisions is highly evolutionarily conserved, and is likely a pivotal biological mechanism underlying stem cell homeostasis. This has immense implications for organismal development, tissue maintenance, and regeneration. In this article, we discuss the roles of E2F factors and PPs in stem cell populations, focusing on mammalian systems. We discuss emerging findings that position the E2F and PP families as widespread and dynamic epigenetic regulators of cell fate decisions. Additionally, we focus on the ever expanding landscape of E2F/PP target genes, and explore the possibility that E2Fs are not simply regulators of general ‘multi-purpose’ cell fate genes but can execute tissue- and cell type-specific gene regulatory programs. PMID:25972892

  15. miR-98 delays skeletal muscle differentiation by down-regulating E2F5.

    PubMed

    Kropp, Jeremie; Degerny, Cindy; Morozova, Nadezda; Pontis, Julien; Harel-Bellan, Annick; Polesskaya, Anna

    2015-02-15

    A genome-wide screen had previously shown that knocking down miR-98 and let-7g, two miRNAs of the let-7 family, leads to a dramatic increase in terminal myogenic differentiation. In the present paper, we report that a transcriptomic analysis of human myoblasts, where miR-98 was knocked down, revealed that approximately 240 genes were sensitive to miR-98 depletion. Among these potential targets of miR-98, we identified the transcriptional repressor E2F5 and showed that it is a direct target of miR-98. Knocking down simultaneously E2F5 and miR-98 almost fully restored normal differentiation, indicating that E2F5 is involved in the regulation of skeletal muscle differentiation. We subsequently show that E2F5 can bind to the promoters of two inhibitors of terminal muscle differentiation, ID1 (inhibitor of DNA binding 1) and HMOX1 (heme oxygenase 1), which decreases their expression in skeletal myoblasts. We conclude that miR-98 regulates muscle differentiation by altering the expression of the transcription factor E2F5 and, in turn, of multiple E2F5 targets. PMID:25422988

  16. Temperature Dependent E2 Raman Modes in the ZnCoO Ternary Alloy

    NASA Technical Reports Server (NTRS)

    Samanta, K.; Bhattacharya, P.; Katiyar, R. S.

    2007-01-01

    The anharmonic properties of low and high frequency E2 modes of ZnO and Co doped ZnO were investigated using Raman scattering spectroscopy. We have determined the behavior of frequency, linewidths, and lifetime of E2 modes in the temperature range from 80 to 800 K. In the case of E2(high) mode the frequency shift towards the lower energy side was analyzed in light of the theory of anharmonic phonon-phonon interaction and thermal expansion of the lattice, and the linewidth behavior was analyzed in terms of anharmonic effect of three-phonon decay mechanism. But in the case of E2(low), the linewidth and frequency behaved practically harmonic with respect to temperature and independent of Co substitutions. It is found that the E2(high) phonon anharmonicity is higher for ZnCoO alloys than in pure ZnO and it increases with the compositional disorder. The low temperature lifetime of E2 phonon in ZnO, 1 % and 3% Co doped ZnO were found to be 1.S2, 1.74, and 1.54 ps, respectively.

  17. Physical and functional interactions of human papillomavirus E2 protein with nuclear receptor coactivators

    SciTech Connect

    Wu, M.-H.; Huang, C.-J.; Liu, S.-T.; Liu, P.-Y.; Ho, C.-L. . E-mail: shihming@ndmctsgh.edu.tw

    2007-05-11

    In addition to the human papillomavirus (HPV)-induced immortalization of epithelial cells, which usually requires integration of the viral DNA into the host cell genome, steroid hormone-activated nuclear receptors (NRs) are thought to bind to specific DNA sequences within transcriptional regulatory regions on the long control region to either increase or suppress transcription of dependent genes. In this study, our data suggest that the NR coactivator function of HPV E2 proteins might be mediated through physical and functional interactions with not only NRs but also the NR coactivators GRIP1 (glucocorticoid receptor-interacting protein 1) and Zac1 (zinc-finger protein which regulates apoptosis and cell cycle arrest 1), reciprocally regulating their transactivation activities. GRIP1 and Zac1 both were able to act synergistically with HPV E2 proteins on the E2-, androgen receptor-, and estrogen receptor-dependent transcriptional activation systems. GRIP1 and Zac1 might selectively function with HPV E2 proteins on thyroid receptor- and p53-dependent transcriptional activation, respectively. Hence, the transcriptional function of E2 might be mediated through NRs and NR coactivators to regulate E2-, NR-, and p53-dependent transcriptional activations.

  18. Cyclin D1 and cyclin E2 are differentially expressed in gastric cancer.

    PubMed

    Kumari, Soni; Puneet; Prasad, Shyam Babu; Yadav, Suresh Singh; Kumar, Mohan; Khanna, A; Dixit, V K; Nath, Gopal; Singh, Sunita; Narayan, Gopeshwar

    2016-05-01

    Cell cycle regulators cyclin D1 and cyclin E2 function in G1/S transition by activating downstream cyclin-dependent kinases. Deregulated expression of these cyclins has been reported in various cancers. However, little is known about their clinical significance in gastric carcinoma. We aimed to explore that whether there is differential expression of these cyclins in clinically distinct gastric cancer patients. In this study we recruited a total of 92 subjects including 20 controls and 72 cases of histopathologically proven gastric carcinoma. Expression profiling at transcript level was done by semiquantitative RT-PCR and of protein by immunohistochemistry. Receiver operator characteristics analysis was done for determining diagnostic utility of cyclin D1 and cyclin E2. We demonstrate that cyclins D1 and E2 are frequently overexpressed in early stages of gastric carcinoma. Interestingly, expression of cyclins D1 and E2 significantly correlates with different clinical parameters such as gender, histological type (intestinal and diffuse), tumor location (proximal, middle, and distal), tumor differentiation (differentiated and undifferentiated), tumor invasion (serosal, lymphatic, and venous) and tumor metastasis (lymph node, peritoneal, ascites, and liver). Cyclin D1 has significantly higher sensitivity and specificity as diagnostic biomarker than cyclin E2. Our results suggest that overexpression of cyclin D1 and cyclin E2 is an early event in gastric carcinogenesis. The differential expression of these cyclins may be useful as diagnostic biomarkers for early detection of gastric carcinoma. PMID:27034264

  19. Analysis of the papillomavirus E2 and bromodomain protein Brd4 interaction using bimolecular fluorescence complementation.

    PubMed

    Helfer, Christine M; Wang, Ranran; You, Jianxin

    2013-01-01

    The human papillomavirus (HPV) vaccines effectively protect against new infections of up to four HPV subtypes. However, these vaccines are not protective against many other clinically relevant HPV subtypes and are ineffective at treating established HPV infections. There is therefore a significant need for antiviral treatments for persistent HPV infections. A promising anti-HPV drug target is the interaction between the HPV E2 protein and cellular bromodomain-containing protein 4 (Brd4) since this protein complex mediates several processes important for the viral life cycle including viral genome maintenance, replication, and transcription. Using bimolecular fluorescence complementation (BiFC) technology, we demonstrate the E2 and Brd4 interaction on both interphase chromatin and mitotic chromosomes throughout mitosis. The E2-Brd4 BiFC was significantly diminished by mutating the Brd4 binding sites in E2 or by a dominant negative inhibitor of the E2-Brd4 interaction, demonstrating the potential of BiFC for identifying inhibitors of this important virus-host interaction. Importantly, when Brd4 was released from chromatin using the bromodomain inhibitor JQ1(+), the E2-Brd4 interacting complex relocated into foci that no longer associate with mitotic chromosomes, pointing to JQ1(+) as a promising antiviral inhibitor of HPV genome maintenance during HPV persistent infection. PMID:24205059

  20. Formation mechanism of superconducting phase and its three-dimensional architecture in pseudo-single-crystal KxF e2 -yS e2

    NASA Astrophysics Data System (ADS)

    Liu, Yong; Xing, Qingfeng; Straszheim, Warren E.; Marshman, Jeff; Pedersen, Pal; McLaughlin, Richard; Lograsso, Thomas A.

    2016-02-01

    We report how the superconducting phase forms in pseudo-single-crystal KxF e2 -yS e2 . In situ scanning electron microscopy (SEM) observation reveals that, as an order-disorder transition occurs, on cooling, most of the high-temperature iron-vacancy-disordered phase gradually changes into the iron-vacancy-ordered phase, whereas a small quantity of the high-temperature phase retains its structure and aggregates to the stripes with more iron concentration but less potassium concentration compared to the iron-vacancy-ordered phase. The stripes that are generally recognized as the superconducting phase are actually formed as a remnant of the high-temperature phase with a compositional change after an "imperfect" order-disorder transition. It should be emphasized that the phase separation in pseudo-single-crystal KxF e2 -yS e2 is caused by the iron-vacancy order-disorder transition. The shrinkage of the high-temperature phase and the expansion of the newly created iron-vacancy-ordered phase during the phase separation rule out the mechanism of spinodal decomposition proposed in an early report [Z. Wang et al., Phys. Rev. B 91, 064513 (2015), 10.1103/PhysRevB.91.064513]. Since the formation of the superconducting phase relies on the occurrence of the iron-vacancy order-disorder transition, it is impossible to synthesize a pure superconducting phase by a conventional solid state reaction or melt growth. By focused ion beam scanning electron microscopy, we further demonstrate that the superconducting phase forms a contiguous three-dimensional architecture composed of parallelepipeds that have a coherent orientation relationship with the iron-vacancy-ordered phase.

  1. UHMWPE carrying estradiol to treat the particle-induced osteolysis-Processing and characterizing.

    PubMed

    Liu, Aiqin; Qu, Shuxin; Chao, Mengmeng; Zhu, Minhao; Weng, Jie; Zhou, Zhongrong

    2009-08-01

    The objective of this study was to explore the possibility of UHMWPE implant used as the drug carrier to treat particle-induced osteolysis. 17beta-estradiol (E2), which had the potential application on osteolysis treatment and the high melting point, was added into UHMWPE powder to produce UHMWPE-E2 composites through hot press processing. The hydrophobicity, crystallinity, mechanical properties, and wear performance of the UHMWPE-E2 were characterized compared with the control UHMWPE. The thermal analysis and Fourier Transform Infrared Spectroscopy results demonstrated that the hot press processing would not alter the functional groups of E2 in this study. There were no significant differences in the hydrophobicity and crystallinity between the UHMWPE-E2 and UHMWPE. The UHMWPE-E2 showed satisfying mechanical properties, including ultimate tensile strength (47.2 +/- 3.6 MPa), yield strength (25.0 +/- 0.6 MPa) and elongation at break (320 +/- 25.5 %), which were similar with the control UHMWPE. The friction coefficients and worn scars were similar between the UHMWPE-E2 and the control UHMWPE. The wear mechanism of the UHMWPE-E2 and UHMWPE both were abrasive wear under dry friction. The UHMWPE-E2 possesses the approving mechanical properties and wear performance compared with the control UHMWPE, which might be used as the potential implanted drug carrier to prevent the particle-induced osteolysis in joint replacements. PMID:18563828

  2. Tissue-specific targeting of cell fate regulatory genes by E2f factors.

    PubMed

    Julian, L M; Liu, Y; Pakenham, C A; Dugal-Tessier, D; Ruzhynsky, V; Bae, S; Tsai, S-Y; Leone, G; Slack, R S; Blais, A

    2016-04-01

    Cell cycle proteins are important regulators of diverse cell fate decisions, and in this capacity have pivotal roles in neurogenesis and brain development. The mechanisms by which cell cycle regulation is integrated with cell fate control in the brain and other tissues are poorly understood, and an outstanding question is whether the cell cycle machinery regulates fate decisions directly or instead as a secondary consequence of proliferative control. Identification of the genes targeted by E2 promoter binding factor (E2f) transcription factors, effectors of the pRb/E2f cell cycle pathway, will provide essential insights into these mechanisms. We identified the promoter regions bound by three neurogenic E2f factors in neural precursor cells in a genome-wide manner. Through bioinformatic analyses and integration of published genomic data sets we uncovered hundreds of transcriptionally active E2f-bound promoters corresponding to genes that control cell fate processes, including key transcriptional regulators and members of the Notch, fibroblast growth factor, Wnt and Tgf-β signaling pathways. We also demonstrate a striking enrichment of the CCCTC binding factor transcription factor (Ctcf) at E2f3-bound nervous system-related genes, suggesting a potential regulatory co-factor for E2f3 in controlling differentiation. Finally, we provide the first demonstration of extensive tissue specificity among E2f target genes in mammalian cells, whereby E2f3 promoter binding is well conserved between neural and muscle precursors at genes associated with cell cycle processes, but is tissue-specific at differentiation-associated genes. Our findings implicate the cell cycle pathway as a widespread regulator of cell fate genes, and suggest that E2f3 proteins control cell type-specific differentiation programs by regulating unique sets of target genes. This work significantly enhances our understanding of how the cell cycle machinery impacts cell fate and differentiation, and will importantly drive further discovery regarding the mechanisms of cell fate control and transcriptional regulation in the brain, as well as in other tissues. PMID:25909886

  3. E2F-1-deficient NOD/SCID mice developed showing decreased saliva production.

    PubMed

    Matsui-Inohara, Hikaru; Uematsu, Hiroshi; Narita, Takanori; Satoh, Keitaro; Yonezawa, Hideo; Kuroda, Koichiro; Ito, Tatsuro; Yoneda, Saori; Kawarai, Taketo; Sugiya, Hiroshi; Watanabe, Haruo; Senpuku, Hidenobu

    2009-12-01

    The non-obese diabetic mouse (NOD) is the most characterized model used to study insulin-dependent type 1 diabetes mellitus (IDDM) and Sjogren's syndrome (SS). In a previous report, we found NOD.E2f1(-/-) mice show a greater progressive development to IDDM and SS compared to NOD mice. Our previous data indicated a progressive decrease in regulatory T cells (CD4(+)CD25(+)) and a decrease in the systemic secretion systems for insulin, and saliva was associated with the progression of IDDM and SS. Therefore, to define the mechanism of early-onset IDDM SS in E2F-1 deficient NOD mice required further investigation by producing E2F-1 deficient NOD/SCID mice in which the T and B cells do not develop. The purpose here was to analyze the essential function of the E2F-1 molecule in the development of IDDM and SS; and the dysfunction of the pancreas islet and salivary gland in the NOD background using NOD/SCID mice. We produced NOD/SCID.E2f1(-/-) mice using homologous recombination; determined diabetes development; measured saliva and insulin production; and performed a histological analysis. The deficient mice showed a decreasing volume of saliva; no infiltration of lymphocytes into salivary glands; no development of diabetes; and no protein localization of FGFR-2b in the ducts of the salivary gland that regulates submandibular gland proliferation and morphogenesis. Therefore, we considered a deficiency in E2F-1 induces a decrease in regulatory T cells and an increase in auto-reactive T cells; however, the E2F-1 deficiency is not associated with T and B cells-independent dysfunction of pancreatic beta cell in insulin secretion. Further, the E2F-1 deficiency is associated with T and B cells-independent dysfunction of the salivary gland exhibits a decrease in saliva production volume. We suggest E2F-1 may be also associated with the differentiation of exocrine cells in the duct where FGFR-2b is expressed in the salivary gland. The E2F-1 deficient NOD/SCID mouse model is useful for showing the development of the salivary gland; and is also useful for various experiments in humanized mice. PMID:19934373

  4. Inactivation of Rb and E2f8 Synergizes To Trigger Stressed DNA Replication during Erythroid Terminal Differentiation

    PubMed Central

    Ghazaryan, Seda; Sy, Chandler; Hu, Tinghui; An, Xiuli; Mohandas, Narla; Fu, Haiqing; Aladjem, Mirit I.; Chang, Victor T.; Opavsky, Rene

    2014-01-01

    Rb is critical for promoting cell cycle exit in cells undergoing terminal differentiation. Here we show that during erythroid terminal differentiation, Rb plays a previously unappreciated and unorthodox role in promoting DNA replication and cell cycle progression. Specifically, inactivation of Rb in erythroid cells led to stressed DNA replication, increased DNA damage, and impaired cell cycle progression, culminating in defective terminal differentiation and anemia. Importantly, all of these defects associated with Rb loss were exacerbated by the concomitant inactivation of E2f8. Gene expression profiling and chromatin immunoprecipitation (ChIP) revealed that Rb and E2F8 cosuppressed a large array of E2F target genes that are critical for DNA replication and cell cycle progression. Remarkably, inactivation of E2f2 rescued the erythropoietic defects resulting from Rb and E2f8 deficiencies. Interestingly, real-time quantitative PCR (qPCR) on E2F2 ChIPs indicated that inactivation of Rb and E2f8 synergizes to increase E2F2 binding to its target gene promoters. Taken together, we propose that Rb and E2F8 collaborate to promote DNA replication and erythroid terminal differentiation by preventing E2F2-mediated aberrant transcriptional activation through the ability of Rb to bind and sequester E2F2 and the ability of E2F8 to compete with E2F2 for E2f-binding sites on target gene promoters. PMID:24865965

  5. Structure-function analysis of hepatitis C virus envelope glycoproteins E1 and E2.

    PubMed

    Nayak, Aparajita; Pattabiraman, Nagarajan; Fadra, Numrah; Goldman, Radoslav; Kosakovsky Pond, Sergei L; Mazumder, Raja

    2015-01-01

    Hepatitis C virus (HCV) is the leading cause of chronic liver disease in humans. The envelope proteins of HCV are potential candidates for vaccine development. The absence of three-dimensional (3D) structures for the functional domain of HCV envelope proteins [E1.E2] monomer complex has hindered overall understanding of the virus infection, and also structure-based drug design initiatives. In this study, we report a 3D model containing both E1 and E2 proteins of HCV using the recently published structure of the core domain of HCV E2 and the functional part of E1, and investigate immunogenic implications of the model. HCV [E1.E2] molecule is modeled by using aa205-319 of E1 to aa421-716 of E2. Published experimental data were used to further refine the [E1.E2] model. Based on the model, we predict 77 exposed residues and several antigenic sites within the [E1.E2] that could serve as vaccine epitopes. This study identifies eight peptides which have antigenic propensity and have two or more sequentially exposed amino acids and 12 singular sites are under negative selection pressure that can serve as vaccine or therapeutic targets. Our special interest is 285FLVGQLFTFSPRRHW299 which has five negatively selected sites (L286, V287, G288, T292, and G303) with three of them sequential and four amino acids exposed (F285, L286, T292, and R296). This peptide in the E1 protein maps to dengue envelope vaccine target identified previously by our group. Our model provides for the first time an overall view of both the HCV envelope proteins thereby allowing researchers explore structure-based drug design approaches. PMID:25245635

  6. Glucocorticoid regulation of branched-chain alpha-ketoacid dehydrogenase E2 subunit gene expression.

    PubMed Central

    Costeas, P A; Chinsky, J M

    2000-01-01

    Regulation of the mammalian branched-chain alpha-ketoacid dehydrogenase complex (BCKAD) occurs under a variety of stressful conditions associated with changes in circulating glucocorticoids. Multiple levels of regulation in hepatocytes, including alteration of the levels of the structural subunits available for assembly (E1, alpha-ketoacid decarboxylase; E2, dihydrolipoamide acyltransferase; and E3, dihydrolipoamide dehydrogenase), as well as BCKAD kinase, which serves to phosphorylate the E1alpha subunit and inactivate complex activity, have been proposed. The direct role of glucocorticoids in regulating the expression of the murine gene encoding the major BCKAD subunit E2, upon which the other BCKAD subunits assemble, was therefore examined. Deletion analysis of the 5' proximal 7.0 kb of the murine E2 promoter sequence, using E2 promoter/luciferase expression minigene plasmids introduced into the hepatic H4IIEC3 cell line, suggested a promoter proximal region responsive to glucocorticoid regulation. Linker-scanning mutagenesis combined with deletion analysis established this functional glucocorticoid-responsive unit (GRU) to be located near the murine E2 proximal promoter site at -140 to -70 bp upstream from the transcription initiation site. The presence of this region in plasmid minigenes, containing varying amounts of the murine genomic sequence 5' upstream from proximal E2 promoter sequences, conferred 2-10 fold increases in luciferase reporter gene expression in H4IIEC3 cells, whether introduced by transient transfection or following co-selection for stable transfectants. The GRU region itself appeared to contain multiple interacting elements that combine to regulate overall E2 promoter activity in response to changing physiological conditions associated with varying concentrations of glucocorticoids and likely other hormonal effectors. PMID:10749674

  7. Expression of E2F-4 in invasive breast carcinomas is associated with poor prognosis.

    PubMed

    Rakha, Emad A; Pinder, Sarah E; Paish, E Claire; Robertson, John F; Ellis, Ian O

    2004-07-01

    The E2F family of transcription factors plays a key role in the control of cellular proliferation and apoptosis. Some family members act as oncogenes and others act as tumour suppressor genes (TSGs), behaviour which appears to be tissue-specific. E2F-4 is a member of the E2F family, located at chromosome band 16q22.1, that shows frequent deletion in breast cancer, suggesting that it may function as a TSG in breast carcinogenesis. In the present study, the expression of E2F-4 was assessed immunohistochemically on paraffin wax sections from 265 breast carcinomas and expression was compared with both clinicopathological variables and disease outcome in an attempt to identify its possible role as a TSG and to assess its prognostic value, if any, in breast cancer. E2F-4 protein expression was detected in the nuclei and in the cytoplasm of normal and malignant breast epithelial cells. In the malignant tissues, no significant loss or decrease of expression was seen in association with any specific tumour type. There was a correlation between increased nuclear expression of E2F-4 and indicators of poor prognosis including larger tumour size (p = 0.000), grade 3 lesions (p = 0.033), lymph node stage (p = 0.037), and poorer Nottingham prognostic index group (p = 0.003). Increased immunoreactivity was also seen in association with the development of recurrent disease (p = 0.004), distant metastasis (p = 0.001), and poorer outcome including poorer overall survival time (p = 0.002) and shorter disease-free interval (p = 0.001). In multivariate analysis, E2F-4 was of independent prognostic significance along with grade and lymph node stage. These results suggest that E2F-4 may play a role in breast cancer progression and that increased nuclear expression is associated with more advanced tumours with poor outcomes. E2F-4 appears to have an oncogenic role rather than a tumour suppressor role in breast carcinogenesis and, hence, it is not the gene targeted by 16q22.1 loss in breast carcinoma. PMID:15221934

  8. Detergent-Resistant Membrane Association of NS2 and E2 during Hepatitis C Virus Replication

    PubMed Central

    Shanmugam, Saravanabalaji; Saravanabalaji, Dhanaranjani

    2015-01-01

    ABSTRACT Previously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-?-cyclodextrin (M?CD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by M?CD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production. IMPORTANCE The biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these membranes. We then showed that NS2 regulates E2 localization to the DRM, consistent with its role in recruiting E2 to the virus assembly sites. We also showed that short-term treatment with the cholesterol-extracting agent methyl-?-cyclodextrin (M?CD) not only disrupted the DRM localization of Core, NS2, and E2 but also specifically inhibited intracellular virus assembly without affecting HCV RNA replication. Thus, our data support the role of the DRM as a platform for particle assembly process. PMID:25673706

  9. ModelE2-TOMAS development and evaluation using aerosol optical depths, mass and number concentrations

    NASA Astrophysics Data System (ADS)

    Lee, Y. H.; Adams, P. J.; Shindell, D. T.

    2014-09-01

    The TwO-Moment Aerosol Sectional microphysics model (TOMAS) has been integrated into the state-of-the-art general circulation model, GISS ModelE2. TOMAS has the flexibility to select a size resolution as well as the lower size cutoff. A computationally efficient version of TOMAS is used here, which has 15 size bins covering 3 nm to 10 μm aerosol dry diameter. For each bin, it simulates the total aerosol number concentration and mass concentrations of sulphate, pure elementary carbon (hydrophobic), mixed elemental carbon (hydrophilic), hydrophobic organic matter, hydrophilic organic matter, sea salt, mineral dust, ammonium, and aerosol-associated water. This paper provides a detailed description of the ModelE2-TOMAS model and evaluates the model against various observations including aerosol precursor gas concentrations, aerosol mass and number concentrations, and aerosol optical depths. Additionally, global budgets in ModelE2-TOMAS are compared with those of other global aerosol models, and the TOMAS model is compared to the default aerosol model in ModelE2, which is a bulk aerosol model. Overall, the ModelE2-TOMAS predictions are within the range of other global aerosol model predictions, and the model has a reasonable agreement with observations of sulphur species and other aerosol components as well as aerosol optical depth. However, ModelE2-TOMAS (as well as the bulk aerosol model) cannot capture the observed vertical distribution of sulphur dioxide over the Pacific Ocean possibly due to overly strong convective transport. The TOMAS model successfully captures observed aerosol number concentrations and cloud condensation nuclei concentrations. Anthropogenic aerosol burdens in the bulk aerosol model running in the same host model as TOMAS (ModelE2) differ by a few percent to a factor of 2 regionally, mainly due to differences in aerosol processes including deposition, cloud processing, and emission parameterizations. Larger differences are found for naturally emitted aerosols such as sea salt and mineral dust. With TOMAS, ModelE2 has three different aerosol models (the bulk aerosol model and modal-based aerosol microphysics model, MATRIX) and allows exploration of the uncertainties associated with aerosol modelling within the same host model, NASA GISS ModelE2.

  10. Prolactin and aging: X-irradiated and estrogen-induced rat mammary tumorigenesis

    SciTech Connect

    Ito, A.; Naito, M.; Watanabe, H.; Yokoro, K.

    1984-07-01

    Both sexes of inbred WF rats at either 8 or 28-60 weeks of age were exposed to 200 rad whole-body radiation, 2.5 or 5.0 mg 17 beta-estradiol (E2), or both agents The female rats treated with E2 alone or with both X-rays and E2 at 8 weeks of age showed a high incidence of mammary carcinomas (MCA), a large increase in pituitary weight, and a rise in serum prolactin (PRL) levels. However, the same treatments to males did not induce MCA despite a moderate increase in both pituitary weight and serum PRL. Ovariectomy prior to E2 treatment failed to modify the occurrence of MCA or pituitary tumors. When X-rays and E2 were given to female rats at 28-60 weeks of age, pituitary weight, serum PRL levels, and the incidence of MCA were unaffected. When the E2 pellet was kept for the first 24 weeks and withdrawn during the last 12 weeks, the incidence of MCA, pituitary weight, and serum PRL was low. It was concluded that: 1) the pituitary glands of young female rats were susceptible to E2 treatment but were insensitive in older females, and 2) the occurrence of MCA in female rats appeared to be promoted by elevated PRL levels secreted by E2-induced pituitary tumors. Mammary tissue of male rats was less sensitive to PRL levels in the development of MCA.

  11. Role of E2-Ub-conjugating enzymes during skeletal muscle atrophy

    PubMed Central

    Polge, Cecile; Attaix, Didier; Taillandier, Daniel

    2015-01-01

    The Ubiquitin Proteasome System (UPS) is a major actor of muscle wasting during various physio-pathological situations. In the past 15 years, increasing amounts of data have depicted a picture, although incomplete, of the mechanisms implicated in myofibrillar protein degradation, from the discovery of muscle-specific E3 ligases to the identification of the signaling pathways involved. The targeting specificity of the UPS relies on the capacity of the system to first recognize and then label the proteins to be degraded with a poly-ubiquitin (Ub) chain. It is fairly assumed that the recognition of the substrate is accomplished by the numerous E3 ligases present in mammalian cells. However, most E3s do not possess any catalytic activity and E2 enzymes may be more than simple Ub-providers for E3s since they are probably important actors in the ubiquitination machinery. Surprisingly, most authors have tried to characterize E3 substrates, but the exact role of E2s in muscle protein degradation is largely unknown. A very limited number of the 35 E2s described in humans have been studied in muscle protein breakdown experiments and the vast majority of studies were only descriptive. We review here the role of E2 enzymes in skeletal muscle and the difficulties linked to their study and provide future directions for the identification of muscle E2s responsible for the ubiquitination of contractile proteins. PMID:25805999

  12. E2-c-Cbl recognition is necessary but not sufficient for ubiquitination activity.

    PubMed

    Huang, Anding; de Jong, Rob N; Wienk, Hans; Winkler, G Sebastiaan; Timmers, H Th Marc; Boelens, Rolf

    2009-01-16

    The E2 ubiquitin-conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING (really interesting new gene) domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. Here, we found that neither structural changes nor subtle differences in the E2-E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5B-Ub thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7-Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required but not sufficient for transfer of ubiquitin to potential targets. PMID:18996392

  13. Redeployment of Myc and E2f1-3 drives Rb deficient cell cycles

    PubMed Central

    Liu, Huayang; Tang, Xing; Srivastava, Arunima; Pécot, Thierry; Daniel, Piotr; Hemmelgarn, Benjamin; Reyes, Stephan; Fackler, Nicholas; Bajwa, Amneet; Kladney, Raleigh; Koivisto, Christopher; Chen, Zhong; Wang, Qianben; Huang, Kun; Machiraju, Raghu; Sáenz-Robles, Maria Teresa; Cantalupo, Paul; Pipas, James M.; Leone, Gustavo

    2015-01-01

    Robust mechanisms to control cell proliferation have evolved to maintain the integrity of organ architecture. Here, we investigated how two critical proliferative pathways, Myc and E2f, are integrated to control cell cycles in normal and Rb deficient cells using a murine intestinal model. We show that Myc and E2f1-3 have little impact on normal G1-S transitions. Instead, they synergistically control an S-G2 transcriptional program required for normal cell divisions and maintaining crypt-villus integrity. Surprisingly, Rb deficiency results in the Myc-dependent accumulation of E2f3 protein and chromatin repositioning of both Myc and E2f3, leading to the ‘super activation’ of a G1-S transcriptional program, ectopic S phase entry and rampant cell proliferation. These findings reveal that Rb deficient cells hijack and redeploy Myc and E2f3 from an S-G2 program essential for normal cell cycles to a G1-S program that re-engages ectopic cell cycles, exposing an unanticipated addiction of Rb-null cells on Myc. PMID:26192440

  14. Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

    SciTech Connect

    Kasim, Vivi; Huang, Can; Zhang, Jing; Jia, Huizhen; Wang, Yunxia; Yang, Li; Miyagishi, Makoto; Wu, Shourong

    2014-07-04

    Highlights: • MDM2 is a novel positive regulator of TAp73 transcriptional activity. • MDM2 colocalizes together and physically interacts with E2F1. • Synergistic cooperation of MDM2 and E2F1 is crucial for TAp73 transcription. • MDM2 regulates TAp73 transcriptional activity in a p53-independent manner. - Abstract: TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. We further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73.

  15. Transcriptome regulation and chromatin occupancy by E2F3 and MYC in mice

    PubMed Central

    Tang, Xing; Liu, Huayang; Srivastava, Arunima; Pécot, Thierry; Chen, Zhong; Wang, Qianben; Huang, Kun; Sáenz-Robles, Maria Teresa; Cantalupo, Paul; Pipas, James; Leone, Gustavo

    2016-01-01

    E2F3 and MYC are transcription factors that control cellular proliferation. To study their mechanism of action in the context of a regenerating tissue, we isolated both proliferating (crypts) and non-dividing (villi) cells from wild-type and Rb depleted small intestines of mice and performed ChIP-exo-seq (chromatin immunoprecipitation combined with lambda exonuclease digestion followed by high-throughput sequencing). The genome-wide chromatin occupancy of E2F3 and MYC was determined by mapping sequence reads to the genome and predicting preferred binding sites (peaks). Binding sites could be accurately identified within small regions of only 24 bp-28 bp long, highlighting the precision to which binding peaks can be identified by ChIP-exo-seq. Forty randomly selected E2F3- and MYC-specific binding sites were validated by ChIP-PCR. In addition, we also presented gene expression data sets from wild type, Rb-, E2f3- and Myc-depleted crypts and villi within this manuscript. These represent comprehensive and validated datasets that can be integrated to identify putative direct targets of E2F3 and MYC involved in the control of cellular proliferation in normal and Rb-deficient small intestines. PMID:26881867

  16. Transcriptome regulation and chromatin occupancy by E2F3 and MYC in mice.

    PubMed

    Tang, Xing; Liu, Huayang; Srivastava, Arunima; Pécot, Thierry; Chen, Zhong; Wang, Qianben; Huang, Kun; Sáenz-Robles, Maria Teresa; Cantalupo, Paul; Pipas, James; Leone, Gustavo

    2016-01-01

    E2F3 and MYC are transcription factors that control cellular proliferation. To study their mechanism of action in the context of a regenerating tissue, we isolated both proliferating (crypts) and non-dividing (villi) cells from wild-type and Rb depleted small intestines of mice and performed ChIP-exo-seq (chromatin immunoprecipitation combined with lambda exonuclease digestion followed by high-throughput sequencing). The genome-wide chromatin occupancy of E2F3 and MYC was determined by mapping sequence reads to the genome and predicting preferred binding sites (peaks). Binding sites could be accurately identified within small regions of only 24 bp-28 bp long, highlighting the precision to which binding peaks can be identified by ChIP-exo-seq. Forty randomly selected E2F3- and MYC-specific binding sites were validated by ChIP-PCR. In addition, we also presented gene expression data sets from wild type, Rb-, E2f3- and Myc-depleted crypts and villi within this manuscript. These represent comprehensive and validated datasets that can be integrated to identify putative direct targets of E2F3 and MYC involved in the control of cellular proliferation in normal and Rb-deficient small intestines. PMID:26881867

  17. Relationship between vitamin D, IFN-?, and E2 levels in systemic lupus erythematosus.

    PubMed

    Kokic, V; Martinovic Kaliterna, D; Radic, M; Perkovic, D; Cvek, M; Capkun, V

    2016-03-01

    In this study, we investigated the relationship between vitamin D, interferon-gamma (IFN-?), and estradiol (E2) in females of childbearing age with inactive systemic lupus erythematosus (SLE). The study included 22 SLE patients, and 21 age- and gender-matched healthy individuals. Serum concentrations of 25-hydroxyvitamin D3 (25(OH)D3), E2, and IFN-? were measured by radioimmunoassay using the gamma-counter and ELISA. Patients and control subjects were divided into two groups based on their vitamin D levels (25(OH)D3???20?ng/mL; 25(OH)D3?>?20?ng/mL). The median values of IFN-? and E2 were higher in SLE patients compared to the controls, irrespective of vitamin D level (p?=?0.001, p?=?0.009, p?=?0.003, and p?=?0.003, respectively). In SLE patients, there was a negative correlation between IFN-? and 25(OH)D3 (rs?=?-0.330; p?=?0.03) and a positive correlation between IFN-? and E2 (rs?=?0.404; p?=?0.007). This study demonstrates an interesting interplay between vitamin D, INF-?, and E2 in SLE patients with inactive disease. PMID:26405019

  18. Identification of E2F1 as a positive transcriptional regulator for {delta}-catenin

    SciTech Connect

    Kim, Kwonseop; Oh, Minsoo; Ki, Hyunkyoung; Wang Tao; Bareiss, Sonja; Fini, M. Elizabeth.; Li Dawei; Lu Qun

    2008-05-02

    {delta}-Catenin is upregulated in human carcinomas. However, little is known about the potential transcriptional factors that regulate {delta}-catenin expression in cancer. Using a human {delta}-catenin reporter system, we have screened several nuclear signaling modulators to test whether they can affect {delta}-catenin transcription. Among {beta}-catenin/LEF-1, Notch1, and E2F1, E2F1 dramatically increased {delta}-catenin-luciferase activities while {beta}-catenin/LEF-1 induced only a marginal increase. Rb suppressed the upregulation of {delta}-catenin-luciferase activities induced by E2F1 but did not interact with {delta}-catenin. RT-PCR and Western blot analyses in 4 different prostate cancer cell lines revealed that regulation of {delta}-catenin expression is controlled mainly at the transcriptional level. Interestingly, the effects of E2F1 on {delta}-catenin expression were observed only in human cancer cells expressing abundant endogenous {delta}-catenin. These studies identify E2F1 as a positive transcriptional regulator for {delta}-catenin, but further suggest the presence of strong negative regulator(s) for {delta}-catenin in prostate cancer cells with minimal endogenous {delta}-catenin expression.

  19. Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine

    PubMed Central

    Das, Ranabir; Liang, Yu-He; Mariano, Jennifer; Li, Jess; Huang, Tao; King, Aaren; Tarasov, Sergey G; Weissman, Allan M; Ji, Xinhua; Byrd, R Andrew

    2013-01-01

    RING finger proteins constitute the large majority of ubiquitin ligases (E3s) and function by interacting with ubiquitin-conjugating enzymes (E2s) charged with ubiquitin. How low-affinity RING–E2 interactions result in highly processive substrate ubiquitination is largely unknown. The RING E3, gp78, represents an excellent model to study this process. gp78 includes a high-affinity secondary binding region for its cognate E2, Ube2g2, the G2BR. The G2BR allosterically enhances RING:Ube2g2 binding and ubiquitination. Structural analysis of the RING:Ube2g2:G2BR complex reveals that a G2BR-induced conformational effect at the RING:Ube2g2 interface is necessary for enhanced binding of RING to Ube2g2 or Ube2g2 conjugated to Ub. This conformational effect and a key ternary interaction with conjugated ubiquitin are required for ubiquitin transfer. Moreover, RING:Ube2g2 binding induces a second allosteric effect, disrupting Ube2g2:G2BR contacts, decreasing affinity and facilitating E2 exchange. Thus, gp78 is a ubiquitination machine where multiple E2-binding sites coordinately facilitate processive ubiquitination. PMID:23942235

  20. E2f6 and Bmi1 cooperate in axial skeletal development

    PubMed Central

    Courel, Maria; Friesenhahn, Laurie; Lees, Jacqueline A.

    2009-01-01

    Bmi1 is a Polycomb Group protein that functions as a component of Polycomb Repressive Complex 1 (PRC1) to control axial skeleton development through Hox gene repression. Bmi1 also represses transcription of the Ink4a-Arf locus and is consequently required to maintain the proliferative and self-renewal properties of hematopoietic and neural stem cells. Previously, one E2F family member, E2F6, has been shown to interact with Bmi1 and other known PRC1 components. However, the biological relevance of this interaction is unknown. In this study, we use mouse models to investigate the interplay between E2F6 and Bmi1. This analysis shows that E2f6 and Bmi1 cooperate in the regulation of Hox genes, and consequently axial skeleton development, but not in the repression of the Ink4a-Arf locus. These findings underscore the significance of the E2F6-Bmi1 interaction in vivo and suggest that the Hox and Ink4a-Arf loci are regulated by somewhat different mechanisms. PMID:18366140

  1. Redeployment of Myc and E2f1-3 drives Rb-deficient cell cycles.

    PubMed

    Liu, Huayang; Tang, Xing; Srivastava, Arunima; Pcot, Thierry; Daniel, Piotr; Hemmelgarn, Benjamin; Reyes, Stephan; Fackler, Nicholas; Bajwa, Amneet; Kladney, Raleigh; Koivisto, Christopher; Chen, Zhong; Wang, Qianben; Huang, Kun; Machiraju, Raghu; Senz-Robles, Maria Teresa; Cantalupo, Paul; Pipas, James M; Leone, Gustavo

    2015-08-01

    Robust mechanisms to control cell proliferation have evolved to maintain the integrity of organ architecture. Here, we investigated how two critical proliferative pathways, Myc and E2f, are integrated to control cell cycles in normal and Rb-deficient cells using a murine intestinal model. We show that Myc and E2f1-3 have little impact on normal G1-S transitions. Instead, they synergistically control an S-G2 transcriptional program required for normal cell divisions and maintaining crypt-villus integrity. Surprisingly, Rb deficiency results in the Myc-dependent accumulation of E2f3 protein and chromatin repositioning of both Myc and E2f3, leading to the 'super activation' of a G1-S transcriptional program, ectopic S phase entry and rampant cell proliferation. These findings reveal that Rb-deficient cells hijack and redeploy Myc and E2f3 from an S-G2 program essential for normal cell cycles to a G1-S program that re-engages ectopic cell cycles, exposing an unanticipated addiction of Rb-null cells on Myc. PMID:26192440

  2. Identification of E2F1 as a positive transcriptional regulator for ?-catenin

    PubMed Central

    Kim, Kwonseop; Oh, Minsoo; Ki, Hyunkyoung; Wang, Tao; Bareiss, Sonja; Fini, M. Elizabeth; Li, Dawei; Lu, Qun

    2008-01-01

    ?-Catenin is upregulated in human carcinomas. However, little is known about the potential transcriptional factors that regulate ?-catenin expression in cancer. Using a human ?-catenin reporter system, we have screened several nuclear signaling modulators to test whether they can affect ?-catenin transcription. Among ?-catenin/LEF-1, Notch1, and E2F1, E2F1 dramatically increased ?-catenin-luciferase activities while ?-catenin/LEF-1 induced only a marginal increase. Rb suppressed the upregulation of ?-catenin-luciferase activities induced by E2F1 but did not interact with ?-catenin. RT-PCR and Western blot analyses in 4 different prostate cancer cell lines revealed that regulation of ?-catenin expression is controlled mainly at the transcriptional level. Interestingly, the effects of E2F1 on ?-catenin expression were observed only in human cancer cells expressing abundant endogenous ?-catenin. These studies identify E2F1 as a positive transcriptional regulator for ?-catenin, but further suggest the presence of strong negative regulator(s) for ?-catenin in prostate cancer cells with minimal endogenous ?-catenin expression. PMID:18302937

  3. HEB and E2A function as SMAD/FOXH1 cofactors

    PubMed Central

    Yoon, Se-Jin; Wills, Andrea E.; Chuong, Edward; Gupta, Rakhi; Baker, Julie C.

    2011-01-01

    Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. We identified a new motif, termed SMAD complex-associated (SCA), that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that two basic helix–loop–helix (bHLH) proteins—HEB and E2A—bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Furthermore, we show that HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E proteins are novel Nodal signaling cofactors that associate with SMAD2/3 and FOXH1 and are necessary for mesendoderm differentiation. PMID:21828274

  4. Homogeneous linewidth broadening and exciton dephasing mechanism in MoT e2

    NASA Astrophysics Data System (ADS)

    Koirala, Sandhaya; Mouri, Shinichiro; Miyauchi, Yuhei; Matsuda, Kazunari

    2016-02-01

    Spectroscopic studies of mechanically exfoliated monolayer MoT e2 were performed over a wide temperature range from 4.2 to 300 K. At a low temperature, the photoluminescence spectra for monolayer MoT e2 showed two sharp peaks for excitons and charged excitons (trions). The homogeneous linewidth of the exciton peak broadened linearly as the temperature increased. This linear linewidth broadening was caused by acoustic-phonon scattering of the exciton, i.e., shortening of exciton dephasing. The broadening factor due to exciton-acoustic-phonon interactions was found to be 0.11 meV K-1 . This small value for the exciton-phonon coupling coefficient and the lack of a Stokes shift suggest that exciton-phonon interactions in monolayer MoT e2 are in the weak coupling regime.

  5. Purification, crystallization and initial crystallographic characterization of brazil-nut allergen Ber e 2

    SciTech Connect

    Guo, Feng; Jin, Tengchuan; Howard, Andrew; Zhang, Yu-Zhu

    2007-11-01

    The crystallization of the brazil nut allergen Ber e 2 is reported. Peanut and tree-nut allergies have attracted considerable attention because of their frequency and their lifelong persistence. Brazil-nut (Bertholletia excelsa) allergies have been well documented and the 11S legumin-like seed storage protein Ber e 2 (excelsin) is one of the two known brazil-nut allergens. In this study, Ber e 2 was extracted from brazil-nut kernels and purified to high purity by crystalline precipitation and gel-filtration chromatography. Well diffracting single crystals were obtained using the hanging-drop vapour-diffusion method. A molecular-replacement structural solution has been obtained. Refinement of the structure is currently under way.

  6. Decreased brain infarct following focal ischemia in mice lacking the transcription factor E2F1.

    PubMed

    MacManus, J P; Koch, C J; Jian, M; Walker, T; Zurakowski, B

    1999-09-01

    E2F1+/- mice subjected to 2 h middle cerebral artery occlusion developed an infarct of 77.0 +/- 3.2 mm3 (mean +/- s.e.m., n = 15) in the ischemic hemisphere after 24 h reperfusion. A significantly smaller infarct of 58.8 +/- 4.8 mm3 (n = 15; p < 0.01) was found in E2F1-/- animals. Both deficient and normal mice had similar cerebral angioarchitecture and intra-ischemic decreases in regional blood flow. Similar areas of hypoxia in both groups of ischemic animals were demonstrated directly by immunohistochemical detection of nitroimidazole adducts. It was concluded that all animals received the same ischemic insult, yet the subsequent damage was different in the mutant mice. This is the first indication that the E2F1 gene plays a role in ischemic death of post-mitotic neurons. PMID:10511428

  7. The challenges of classical swine fever control: modified live and E2 subunit vaccines.

    PubMed

    Huang, Yu-Liang; Deng, Ming-Chung; Wang, Fun-In; Huang, Chin-Cheng; Chang, Chia-Yi

    2014-01-22

    Classical swine fever (CSF) is an economically important, highly contagious disease of swine worldwide. CSF is caused by classical swine fever virus (CSFV), and domestic pigs and wild boars are its only natural hosts. The two main strategies used to control CSF epidemic are systematic prophylactic vaccination and a non-vaccination stamping-out policy. This review compares the protective efficacy of the routinely used modified live vaccine (MLV) and E2 subunit vaccines and summarizes the factors that influence the efficacy of the vaccines and the challenges that both vaccines face to CSF control. Although MLV provide earlier and more complete protection than E2 subunit vaccines, it has the drawback of not allowing differentiation between infected and vaccinated animals (DIVA). The marker vaccine of E2 protein with companion discriminatory test to detect antibodies against E(rns) allows DIVA and is a promising strategy for future control and eradication of CSF. Maternal derived antibody (MDA) is the critical factor in impairing the efficacy of both MLV and E2 subunit vaccines, so the well-designed vaccination programs of sows and piglets should be considered together. Because of the antigen variation among various genotypes of CSFV, antibodies raised by either MLV or subunit vaccine neutralize genotypically homologous strains better than heterologous ones. However, although this is not a major concern for MLV as the induced immune responses can protect pigs against the challenge of various genotypes of CSFVs, it is critical for E2 subunit vaccines. It is thus necessary to evaluate whether the E2 subunit vaccine can completely protect against the current prevalent strains in the field. An ideal new generation of vaccine should be able to maintain the high protective efficiency of MLV and overcome the problem of antigenic variations while allowing for DIVA. PMID:24211665

  8. RNF126 promotes homologous recombination via regulation of E2F1-mediated BRCA1 expression.

    PubMed

    Wang, Y; Deng, O; Feng, Z; Du, Z; Xiong, X; Lai, J; Yang, X; Xu, M; Wang, H; Taylor, D; Yan, C; Chen, C; Difeo, A; Ma, Z; Zhang, J

    2016-03-17

    RNF126 is an E3 ubiquitin ligase. The deletion of RNF126 gene was observed in a wide range of human cancers and is correlated with improved disease-free and overall survival. These data highlight the clinical relevance of RNF126 in tumorigenesis and cancer therapy. However, the specific functions of RNF126 remain largely unknown. Homologous recombination (HR)-mediated DNA double-strand break repair is important for tumor suppression and cancer therapy resistance. Here, we demonstrate that RNF126 facilitates HR by promoting the expression of BRCA1, in a manner independent of its E3 ligase activity but depending on E2F1, a well-known transcription factor of BRCA1 promoter. In support of this result, RNF126 promotes transactivation of BRCA1 promoter by directly binding to E2F1. Most importantly, an RNF126 mutant lacking 11 amino acids that is responsible for the interaction with E2F1 has a dominant-negative effect on BRCA1 expression and HR by suppressing E2F1-mediated transactivation of BRCA1 promoter and blocking the enrichment of E2F1 on BRCA1 promoter. Lastly, RNF126 depletion leads to the increased sensitivity to ionizing radiation and poly (ADP-ribose) polymerase inhibition. Collectively, our results suggest a novel role of RNF126 in promoting HR-mediated repair through positive regulation on BRCA1 expression by direct interaction with E2F1. This study not only offers novel insights into our current understanding of the biological functions of RNF126 but also provides a potential therapeutic target for cancer treatment. PMID:26234677

  9. Comparative genomics reveals multistep pathogenesis of E2A-PBX1 acute lymphoblastic leukemia

    PubMed Central

    Duque-Afonso, Jesús; Feng, Jue; Scherer, Florian; Lin, Chiou-Hong; Wong, Stephen H.K.; Wang, Zhong; Iwasaki, Masayuki; Cleary, Michael L.

    2015-01-01

    Acute lymphoblastic leukemia (ALL) is the most common childhood cancer; however, its genetic diversity limits investigation into the molecular pathogenesis of disease and development of therapeutic strategies. Here, we engineered mice that conditionally express the E2A-PBX1 fusion oncogene, which results from chromosomal translocation t(1;19) and is present in 5% to 7% of pediatric ALL cases. The incidence of leukemia in these mice varied from 5% to 50%, dependent on the Cre-driving promoter (Cd19, Mb1, or Mx1) used to induce E2A-PBX1 expression. Two distinct but highly similar subtypes of B cell precursor ALLs that differed by their pre–B cell receptor (pre-BCR) status were induced and displayed maturation arrest at the pro-B/large pre–B II stages of differentiation, similar to human E2A-PBX1 ALL. Somatic activation of E2A-PBX1 in B cell progenitors enhanced self-renewal and led to acquisition of multiple secondary genomic aberrations, including prominent spontaneous loss of Pax5. In preleukemic mice, conditional Pax5 deletion cooperated with E2A-PBX1 to expand progenitor B cell subpopulations, increasing penetrance and shortening leukemia latency. Recurrent secondary activating mutations were detected in key signaling pathways, most notably JAK/STAT, that leukemia cells require for proliferation. These data support conditional E2A-PBX1 mice as a model of human ALL and suggest targeting pre-BCR signaling and JAK kinases as potential therapeutic strategies. PMID:26301816

  10. ApoE2 Exaggerates PTSD-Related Behavioral, Cognitive, and Neuroendocrine Alterations.

    PubMed

    Johnson, Lance A; Zuloaga, Damian G; Bidiman, Erin; Marzulla, Tessa; Weber, Sydney; Wahbeh, Helane; Raber, Jacob

    2015-09-01

    Apolipoprotein E (apoE) is an essential component of lipoprotein particles in both the brain and periphery, and exists in three isoforms in the human population: E2, E3, and E4. ApoE has numerous, well-established roles in neurobiology. Most notably, E4 is associated with earlier onset and increased risk of Alzheimer's disease (AD). Although possession of E2 is protective in the context of AD, E2 appears to confer an increased incidence and severity of posttraumatic stress disorder (PTSD). However, the biological processes underlying this link remain unclear. In this study, we began to elucidate these associations by examining the effects of apoE on PTSD severity in combat veterans, and on PTSD-like behavior in mice with human apoE. In a group of 92 veterans with PTSD, we observed significantly higher Clinician-Administered PTSD Scale and PTSD Checklist scores in E2+ individuals, as well as alterations in salivary cortisol levels. Furthermore, we measured behavioral and biological outcomes in mice expressing human apoE after a single stressful event as well as following a period of chronic variable stress, a model of combat-related trauma. Mice with E2 showed impairments in fear extinction, and behavioral, cognitive, and neuroendocrine alterations following trauma. To the best of our knowledge, these data constitute the first translational demonstration of PTSD severity in men and PTSD-like symptoms in mice with E2, and point to apoE as a novel biomarker of susceptibility, and potential therapeutic target, for PTSD. PMID:25857685

  11. Second-order Born effect in coplanar doubly symmetric (e,2e) collisions for sodium

    NASA Astrophysics Data System (ADS)

    Wang, Yang; Jiao, Liguang; Zhou, Yajun

    2012-06-01

    The second-order distorted wave Born approximation (DWBA) method is employed to investigate the triple differential cross sections (TDCS) of coplanar doubly symmetric (e,2e) collisions for alkali target sodium at excess energies of 6-60 eV. Comparing with the first-order DWBA calculations, the inclusion of second-order Born term in the scattering amplitude improves the degree of agreement with experiments, especially for backward scattering region of TDCS. This indicates the present second-order Born term is capable to give a reasonable correction to DWBA model in studying coplanar symmetric (e,2e) problems in low and intermediate energy range.

  12. Discovery and Classification of the z=1.86 SLSNe: DES15E2mlf

    NASA Astrophysics Data System (ADS)

    Pan, Y.-C.; Foley, R. J.; Galbany, L.; Gonzalez-Gaitan, S.; Forster, F.; Hamuy, M.; Prieto, J. L.; Yuan, F.; Tucker, B. E.; Lidman, C.; Martini, P.; Gshwend, Julia; Moller, A.; Zhang, B.; Desai, S.; Paech, K.; Smith, R. C.; Schubnell, M.; Kessler, R.; Lasker, J.; Scolnic, D.; Brout, D. J.; Gladney, L.; Sako, M.; Wolf, R. C.; Brown, P. J.; Krisciunas, K.; Suntzeff, N.; Nichol, R.; Papadopoulos, A.; Childress, M.; D'Andrea, C.; Prajs, S.; Smith, M.; Sullivan, M.; Maartens, R.; Gupta, R.; Kovacs, E.; Kuhlmann, S.; Spinka, H.; Ahn, E.; Finley, D. A.; Frieman, J.; Marriner, J.; Wester, W.; Aldering, G.; Kim, A. G.; Thomas, R. C.; Barbary, K.; Bloom, J. S.; Goldstein, D.; Nugent, P.; Perlmutter, S.; Casas, R.; Castander, F. J.

    2015-12-01

    We report the spectroscopic classification of DES15E2mlf as a superluminous supernova (SLSN) discovered by the Dark Energy Survey (ATEL #4668). DES15E2mlf was discovered on 7 November 2015 at R.A. = 00:41:33.40, Decl = -43:27:17.2 with r = 24.1 mag. We obtained spectra using GMOS on Gemini-South (520-990nm) on 06 December 2015 which indicated a redshift of z = 1.86 from Mg II 2800 absorption.

  13. Theory of Deep Minima in (e,2e) Measurements of Triply Differential Cross Sections

    NASA Astrophysics Data System (ADS)

    Macek, J. H.; Sternberg, J. B.; Ovchinnikov, S. Y.; Briggs, J. S.

    2010-01-01

    Deep minima in He(e,2e)He+ triply differential cross sections are traced to vortices in atomic wave functions. Such vortices have been predicted earlier, but the present calculations show that they have also been observed experimentally, although not recognized as vortices. Their observation in (e,2e) measurements shows that vortices play an important role in electron correlations related to the transfer of angular momentum between incident and ejected electrons. The vortices significantly extend the list of known features that summarize the general picture of electron correlations in impact ionization.

  14. YB-1, the E2F Pathway, and Regulation of Tumor Cell Growth

    PubMed Central

    Samuel, Weini; Cao, Helen; Patel, Rachna; Mehta, Reena; Stern, J. Lewis; Reid, Glen; Woolley, Adele G.; Miller, Lance D.; Black, Michael A; Shelling, Andrew N.; Print, Cristin G.; Braithwaite, Antony W.

    2012-01-01

    Background Y-box binding factor 1 (YB-1) has been associated with prognosis in many tumor types. Reduced YB-1 expression inhibits tumor cell growth, but the mechanism is unclear. Methods YB-1 mRNA levels were compared with tumor grade and histology using microarray data from 771 breast cancer patients and with disease-free survival and distant metastasis–free survival using data from 375 of those patients who did not receive adjuvant therapy. Microarrays were further searched for genes that had correlated expression with YB-1 mRNA. Small interfering RNA (siRNA) was used to study the effects of reduced YB-1 expression on growth of three tumor cell lines (MCF-7 breast, HCT116 colon, and A549 lung cancer cells), on tumorigenesis by A549 cells in nude mice, and on global transcription in the three cancer cell lines. Reporter gene assays were used to determine whether YB-1 siRNAs affected the expression of E2F1, and chromatin immunoprecipitation was used to determine whether YB-1 bound to various E2F promoters as well as E2F1-regulated promoters. All P values were from two-sided tests. Results YB-1 levels were elevated in more aggressive tumors and were strongly associated with poor disease-free survival and distant metastasis–free survival. YB-1 expression was often associated with the expression of genes with E2F sites in their promoters. Cells expressing YB-1 siRNA grew substantially more slowly than control cells and formed tumors less readily in nude mice. Transcripts that were altered in cancer cell lines with YB-1 siRNA included 32 genes that are components of prognostic gene expression signatures. YB-1 regulated expression of an E2F1 promoter–reporter construct in A549 cells (eg, relative E2F1 promoter activity with control siRNA = 4.04; with YB-1 siRNA = 1.40, difference= −2.64, 95% confidence interval = −3.57 to −1.71, P < .001) and bound to the promoters of several well-defined E2F1 target genes. Conclusion YB-1 expression is associated with the activity of E2F transcription factors and may control tumor cell growth by this mechanism. PMID:22205655

  15. Purification, crystallization and initial crystallographic characterization of brazil-nut allergen Ber e 2.

    PubMed

    Guo, Feng; Jin, Tengchuan; Howard, Andrew; Zhang, Yu Zhu

    2007-11-01

    Peanut and tree-nut allergies have attracted considerable attention because of their frequency and their lifelong persistence. Brazil-nut (Bertholletia excelsa) allergies have been well documented and the 11S legumin-like seed storage protein Ber e 2 (excelsin) is one of the two known brazil-nut allergens. In this study, Ber e 2 was extracted from brazil-nut kernels and purified to high purity by crystalline precipitation and gel-filtration chromatography. Well diffracting single crystals were obtained using the hanging-drop vapour-diffusion method. A molecular-replacement structural solution has been obtained. Refinement of the structure is currently under way. PMID:18007055

  16. Direct observation of distorted wave effects in ethylene using the (e,2e) reaction.

    PubMed

    Ren, X G; Ning, C G; Deng, J K; Zhang, S F; Su, G L; Huang, F; Li, G Q

    2005-04-29

    We report here the direct measurements of electron momentum distributions for ethylene using the (e,2e) reaction at different impact energies from 400 to 2400 eV. The "turn up" effects in the (e,2e) cross sections of the 1b(3g) orbital compared with the plane-wave impulse approximation calculations were observed at low and high momentum regions, and such discrepancies become smaller with the increase of the impact electron energies. It is suggested that the observed discrepancies are due to the distorted-wave effects in molecules, while appropriate theoretical calculations using distorted waves in molecules could not be achieved until now. PMID:15904222

  17. Estradiol uptake, toxicity, metabolism, and adverse effects on cadmium-treated amphibian embryos.

    PubMed Central

    Fridman, Osvaldo; Corró, Lucrecia; Herkovits, Jorge

    2004-01-01

    The exposure of Bufo arenarum embryos to 25 micromol/L 17beta-estradiol (E2) resulted in 100% lethality within 48 hr, whereas 10 micromol//L E2 was the no observed effect concentration value for short-term chronic (7 days) exposure. The toxicity profile curves show that lethal effects were proportional to the E2 concentration and the time of exposure. The E2 uptake resulted in 20.1 ng E2/mg embryo at 8 hr posttreatment, but 67.3% of this value was achieved during the first 30 min of incubation with this estrogen. Regarding metabolism, the embryos synthesize estrone (E1) from E2 by means of 17beta-hydroxysteroid dehydrogenase. Simultaneous treatments of Bufo arenarum embryos with 1 mg/L Cd2+ and 0.1, 1, or 10 micromol/L E2 enhanced the lethality exerted by cadmium in 76.7, 80, and 83.3% of embryos, respectively. The results indicate that estrogenic endocrine disruptors could have an adverse effect on amphibian embryos and enhance the toxic effect of Cd on amphibian embryos. This study points to the possibility of using the AMPHITOX test as a screening method for potential endocrine disruption as well as the combined effects of chemical mixtures. PMID:15175173

  18. Degradation of estradiol and ethinyl estradiol by activated sludge and by a defined mixed culture.

    PubMed

    Weber, Stefanie; Leuschner, Prisca; Kmpfer, Peter; Dott, Wolfgang; Hollender, Juliane

    2005-04-01

    The aerobic degradation of the natural hormone 17-beta-estradiol (E2) and the synthetic hormone 17-alpha-ethinyl estradiol (EE2) was investigated in batch experiments with activated sludge from a conventional and a membrane sewage treatment plant. E2 was converted to estrone (E1), the well known metabolite, and further completely transformed within 3 days. The turnover rates of E2 did not differ greatly between conventional and membrane activated sludge. EE2 was persistent in both sludges. By several transfers into fresh E2-medium an enrichment culture could be selected that used E2 as growth substrate. Further enrichment and isolation led to a defined mixed culture consisting of two strains, which were identified by a polyphasic approach as Achromobacter xylosoxidans and Ralstonia sp., respectively. The culture used E2 and E1 as growth substrates and transformed estriol (E3) and 16-alpha-hydroxyestrone but not the xenoestrogens bisphenol A, alpha-zearalenol, mestranol or EE2. The turnover rates of E2 were 0.025-0.1 microg h(-1) cfu(-1) and did not depend on the steroid concentration. PMID:15290133

  19. The effect of estrogen synthesis inhibition on hippocampal memory.

    PubMed

    Bayer, Janine; Rune, Gabriele; Schultz, Heidrun; Tobia, Michael J; Mebes, Imke; Katzler, Olaf; Sommer, Tobias

    2015-06-01

    17-Beta-estradiol (E2) facilitates long term-potentiation (LTP) and increases spine synapse density in hippocampal neurons of ovariectomized rodents. Consistent with these beneficial effects on the cellular level, E2 improves hippocampus-dependent memory. A prominent approach to study E2 effects in rodents is the inhibition of its synthesis by letrozole, which reduces LTPs and spine synapse density. In the current longitudinal functional magnetic resonance imaging (fMRI) study, we translated this approach to humans and compared the impact of E2 synthesis inhibition on memory performance and hippocampal activity in post-menopausal women taking letrozole (n = 21) to controls (n = 24). In particular, we employed various behavioral memory paradigms that allow the disentanglement of hippocampus-dependent and -independent memory. Consistent with the literature on rodents, E2 synthesis inhibition specifically impaired hippocampus-dependent memory, however, this did not apply to the same degree to all of the employed paradigms. On the neuronal level, E2 depletion tended to decrease hippocampal activity during encoding, whereas it increased activity in the anterior cingulate and the dorsolateral prefrontal cortex. We thus infer that the inhibition of E2 synthesis specifically impairs hippocampal functioning in humans, whereas the increased prefrontal activity presumably reflects a compensatory mechanism, which is already known from studies on cognitive aging and Alzheimer's disease. PMID:25863445

  20. In vitro effect of vitellogenin on steroid production by ovarian follicles of greenback flounder Rhombosolea tapirina.

    PubMed

    Sun, B; Pankhurst, N W

    2006-05-01

    Ovarian follicles from vitellogenic greenback flounder (Rhombosolea tapirina) were incubated in L15 medium alone, or containing human chorionic gonadotropin (hCG), dibutyryl cyclic AMP (dbcAMP) or the steroid precursors testosterone (T), 17-hydroxyprogesterone (17P) and androstenedione (A) in the presence of vitellogenin (Vtg) at 0.1-5.0mg mL(-)(1). Medium concentrations of 17beta-estradiol (E(2)) and T were measured by radioimmunoassay. HCG generally stimulated follicular E(2) but not T production, whereas 17P, A and T stimulated production of E(2), T, and E(2) respectively. Treatment of follicles with dbcAMP inhibited follicular E(2) production, but increased follicular T production at high doses. The effect of low concentrations of Vtg on follicular steroid production was variable; however, higher doses of Vtg significantly suppressed basal, hCG-, dbcAMP- and steroid precursor-stimulated follicular E(2) and T production. The results of this study show that high concentrations of Vtg may suppress follicular steroid production by interfering in the steroidogenic pathway. This suggests that Vtg may regulate its own production by limiting the ovarian production of E(2). PMID:16580856

  1. 40 CFR Table E-2 to Subpart E of... - Spectral Energy Distribution and Permitted Tolerance for Conducting Radiative Tests

    Code of Federal Regulations, 2010 CFR

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  2. 26 CFR 1.401(e)-2 - General rules relating to plans covering self-employed individuals.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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    Code of Federal Regulations, 2011 CFR

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    ...-employed individuals. 1.401(e)-2 Section 1.401(e)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(e)-2 General rules relating to plans covering self-employed...

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    Code of Federal Regulations, 2013 CFR

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    Code of Federal Regulations, 2010 CFR

    2010-04-01

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  6. Loss of E2F1 Extends Survival and Accelerates Oral Tumor Growth in HPV-Positive Mice

    PubMed Central

    Zhong, Rong; Bechill, John; Spiotto, Michael T.

    2015-01-01

    The Human Papillomavirus (HPV) is associated with several human cancers, including head and neck squamous cell carcinomas (HNSCCs). HPV expresses the viral oncogene E7 that binds to the retinoblastoma protein (RB1) in order to activate the E2F pathway. RB1 can mediate contradictory pathwayscell growth and cell death via E2F family members. Here, we assessed the extent to which E2F1 mediates lethality of HPV oncogenes. Ubiquitous expression of the HPV oncogenes E6 and E7 caused lethality in mice that was associated with focal necrosis in hepatocytes and pancreatic tissues. Furthermore, all organs expressing HPV oncogenes displayed up-regulation of several E2F1 target genes. The E2F1 pathway mediated lethality in HPV-positive mice because deletion of E2F1 increased survival of mice ubiquitously expressing HPV oncogenes. E2F1 similarly functioned as a tumor suppressor in HPV-positive oral tumors as tumors grew faster with homozygous loss of E2F1 compared to tumors with heterozygous loss of E2F1. Re-expression of E2F1 caused decreased clonogenicity in HPV-positive cancer cells. Our results indicate that HPV oncogenes activated the E2F1 pathway to cause lethality in normal mice and to suppress oral tumor growth. These results suggest that selective modulation of the E2F1 pathway, which is activated in HPV tumors, may facilitate tumor regression. PMID:26670255

  7. Estradiol prevents olfactory dysfunction induced by A-β 25–35 injection in hippocampus

    PubMed Central

    2013-01-01

    Background Some neurodegenerative diseases, such as Alzheimer and Parkinson, present an olfactory impairment in early stages, and sometimes even before the clinical symptoms begin. In this study, we assess the role of CA1 hippocampus (structure highly affected in Alzheimer disease) subfield in the rats’ olfactory behavior, and the neuroprotective effect of 17 beta estradiol (E2) against the oxidative stress produced by the injection of amyloid beta 25–35. Results 162 Wistar rats were ovariectomized and two weeks after injected with 2 μl of amyloid beta 25–35 (A-β25–35) in CA1 subfield. Olfactory behavior was evaluated with a social recognition test, odor discrimination, and search tests. Oxidative stress was evaluated with FOX assay and Western Blot against 4-HNE, Fluoro Jade staining was made to quantify degenerated neurons; all these evaluations were performed 24 h, 8 or 15 days after A-β25–35 injection. Three additional groups treated with 17 beta estradiol (E2) were also evaluated. The injection of A-β25–35 produced an olfactory impairment 24 h and 8 days after, whereas a partial recovery of the olfactory behavior was observed at 15 days. A complete prevention of the olfactory impairment was observed with the administration of E2 two weeks before the amyloid injection (A-β25–35 24 h + E2) and one or two weeks after (groups 8 A-β +E2 and 15 A-β +E2 days, respectively); a decrease of the oxidative stress and neurodegeneration were also observed. Conclusions Our finding shows that CA1 hippocampus subfield plays an important role in the olfactory behavior of the rat. The oxidative stress generated by the administration of A-β25–35 is enough to produce an olfactory impairment. This can be prevented with the administration of E2 before and after amyloid injection. This suggests a possible therapeutic use of estradiol in Alzheimer’s disease. PMID:24059981

  8. TGF{beta}-mediated formation of pRb-E2F complexes in human myeloid leukemia cells

    SciTech Connect

    Hu Xiaotang

    2008-05-02

    TGF{beta} is well known for its inhibitory effect on cell cycle G1 checkpoint kinases. However, its role in the control of pRb-E2F complexes is not well established. TGF{beta} inhibits phosphorylation of pRb at several serine and threonine residues and regulates the association of E2F transcription factors with pRb family proteins. Recent studies found that predominantly E2F-4, p130, and histone deacetylase (HDAC) are found to bind to corresponding E2F-responsive promoters in G0/G1 phase. As cells progress through mid-G1, p130-E2F4 complex are replaced by p107-E2F4 followed by activators E2F1, 2, and 3. pRb was not detectable in the promoters containing the E2F-responsive site in cycling cells but was associated with E2F4-p130 complexes or E2F4-p107 complexes during G0/G1 phase. In human myeloid leukemia cell line, MV4-11, TGF{beta} upregulated pRb-E2F-4 and p130-E2F-4, and downregulated p107-E2F-4 complexes. However, pRB-E2F1 and pRb-E2F3 complexes were found in proliferating cells but not in TGF{beta} arrested G1 cells. In addition, electrophoretic gel mobility shift assay (EMSA) could not detect pRb-E2F DNA-binding activities either in S or G1 phase but exhibited the existence of p107-E2F4 in proliferating cells and p130-E2F4 complexes in TGF{beta}-arrested G1 cells, respectively. Our data suggest that p107 and p130, but not pRb, and the repressor E2F, but not activator E2Fs, play a critical role in regulating E2F-responsive gene expression in TGF{beta}-mediated cell cycle control in human myeloid leukemia cells.

  9. Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain.

    PubMed

    Ogunjimi, Abiodun A; Briant, Douglas J; Pece-Barbara, Nadia; Le Roy, Christine; Di Guglielmo, Gianni M; Kavsak, Peter; Rasmussen, Richele K; Seet, Bruce T; Sicheri, Frank; Wrana, Jeffrey L

    2005-08-01

    The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins. PMID:16061177

  10. E2A Antagonizes PU.1 Activity through Inhibition of DNA Binding

    PubMed Central

    Rogers, Jason H.; Owens, Kristin S.; Kurkewich, Jeffrey; Klopfenstein, Nathan; Iyer, Sangeeta R.; Simon, M. Celeste; Dahl, Richard

    2016-01-01

    Antagonistic interactions between transcription factors contribute to cell fate decisions made by multipotent hematopoietic progenitor cells. Concentration of the transcription factor PU.1 affects myeloid/lymphoid development with high levels of PU.1 directing myeloid cell fate acquisition at the expense of B cell differentiation. High levels of PU.1 may be required for myelopoiesis in order to overcome inhibition of its activity by transcription factors that promote B cell development. The B cell transcription factors, E2A and EBF, are necessary for commitment of multipotential progenitors and lymphoid primed multipotential progenitors to lymphocytes. In this report we hypothesized that factors required for early B cell commitment would bind to PU.1 and antagonize its ability to induce myeloid differentiation. We investigated whether E2A and/or EBF associate with PU.1. We observed that the E2A component, E47, but not EBF, directly binds to PU.1. Additionally E47 represses PU.1-dependent transactivation of the MCSFR promoter through antagonizing PU.1's ability to bind to DNA. Exogenous E47 expression in hematopoietic cells inhibits myeloid differentiation. Our data suggest that E2A antagonism of PU.1 activity contributes to its ability to commit multipotential hematopoietic progenitors to the lymphoid lineages. PMID:26942192

  11. E2A Antagonizes PU.1 Activity through Inhibition of DNA Binding.

    PubMed

    Rogers, Jason H; Owens, Kristin S; Kurkewich, Jeffrey; Klopfenstein, Nathan; Iyer, Sangeeta R; Simon, M Celeste; Dahl, Richard

    2016-01-01

    Antagonistic interactions between transcription factors contribute to cell fate decisions made by multipotent hematopoietic progenitor cells. Concentration of the transcription factor PU.1 affects myeloid/lymphoid development with high levels of PU.1 directing myeloid cell fate acquisition at the expense of B cell differentiation. High levels of PU.1 may be required for myelopoiesis in order to overcome inhibition of its activity by transcription factors that promote B cell development. The B cell transcription factors, E2A and EBF, are necessary for commitment of multipotential progenitors and lymphoid primed multipotential progenitors to lymphocytes. In this report we hypothesized that factors required for early B cell commitment would bind to PU.1 and antagonize its ability to induce myeloid differentiation. We investigated whether E2A and/or EBF associate with PU.1. We observed that the E2A component, E47, but not EBF, directly binds to PU.1. Additionally E47 represses PU.1-dependent transactivation of the MCSFR promoter through antagonizing PU.1's ability to bind to DNA. Exogenous E47 expression in hematopoietic cells inhibits myeloid differentiation. Our data suggest that E2A antagonism of PU.1 activity contributes to its ability to commit multipotential hematopoietic progenitors to the lymphoid lineages. PMID:26942192

  12. 40 CFR Figure E-2 to Subpart E of... - Product Manufacturing Checklist

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Sections of 40 CFR Part 53 or 40 CFR Part 50, Appendix L Verification Comments (Includes documentation of... 40 Protection of Environment 5 2010-07-01 2010-07-01 false Product Manufacturing Checklist E Figure E-2 to Subpart E of Part 53 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY...

  13. Triaxial rotor model description of E2 properties in {sup 186,188,190,192}Os

    SciTech Connect

    Allmond, J. M.; Zaballa, R.; Oros-Peusquens, A. M.; Kulp, W. D.; Wood, J. L.

    2008-07-15

    The triaxial rotor model with independent inertia and electric quadrupole tensors is applied to the description of the extensive set of E2 matrix elements available for {sup 186,188,190,192}Os. Most large and medium transition E2 matrix elements can be reproduced to within {approx}10%, and most diagonal elements to within {approx}30%. Most small transition matrix elements can be reproduced to within {approx}30%, and they support the interference effect exhibited by the model between the inertia and E2 tensors: this is a new feature of quantum rotor models. The diagonal E2 matrix elements at higher spins in the K=2 band are extremely sensitive to admixtures of higher K values: the low experimental values in {sup 190,192}Os indicate significant admixtures of K=4 components. Attention is given to the K{sup {pi}}=4{sup +} bands in these nuclei and the controversial issue of whether they are of quadrupole or hexadecapole nature.

  14. The E2 Domains of APP and APLP1 Share a Conserved Mode of Dimerization

    SciTech Connect

    S Lee; Y Xue; J Hulbert; Y Wang; X Liu; B Demeler; Y Ha

    2011-12-31

    Amyloid precursor protein (APP) is genetically linked to Alzheimer's disease. APP is a type I membrane protein, and its oligomeric structure is potentially important because this property may play a role in its function or affect the processing of the precursor by the secretases to generate amyloid {beta}-peptide. Several independent studies have shown that APP can form dimers in the cell, but how it dimerizes remains controversial. At least three regions of the precursor, including a centrally located and conserved domain called E2, have been proposed to contribute to dimerization. Here we report two new crystal structures of E2, one from APP and the other from APLP1, a mammalian APP homologue. Comparison with an earlier APP structure, which was determined in a different space group, shows that the E2 domains share a conserved and antiparallel mode of dimerization. Biophysical measurements in solution show that heparin binding induces E2 dimerization. The 2.1 {angstrom} resolution electron density map also reveals phosphate ions that are bound to the protein surface. Mutational analysis shows that protein residues interacting with the phosphate ions are also involved in heparin binding. The locations of two of these residues, Arg-369 and His-433, at the dimeric interface suggest a mechanism for heparin-induced protein dimerization.

  15. "Expectations to Change" ((E2C): A Participatory Method for Facilitating Stakeholder Engagement with Evaluation Findings

    ERIC Educational Resources Information Center

    Adams, Adrienne E.; Nnawulezi, Nkiru A.; Vandenberg, Lela

    2015-01-01

    From a utilization-focused evaluation perspective, the success of an evaluation is rooted in the extent to which the evaluation was used by stakeholders. This paper details the "Expectations to Change" (E2C) process, an interactive, workshop-based method designed to engage primary users with their evaluation findings as a means of…

  16. Synthesis of monoclinic IrT e2 under high pressure and its physical properties

    NASA Astrophysics Data System (ADS)

    Li, X.; Yan, J.-Q.; Singh, D. J.; Goodenough, J. B.; Zhou, J.-S.

    2015-10-01

    In a pressure-temperature (P -T ) diagram for synthesizing IrT e2 compounds, the well-studied trigonal (H ) phase with the Cd I2 -type structure is stable at low pressures. The superconducting cubic (C ) phase can be synthesized under higher temperatures and pressures. A rhombohedral phase with the crystal structure similar to the C phase can be made at ambient pressure; but the phase contains a high concentration of Ir deficiency. In this paper we report that a rarely studied monoclinic (M ) phase can be stabilized in narrow ranges of pressure and temperature in this P -T diagram. The peculiar crystal structure of the M -IrT e2 eliminates the tendency to form Ir-Ir dimers found in the H phase. The M phase has been fully characterized by structural determination and measurements of electrical resistivity, thermoelectric power, DC magnetization, and specific heat. These physical properties have been compared with those in the H and C phases of I r1 -xT e2 . Moreover, magnetic and transport properties and specific heat of the M -IrT e2 can be fully justified by calculations with the density-functional theory presented in this paper.

  17. DOE Challenge Home Case Study: e2 Homes – Winter Park, Florida

    SciTech Connect

    none,

    2013-01-01

    This Challenge Home case study describes the first certified DOE Challenge Home as constructed by e2 Homes. Completed in May 2012, the “Wilson Residence” in Winter Park, Florida, is a 4,305-ft2 custom home that scores a HERS 57 without solar and a better than zero net-energy HERS -7 with solar.

  18. Intramolecular diffraction in (e, 2e) reactions of CX4 (X=F, Cl, Br)

    NASA Astrophysics Data System (ADS)

    Ning, Chuangang; Zhu, Jingsheng; Deng, Jingkang; Miao, Yurun

    2014-04-01

    The remarkable discrepancies between the experimental momentum distributions and the calculated distributions within the plane wave impulse approximation (PWIA) were observed in (e, 2e) reaction of CF4, CCl4, and CBr4. The discrepancies evidently depend on the impact energy of electrons. One possible explanation is the intramolecular diffraction.

  19. Viral-mediated noisy gene expression reveals biphasic E2f1 response to MYC

    PubMed Central

    Wong, Jeffrey V.; Yao, Guang; Nevins, Joseph R.; You, Lingchong

    2011-01-01

    Gene expression mediated by viral vectors is subject to cell-to-cell variability, which limits the accuracy of gene delivery. When coupled with single-cell measurements, however, such variability provides an efficient means to quantify signaling dynamics in mammalian cells. Here, we illustrate the utility of this approach by mapping the E2f1 response to MYC, serum stimulation, or both. Our results revealed an underappreciated mode of gene regulation: E2f1 expression first increased then decreased as MYC input increased. This biphasic pattern was also reflected in other nodes of the network including the miR-17-92 micro RNA cluster and p19Arf. A mathematical model of the network successfully predicted modulation of the biphasic E2F response by serum and a CDK inhibitor. In addition to demonstrating how noise can be exploited to probe signaling dynamics, our results reveal how coordination of the MYC/RB/E2F pathway enables dynamic discrimination of aberrant and normal levels of growth stimulation. PMID:21292160

  20. Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses

    PubMed Central

    Weger-Lucarelli, James; Aliota, Matthew T.; Kamlangdee, Attapon; Osorio, Jorge E.

    2015-01-01

    Background Chikungunya virus (CHIKV) and other alphaviruses are the etiologic agents of numerous diseases in both humans and animals. Despite this, the viral mediators of protective immunity against alphaviruses are poorly understood, highlighted by the lack of a licensed human vaccine for any member of this virus genus. The alphavirus E2, the receptor-binding envelope protein, is considered to be the predominant target of the protective host immune response. Although envelope protein domains have been studied for vaccine and neutralization in flaviviruses, their role in alphaviruses is less characterized. Here, we describe the role of the alphavirus E2 domains in neutralization and protection through the use of chimeric viruses. Methodology/Principal Findings Four chimeric viruses were constructed in which individual E2 domains of CHIKV were replaced with the corresponding domain from Semliki Forest virus (SFV) (ΔDomA/ΔDomB/ΔDomC/ ΔDomA+B). Vaccination studies in mice (both live and inactivated virus) revealed that domain B was the primary determinant of neutralization. Neutralization studies with CHIKV immune serum from humans were consistent with mouse studies, as ΔDomB was poorly neutralized. Conclusions/Significance Using chimeric viruses, it was determined that the alphavirus E2 domain B was the critical target of neutralizing antibodies in both mice and humans. Therefore, chimeric viruses may have more relevance for vaccine discovery than peptide-based approaches, which only detect linear epitopes. This study provides new insight into the role of alphavirus E2 domains on neutralization determinants and may be useful for the design of novel therapeutic technologies. PMID:26473963

  1. Single Cell Analysis to locate the Restriction Point with respect to E2F Expression

    NASA Astrophysics Data System (ADS)

    Pimienta, R.; Johnson, A.

    2011-12-01

    The restriction point is a G1-phase checkpoint that regulates passage through the cell cycle and is misregulated in all known types of cancer. The Rb-E2F switch is thought to be one of the most relevant molecular mechanisms which regulate the restriction point in mammalian cells. However, recent experiments have brought the timing of the restriction point into question. In previous studies, cells were analyzed as populations and this prevented an accurate determination of the restriction point. By creating and analyzing an E2F-GFP reporter in single cells, we can pinpoint the timing of E2F activation and determine whether it coincides with the restriction point. Using calcium phosphate and Fugene,we transfected human embryonic kidney (293T) cells with a CMV-GFP plasmid and an E2F-GFP reporter. Based on our results, it appears that calcium phosphate is more effective than Fugene at transfecting mammalian cells. The calcium phosphate transfection had 9.59% more fluorescent cells than Fugene. However, this result only occurred with the CMV-GFP plasmid and not the E2F-GFP reporter, which was not properly expressed in human embryonic kidney (293T) cells. We will continue troubleshooting to fix this reporter as we proceed with our research. Once the reporter is properly cloned, we will transfect it into retinal pigmented epithelial (RPE1-hTERT) cells using the calcium phosphate method. RPE1-hTERT cells are an immortalized with telomerase and are more close to normal cells than tumor-derived cell lines. Through this research we will better comprehend commitment to the mammalian cell cycle.

  2. Transcriptional activation function is not required for stimulation of DNA replication by bovine papillomavirus type 1 E2.

    PubMed

    Grossel, M J; Sverdrup, F; Breiding, D E; Androphy, E J

    1996-10-01

    Bovine papillomavirus type 1 replication was previously shown to require both the E1 initiator protein and the E2 transactivator protein. We show here that E1, in the absence of E2, is sufficient for low-level bovine papillomavirus type 1 DNA replication in C-33A cells. In addition, studies of genetically isolated E2 point mutants demonstrate that enhancement of replication by E2 does not require its transcriptional activation function. The uncoupling of the E2 functions suggests that stimulation of transcription and replication by enhancer proteins occurs via divergent mechanisms. PMID:8794380

  3. Requirements for dE2F function in proliferating cells and in post-mitotic differentiating cells.

    PubMed Central

    Brook, A; Xie, J E; Du, W; Dyson, N

    1996-01-01

    The transcription factor E2F is a target of the retinoblastoma tumor suppressor protein (pRB) and may mediate pRB regulation of S phase entry in mammalian cells. The recent identification of mutant alleles of the Drosophila E2F gene (dE2F) has shown that dE2F is required for embryogenesis. dE2F-mutant embryos lack a co-ordinated program of gene expression which accompanies S phase entry and DNA synthesis declines to levels that are barely detectable. We have investigated the role of the dE2F gene at later stages of development. dE2F is expressed in several larval tissues and is required for cell proliferation in the eye imaginal disc. Surprisingly, dE2F expression persists in post-mitotic cells of the eye disc of third-instar larvae. The loss of dE2F function in these cells causes a novel phenotype, characterized by loss of photoreceptors and abnormal rhabdomere cell morphology. These results show that dE2F is required at multiple stages of development and suggest that E2F may have an important function in post-mitotic cells in addition to its role during cell proliferation. Images PMID:8670871

  4. Structure of the Human FANCL RING-Ube2T Complex Reveals Determinants of Cognate E3-E2 Selection

    PubMed Central

    Hodson, Charlotte; Purkiss, Andrew; Miles, Jennifer Anne; Walden, Helen

    2014-01-01

    Summary The combination of an E2 ubiquitin-conjugating enzyme with an E3 ubiquitin-ligase is essential for ubiquitin modification of a substrate. Moreover, the pairing dictates both the substrate choice and the modification type. The molecular details of generic E3-E2 interactions are well established. Nevertheless, the determinants of selective, specific E3-E2 recognition are not understood. There are ∼40 E2s and ∼600 E3s giving rise to a possible ∼24,000 E3-E2 pairs. Using the Fanconi Anemia pathway exclusive E3-E2 pair, FANCL-Ube2T, we report the atomic structure of the FANCL RING-Ube2T complex, revealing a specific and extensive network of additional electrostatic and hydrophobic interactions. Furthermore, we show that these specific interactions are required for selection of Ube2T over other E2s by FANCL. PMID:24389026

  5. Estrogen inhibits the response-to-injury in a mouse carotid artery model.

    PubMed Central

    Sullivan, T R; Karas, R H; Aronovitz, M; Faller, G T; Ziar, J P; Smith, J J; O'Donnell, T F; Mendelsohn, M E

    1995-01-01

    The atheroprotective effects of estrogen are well documented, but the mechanisms responsible for these effects are not well understood. To study the role of physiologic (nanomolar) estrogen levels on the arterial response-to-injury, we applied a mouse carotid artery injury model to ovariectomized C57BL/6J mice. Mice were treated with vehicle (-E2, n = 10) or 17 beta-estradiol (+E2, n = 10) for 7 d, subjected to unilateral carotid injury, and 14 d later contralateral (normal = NL) and injured carotids from -E2 and +E2 animals were pressure fixed, harvested, and analyzed by quantitative morphometry. E2 levels in +E2 mice were consistently in the nanomolar range (2.1-2.5 nM) at days 0, 7, and 14. At 14 d, measures of both intimal and medial area were markedly increased in the -E2 group: (-E2 vs NL, P < 0.05 for both), but were unchanged from normal levels in the +E2 group (+E2 vs NL, P = NS and +E2 vs -E2, P < 0.05 for both). Cellular proliferation, as assessed by bromodeoxyuridine (BrdU) labeling, was significantly increased over NL in the -E2 mice, but this increase was markedly attenuated in the estrogen replacement group (total BrdU positive cells/section: NL = 6.4 +/- 4.5; -E2 = 113 +/- 26, +E2 = 40 +/- 3.7; -E2 vs NL, P < 0.05; +E2 vs NL, P = NS; -E2 vs +E2, P < 0.05). These data (a) demonstrate significant suppression of the mouse carotid response-to-injury by physiologic levels of estrogen replacement; (b) support the utility of this model in the study of the biologic effects of estrogen on the vascular-injury response; and (c) suggest a direct effect of estrogen on vascular smooth muscle cell proliferation in injured vessels. Images PMID:7593638

  6. Dose-dependent regulation of the early promoter of human papillomavirus type 18 by the viral E2 protein.

    PubMed Central

    Steger, G; Corbach, S

    1997-01-01

    The activity of the E6/E7 promoter of genital human papillomaviruses (HPVs) is positively and negatively modulated by a complex interplay between a variety of cellular transcription factors and the virally encoded E2 protein. The long control region of genital HPVs contains four E2 binding sites in conserved positions, two of which are very close to the TATA box. Binding of E2 to these two sites has been shown to repress the promoter. To carefully analyze the effect of E2 on the activity of the early promoter P105 of HPV18, we used an in vitro transcription system, which allowed titration of the amount of E2 protein. We found that low amounts of HPV18 E2 stimulated the promoter, whereas increasing amounts resulted in promoter repression. When the affinity was analyzed, it became obvious that E2 bound with highest affinity to E2 binding site 4 (BS-4), located 500 bp upstream of the promoter. The promoter most proximal binding site (BS-1) was the weakest site. Transient transfection assays confirmed that small amounts of HPV type (HPV18) E2 and also of bovine papillomavirus type 1 (BPV1) E2 were able to activate the P105, which was dependent on an intact BS-4. The positive role of BS-4 was also obvious at higher E2 concentrations, since mutation of BS-4 enhanced repression. In contrast to HPV18 E2, BPV1 E2 bound better to BS-1 and, in correlation, was able to more strongly repress the P105 in vivo. Our results suggest a dose-dependent regulation of the HPV18 E6/E7 promoter by E2 due to variable occupancy of its binding sites, which have antagonizing effects on the activity of the E6/E7 promoter. PMID:8985322

  7. The E2F transcription factors regulate tumor development and metastasis in a mouse model of metastatic breast cancer.

    PubMed

    Hollern, Daniel P; Honeysett, Jordan; Cardiff, Robert D; Andrechek, Eran R

    2014-09-01

    While the E2F transcription factors (E2Fs) have a clearly defined role in cell cycle control, recent work has uncovered new functions. Using genomic signature methods, we predicted a role for the activator E2F transcription factors in the mouse mammary tumor virus (MMTV)-polyomavirus middle T oncoprotein (PyMT) mouse model of metastatic breast cancer. To genetically test the hypothesis that the E2Fs function to regulate tumor development and metastasis, we interbred MMTV-PyMT mice with E2F1, E2F2, or E2F3 knockout mice. With the ablation of individual E2Fs, we noted alterations of tumor latency, histology, and vasculature. Interestingly, we noted striking reductions in metastatic capacity and in the number of circulating tumor cells in both the E2F1 and E2F2 knockout backgrounds. Investigating E2F target genes that mediate metastasis, we found that E2F loss led to decreased levels of vascular endothelial growth factor (Vegfa), Bmp4, Cyr61, Nupr1, Plod 2, P4ha1, Adamts1, Lgals3, and Angpt2. These gene expression changes indicate that the E2Fs control the expression of genes critical to angiogenesis, the remodeling of the extracellular matrix, tumor cell survival, and tumor cell interactions with vascular endothelial cells that facilitate metastasis to the lungs. Taken together, these results reveal that the E2F transcription factors play key roles in mediating tumor development and metastasis in addition to their well-characterized roles in cell cycle control. PMID:24934442

  8. Estradiol affects liver mitochondrial function in ovariectomized and tamoxifen-treated ovariectomized female rats

    SciTech Connect

    Moreira, Paula I.; Custodio, Jose B.A.; Nunes, Elsa; Moreno, Antonio; Seica, Raquel; Oliveira, Catarina R.; Santos, Maria S. . E-mail: mssantos@ci.uc.pt

    2007-05-15

    Given the tremendous importance of mitochondria to basic cellular functions as well as the critical role of mitochondrial impairment in a vast number of disorders, a compelling question is whether 17{beta}-estradiol (E2) modulates mitochondrial function. To answer this question we exposed isolated liver mitochondria to E2. Three groups of rat females were used: control, ovariectomized and ovariectomized treated with tamoxifen. Tamoxifen has antiestrogenic effects in the breast tissue and is the standard endocrine treatment for women with breast cancer. However, under certain circumstances and in certain tissues, tamoxifen can also exert estrogenic agonist properties. We observed that at basal conditions, ovariectomy and tamoxifen treatment do not induce any statistical alteration in oxidative phosphorylation system and respiratory chain parameters. Furthermore, tamoxifen treatment increases the capacity of mitochondria to accumulate Ca{sup 2+} delaying the opening of the permeability transition pore. The presence of 25 {mu}M E2 impairs respiration and oxidative phosphorylation system these effects being similar in all groups of animals studied. Curiously, E2 protects against lipid peroxidation and increases the production of H{sub 2}O{sub 2} in energized mitochondria of control females. Our results indicate that E2 has in general deleterious effects that lead to mitochondrial impairment. Since mitochondrial dysfunction is a triggering event of cell degeneration and death, the use of exogenous E2 must be carefully considered.

  9. Time course of the estradiol-dependent induction of oxytocin receptor binding in the ventromedial hypothalamic nucleus of the rat

    SciTech Connect

    Johnson, A.E.; Ball, G.F.; Coirini, H.; Harbaugh, C.R.; McEwen, B.S.; Insel, T.R. )

    1989-09-01

    Oxytocin (OT) transmission is involved in the steroid-dependent display of sexual receptivity in rats. One of the biochemical processes stimulated by the ovarian steroid 17 beta-estradiol (E2) that is relevant to reproduction is the induction of OT receptor binding in the ventromedial hypothalamic nucleus (VMN). The purpose of these experiments was to determine if E2-induced changes in OT receptor binding in the VMN occur within a time frame relevant to cyclic changes in ovarian steroid secretion. OT receptor binding was measured in the VMN of ovariectomized rats implanted for 0-96 h with E2-containing Silastic capsules. The rate of decay of OT receptor binding was measured in another group of animals 6-48 h after capsule removal. Receptors were labeled with the specific OT receptor antagonist ({sup 125}I)d(CH2)5(Tyr(Me)2,Thr4,Tyr-NH2(9))OVT, and binding was measured with quantitative autoradiographic methods. In addition, plasma E2 levels and uterine weights were assessed in animals from each treatment condition. Significant increases in E2-dependent OT receptor binding and uterine weight occurred within 24 h of steroid treatment. After E2 withdrawal, OT receptor binding and uterine weight decreased significantly within 24 h. These results are consistent with the hypothesis that steroid modulation of OT receptor binding is necessary for the induction of sexual receptivity.

  10. Ameliorative effects of Anoectochilus formosanus extract on osteopenia in ovariectomized rats.

    PubMed

    Shih, C C; Wu, Y W; Lin, W C

    2001-10-01

    The purpose of this study was to determine ameliorative effects of crude aqueous extract of Anoectochilus formosanus (AFE) on osteopenia in ovariectomized (OVX) rats. First, all of the rats were divided into sham and OVX groups. The OVX rats were allowed to lose bone for 6 weeks. At 6 weeks post-OVX, the OVX rats were divided into four groups treated with water, 17beta-estradiol (30 microg/kg, daily s.c. injection) or AFE (0.5, 2 g/kg, daily, orally) for 12 weeks. In OVX rats, the increases of body weight and serum total cholesterol were significantly decreased by AFE or 17beta-estradiol treatment. In OVX rats, atrophy of uterus and vagina was preserved by treatment with 17beta-estradiol, but not by AFE. The decreased weight of pituitary was increased by treatment with both 17beta-estradiol and AFE. There were decreases in bone density and calcium content including the right femur and the fourth lumbar vertebra, when compared with the sham control rats. Treatment with either 17beta-estradiol or AFE ameliorated these changes induced by OVX. In addition, ovariectomy increased serum alkaline phosphatase levels. The increases were suppressed by the treatment with 17beta-estradiol and AFE. Our results demonstrated that AEF could ameliorate ovariectomy-induced osteopenia. PMID:11535369

  11. Assessment of the role of in situ generated (E)-2,4-diene-valproic acid in the toxicity of valproic acid and (E)-2-ene-valproic acid in sandwich-cultured rat hepatocytes

    SciTech Connect

    Surendradoss, Jayakumar; Chang, Thomas K.H.; Abbott, Frank S.

    2012-11-01

    Valproic acid (VPA) undergoes cytochrome P450-mediated desaturation to form 4-ene-VPA, which subsequently yields (E)-2,4-diene-VPA by β-oxidation. Another biotransformation pathway involves β-oxidation of VPA to form (E)-2-ene-VPA, which also generates (E)-2,4-diene-VPA by cytochrome P450-mediated desaturation. Although the synthetic form of (E)-2,4-diene-VPA is more hepatotoxic than VPA as shown in various experimental models, there is no conclusive evidence to implicate the in situ generated (E)-2,4-diene-VPA in VPA hepatotoxicity. The present study investigated the effects of modulating the in situ formation of (E)-2,4-diene-VPA on markers of oxidative stress (formation of 2′,7′-dichlorofluorescein; DCF), steatosis (accumulation of BODIPY 558/568 C{sub 12}), necrosis (release of lactate dehydrogenase; LDH), and on cellular total glutathione (GSH) levels in sandwich-cultured rat hepatocytes treated with VPA or (E)-2-ene-VPA. Treatment with either of these chemicals alone increased each of the toxicity endpoints. In VPA-treated hepatocytes, (E)-2,4-diene-VPA was detected only at trace levels, even after phenobarbital (PB) pretreatment and there was no effect on the toxicity of VPA. Furthermore, pretreatment with a cytochrome P450 enzyme inhibitor, 1-aminobenzotriazole (1-ABT), did not influence the extent of VPA toxicity in both PB-pretreated and vehicle-pretreated hepatocytes. However, in (E)-2-ene-VPA-treated hepatocytes, PB pretreatment greatly enhanced the levels of (E)-2,4-diene-VPA and this was accompanied by a further enhancement of the effects of (E)-2-ene-VPA on DCF formation, BODIPY accumulation, LDH release, and GSH depletion. Pretreatment with 1-ABT reduced the concentrations of (E)-2,4-diene-VPA and the extent of (E)-2-ene-VPA toxicity; however, this occurred in PB-pretreated hepatocytes, but not in control hepatocytes. In conclusion, in situ generated (E)-2,4-diene-VPA is not responsible for the hepatocyte toxicity of VPA, whereas it contributes to the toxicity of (E)-2-ene-VPA in PB-pretreated rat hepatocytes. -- Highlights: ► (E)-2,4-diene-valproic acid is a reactive and toxic metabolite of valproic acid (VPA). ► In situ, this metabolite is not responsible for VPA toxicity in rat hepatocytes. ► This metabolite enhances (E)-2-ene-VPA toxicity in PB-pretreated hepatocytes.

  12. E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

    SciTech Connect

    Fang, Zejun; Gong, Chaoju; Liu, Hong; Zhang, Xiaomin; Mei, Lingming; Song, Mintao; Qiu, Lanlan; Luo, Shuchai; Zhu, Zhihua; Zhang, Ronghui; Gu, Hongqian; Chen, Xiang

    2015-08-21

    As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration and invasion of CRC cells by activating RRM2. • E2F1 is upregulated in CRC tissues and positively associated with RRM2 level. • E2F1-mediated RRM2 transcription will provide a new strategy in CRC.

  13. Second-order Born calculation of coplanar symmetric (e, 2e) process on Mg

    NASA Astrophysics Data System (ADS)

    Zhang, Yong-Zhi; Wang, Yang; Zhou, Ya-Jun

    2014-06-01

    The second-order distorted wave Born approximation (DWBA) method is employed to investigate the triple differential cross sections (TDCS) of coplanar doubly symmetric (e, 2e) collisions for magnesium at excess energies of 6 eV-20 eV. Comparing with the standard first-order DWBA calculations, the inclusion of the second-order Born term in the scattering amplitude improves the degree of agreement with experiments, especially for backward scattering region of TDCS. This indicates that the present second-order Born term is capable to give a reasonable correction to DWBA model in studying coplanar symmetric (e, 2e) problems of two-valence-electron target in low energy range.

  14. M1-E2 interference in the Zeeman spectra of Bi I

    SciTech Connect

    Werbowy, S.; Kwela, J.

    2008-02-15

    Studies of the M1-E2 interference effect in the mixed-type forbidden lines 461.5, 647.6, and 875.5 nm of Bi I are reported. A special computer program considering the interference effect was designed to obtain the predicted contours of the Zeeman structures of the lines. By variation of free parameters describing the line shapes and the electric-quadrupole admixtures, the calculated profiles were fitted to the recorded spectra. The E2 admixtures found are (7.84{+-}0.14)%, (17.5{+-}0.4)%, and (0.70{+-}0.11)% for the 461.5, 647.6, and 875.5 nm lines, respectively. Our results are compared with recent theories and other experiments.

  15. Antagonist of prostaglandin E2 receptor 4 induces metabolic alterations in liver of mice.

    PubMed

    Li, Ning; Zhang, Limin; An, Yanpeng; Zhang, Lulu; Song, Yipeng; Wang, Yulan; Tang, Huiru

    2015-03-01

    Prostaglandin E2 receptor 4 (EP4) is one of the receptors for prostaglandin E2 and plays important roles in various biological functions. EP4 antagonists have been used as anti-inflammatory drugs. To investigate the effects of an EP4 antagonist (L-161982) on the endogenous metabolism in a holistic manner, we employed a mouse model, and obtained metabolic and transcriptomic profiles of multiple biological matrixes, including serum, liver, and urine of mice with and without EP4 antagonist (L-161982) exposure. We found that this EP4 antagonist caused significant changes in fatty acid metabolism, choline metabolism, and nucleotide metabolism. EP4 antagonist exposure also induced oxidative stress to mice. Our research is the first of its kind to report information on the alteration of metabolism associated with an EP4 antagonist. This information could further our understanding of current and new biological functions of EP4. PMID:25669961

  16. E2 enzyme inhibition by stabilization of a low affinity interface with ubiquitin

    PubMed Central

    St-Cyr, Daniel J.; Ziemba, Amy; Garg, Pankaj; Plamondon, Serge; Auer, Manfred; Sidhu, Sachdev; Marinier, Anne; Kleiger, Gary; Tyers, Mike; Sicheri, Frank

    2014-01-01

    Weak protein interactions between ubiquitin and the ubiquitin-proteasome system (UPS) enzymes that mediate its covalent attachment to substrates serve to position ubiquitin for optimal catalytic transfer. We show that a small molecule inhibitor of the E2 ubiquitin conjugating enzyme Cdc34A, called CC0651, acts by trapping a weak interaction between ubiquitin and the E2 donor ubiquitin binding site. A structure of the ternary CC0651-Cdc34A-ubiquitin complex reveals that the inhibitor engages a composite binding pocket formed from Cdc34A and ubiquitin. CC0651 also suppresses the spontaneous hydrolysis rate of the Cdc34A-ubiquitin thioester, without overtly affecting the interaction between Cdc34A and the RING domain subunit of the E3 enzyme. Stabilization of the numerous other weak interactions between ubiquitin and UPS enzymes by small molecules may be a feasible strategy to selectively inhibit different UPS activities. PMID:24316736

  17. Vitexin inhibits polyubiquitin synthesis by the ubiquitin-conjugating enzyme E2-25K.

    PubMed

    Helms, Kimberli M; Wilson, Randall C; Ogungbe, Ifedayo V; Setzer, William N; Twigg, Pamela D

    2011-10-01

    An extract of bark from the tropical rainforest plant Byrsonima crassifolia was screened for inhibition of diubiquitin formation by the human ubiquitin-conjugating enzyme E2-25K. Activity assays with both the full-length enzyme and a truncated, active catalytic UBC domain revealed that the extract contained inhibitory properties. Separation of the extract into individual components and additional screens identified vitexin as the active inhibitor. An IC50 for vitexin was calculated to be approximately 0.5 mM. Molecular modeling simulations were used to predict the mode of inhibition and NMR spectra were used to confirm the binding site of vitexin to E2-25K. PMID:22164771

  18. Intermediate-energy Coulomb excitation of 104Sn: Moderate E 2 strength decrease approaching 100Sn

    NASA Astrophysics Data System (ADS)

    Doornenbal, P.; Takeuchi, S.; Aoi, N.; Matsushita, M.; Obertelli, A.; Steppenbeck, D.; Wang, H.; Audirac, L.; Baba, H.; Bednarczyk, P.; Boissinot, S.; Ciemala, M.; Corsi, A.; Furumoto, T.; Isobe, T.; Jungclaus, A.; Lapoux, V.; Lee, J.; Matsui, K.; Motobayashi, T.; Nishimura, D.; Ota, S.; Pollacco, E. C.; Sakurai, H.; Santamaria, C.; Shiga, Y.; Sohler, D.; Taniuchi, R.

    2014-12-01

    The reduced transition probability B (E 2 )↑ of the first excited 2+ state in the nucleus 104Sn was measured via Coulomb excitation in inverse kinematics at intermediate energies. A value of 0.173(28) e2b 2 was extracted from the absolute cross section on a Pb target. Feeding contributions in 104Sn from higher lying states were estimated by a reference measurement of the stable 112Sn. Corresponding only to a moderate decrease of excitation strength relative to the almost constant values observed in the proton-rich, even-A Sn-114106 isotopes, present state-of-the-art shell-model predictions, which include proton and neutron excitations across the N =Z =50 shell closures as well as standard polarization charges, underestimate the experimental findings.

  19. E2 superfamily of ubiquitin-conjugating enzymes: constitutively active or activated through phosphorylation in the catalytic cleft

    PubMed Central

    Valimberti, Ilaria; Tiberti, Matteo; Lambrughi, Matteo; Sarcevic, Boris; Papaleo, Elena

    2015-01-01

    Protein phosphorylation is a modification that offers a dynamic and reversible mechanism to regulate the majority of cellular processes. Numerous diseases are associated with aberrant regulation of phosphorylation-induced switches. Phosphorylation is emerging as a mechanism to modulate ubiquitination by regulating key enzymes in this pathway. The molecular mechanisms underpinning how phosphorylation regulates ubiquitinating enzymes, however, are elusive. Here, we show the high conservation of a functional site in E2 ubiquitin-conjugating enzymes. In catalytically active E2s, this site contains aspartate or a phosphorylatable serine and we refer to it as the conserved E2 serine/aspartate (CES/D) site. Molecular simulations of substrate-bound and -unbound forms of wild type, mutant and phosphorylated E2s, provide atomistic insight into the role of the CES/D residue for optimal E2 activity. Both the size and charge of the side group at the site play a central role in aligning the substrate lysine toward E2 catalytic cysteine to control ubiquitination efficiency. The CES/D site contributes to the fingerprint of the E2 superfamily. We propose that E2 enzymes can be divided into constitutively active or regulated families. E2s characterized by an aspartate at the CES/D site signify constitutively active E2s, whereas those containing a serine can be regulated by phosphorylation. PMID:26463729

  20. Role of an adenovirus E2 promoter binding factor in E1A-mediated coordinate gene control.

    PubMed Central

    Kovesdi, I; Reichel, R; Nevins, J R

    1987-01-01

    A product of the adenovirus gene E1A is responsible for the stimulation of transcription from six viral promoters as well as at least two cellular promoters. We have detected a HeLa cell factor, termed E2 promoter binding factor (E2F), that appears to mediate the transcriptional stimulation of the viral E2 promoter. Competition experiments revealed that E2F did not recognize and bind to the E1B, E3, E4, or major late promoter sequences. Furthermore, three additional promoters stimulated by E1A, heat shock protein 70, beta-globin, and early simian virus 40, do not bind E2F. In contrast, the factor does recognize sequences in the E1A enhancer, and within the E1A enhancer are duplicated binding sites for E2F. Finally, a single E2F binding site from the E1A enhancer can confer increased transcription to a mouse beta-globin promoter, dependent on the action of the E1A gene product. This stimulation requires binding of E2F since methylation of the binding site, which blocks binding in vitro, reduces transcription stimulation in vivo. We, therefore, conclude that E2F is likely to be responsible for the E1A-mediated stimulation of the E1A gene as well as the E2 gene but is not involved in the activation of the other E1A-inducible promoters. Images PMID:2951737

  1. Structural basis for the recognition of the E2F transactivation domain by the retinoblastoma tumor suppressor

    PubMed Central

    Lee, Changwook; Chang, Jeong Ho; Lee, Hyun Sook; Cho, Yunje

    2002-01-01

    Repression of E2F transcription activity by the retinoblastoma (Rb) tumor suppressor through its interaction with the transactivation domain of the E2F transcription factor is one of the central features of G1/S arrest in the mammalian cell cycle. Deregulation of the Rb–E2F interaction results in hyperproliferation, lack of differentiation, and apoptosis, and can lead to cancer. The 2.2-Å crystal structure of the Rb pocket complexed with an 18-residue transactivation-domain peptide of E2F-2 reveals that the boomerang-shaped peptide binds to the highly conserved interface between the A-box and the B-box of the Rb pocket in a bipartite manner. The N-terminal segment of the E2F-2 peptide in an extended β-strand-like structure interacts with helices from the conserved groove at the A–B interface, whereas the C-terminal segment, which contains one 310 helix, binds to a groove mainly formed by A-box helices. The flexibility in the middle of the E2F-2 peptide is essential for the tight association of E2F to the Rb pocket. The binding of Rb to the E2F-2 peptide conceals several conserved residues that are crucial for transcription activation of E2F. We provide the structural basis for the Rb-mediated repression of E2F transcription activity without the requirement of histone-modifying enzymes. PMID:12502741

  2. Oct3/4 directly regulates expression of E2F3a in mouse embryonic stem cells

    SciTech Connect

    Kanai, Dai; Ueda, Atsushi; Akagi, Tadayuki; Yokota, Takashi; Koide, Hiroshi

    2015-04-10

    Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, have a characteristic cell cycle with truncated G1 and G2 phases. Recent findings that suppression of Oct3/4 expression results in a reduced proliferation rate of ES cells suggest the involvement of Oct3/4 in the regulation of ES cell growth, although the underlying molecular mechanism remains unclear. In the present study, we identified E2F3a as a direct target gene of Oct3/4 in ES cells. Oct3/4 directly bound to the promoter region of the E2F3a gene and positively regulated expression of E2F3a in mouse ES cells. Suppression of E2F3a activity by E2F6 overexpression led to the reduced proliferation in ES cells, which was relieved by co-expression of E2F3a. Furthermore, cell growth retardation caused by loss of Oct3/4 was rescued by E2F3a expression. These results suggest that Oct3/4 upregulates E2F3a expression to promote ES cell growth. - Highlights: • Oct3/4 positively regulates E2F3a expression in ES cells. • Oct3/4 binds to the promoter region of the E2F3a gene. • Overexpression of E2F6, an inhibitor of E2F3a, reduces ES cell growth. • E2F3a recovers growth retardation of ES cells caused by Oct3/4 reduction.

  3. Electrophile-modified lipoic derivatives of PDC-E2 elicits anti-mitochondrial antibody reactivity.

    PubMed

    Naiyanetr, Phornnop; Butler, Jeffrey D; Meng, Liping; Pfeiff, Janice; Kenny, Thomas P; Guggenheim, Kathryn G; Reiger, Roman; Lam, Kit; Kurth, Mark J; Ansari, Aftab A; Coppel, Ross L; López-Hoyos, Marcos; Gershwin, M Eric; Leung, Patrick S C

    2011-11-01

    Our laboratory has hypothesized that xenobiotic modification of the native lipoyl moiety of the major mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), may lead to loss of self-tolerance in primary biliary cirrhosis (PBC). This thesis is based on the finding of readily detectable levels of immunoreactivity of PBC sera against extensive panels of protein microarrays containing mimics of the inner lipoyl domain of PDC-E2 and subsequent quantitative structure-activity relationships (QSARs). Importantly, we have demonstrated that murine immunization with one such mimic, 2-octynoic acid coupled to bovine serum albumin (BSA), induces anti-mitochondrial antibodies (AMAs) and cholangitis. Based upon these data, we have focused on covalent modifications of the lipoic acid disulfide ring and subsequent analysis of such xenobiotics coupled to a 15mer of PDC-E2 for immunoreactivity against a broad panel of sera from patients with PBC and controls. Our results demonstrate that AMA-positive PBC sera demonstrate marked reactivity against 6,8-bis(acetylthio)octanoic acid, implying that chemical modification of the lipoyl ring, i.e. disruption of the S-S disulfide, renders lipoic acid to its reduced form that will promote xenobiotic modification. This observation is particularly significant in light of the function of the lipoyl moiety in electron transport of which the catalytic disulfide constantly opens and closes and, thus, raises the intriguing thesis that common electrophilic agents, i.e. acetaminophen or non-steroidal anti-inflammatory drugs (NSAIDs), may lead to xenobiotic modification in genetically susceptible individuals that results in the generation of AMAs and ultimately clinical PBC. PMID:21763105

  4. An (e, 2e + ion) investigation of fragmentation of methane induced by low energy electrons

    NASA Astrophysics Data System (ADS)

    Xu, S.; Ma, X.; Ren, X.; Senftleben, A.; Pflüger, T.; Ullrich, J.; Yan, S.; Zhang, P.; Yang, J.; Dorn, A.

    2014-04-01

    An (e, 2e+ion) investigation of the ionization and dissociation of methane by 54 eV electron impact is performed using the advanced reaction microscope. By measuring two electrons and the ion in the final state in triple coincidence, the species of the ions are identified, and the energies deposited into the target are determined. The species and the kinetic energies of the fragmented ion show strong dependence on the intermediate states of the parent ion.

  5. Chlamydia pneumoniae Decreases Smooth Muscle Cell Proliferation through Induction of Prostaglandin E2 Synthesis

    PubMed Central

    Rödel, Jürgen; Prochnau, Dirk; Prager, Katrin; Baumert, Jürgen; Schmidt, Karl-Hermann; Straube, Eberhard

    2004-01-01

    Chlamydia pneumoniae may modulate the proliferation of smooth muscle cells (SMC) in atherosclerotic plaques. Conditioned medium from C. pneumoniae-infected SMC decreased the proliferation of uninfected SMC. Treatment of infected cells with the cyclooxygenase-2 inhibitor NS-398 {N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide} suppressed the up-regulation of prostaglandin E2 (PGE2) and abolished the antimitogenic effect of conditioned medium, suggesting that C. pneumoniae can decrease SMC proliferation via stimulation of PGE2 synthesis. PMID:15271958

  6. Advanced Stirling Convertor (ASC-E2) Performance Testing at NASA Glenn Research Center

    NASA Technical Reports Server (NTRS)

    Oriti, Salvatore; Wilson, Scott

    2011-01-01

    The National Aeronautics and Space Administration (NASA) Glenn Research Center (GRC) has been supporting development of the Advanced Stirling Radioisotope Generator (ASRG) since 2006. A key element of the ASRG Project is providing life, reliability, and performance testing of the Advanced Stirling Convertor (ASC). For this purpose, four pairs of ASCs capable of operating to 850 C and designated with the model number ASC-E2, were delivered by Sunpower of Athens, Ohio, to GRC in 2010. The ASC-E2s underwent a series of tests that included workmanship vibration testing, performance mapping, and extended operation. Workmanship vibration testing was performed following fabrication of each convertor to verify proper hardware build. Performance mapping consisted of operating each convertor at various conditions representing the range expected during a mission. Included were conditions representing beginning-of-mission (BOM), end-of-mission (EOM), and fueling. This same series of tests was performed by Sunpower prior to ASC-E2 delivery. The data generated during the GRC test were compared to performance before delivery. Extended operation consisted of a 500-hr period of operation with conditions maintained at the BOM point. This was performed to demonstrate steady convertor performance following performance mapping. Following this initial 500-hr period, the ASC-E2s will continue extended operation, controller development and special durability testing, during which the goal is to accumulate tens of thousands of hours of operation. Data collected during extended operation will support reliability analysis. Performance data from these tests is summarized in this paper.

  7. Advanced Stirling Convertor (ASC-E2) Performance Testing at NASA Glenn Research Center

    NASA Technical Reports Server (NTRS)

    Oriti, Salvatore; Wilson, Scott

    2011-01-01

    The National Aeronautics and Space Administration (NASA) Glenn Research Center (GRC) has been supporting development of the Advanced Stirling Radioisotope Generator (ASRG) since 2006. A key element of the ASRG Project is providing life, reliability, and performance testing of the Advanced Stirling Convertor (ASC). For this purpose, four pairs of ASCs capable of operating to 850 C and designated with the model number ASC-E2, were delivered by Sunpower of Athens, OH, to GRC in 2010. The ASC-E2s underwent a series of tests that included workmanship vibration testing, performance mapping, and extended operation. Workmanship vibration testing was performed following fabrication of each convertor to verify proper hardware build. Performance mapping consisted of operating each convertor at various conditions representing the range expected during a mission. Included were conditions representing beginning-of-mission (BOM), end-of-mission (EOM), and fueling. This same series of tests was performed by Sunpower prior to ASC-E2 delivery. The data generated during the GRC test were compared to performance before delivery. Extended operation consisted of a 500-hour period of operation with conditions maintained at the BOM point. This was performed to demonstrate steady convertor performance following performance mapping. Following this initial 500-hour period, the ASC-E2s will continue extended operation, controller development and special durability testing, during which the goal is to accumulate tens of thousands of hours of operation. Data collected during extended operation will support reliability analysis. Performance data from these tests is summarized in this paper.

  8. Efficacy of CSF vaccine CP7_E2alf in piglets with maternally derived antibodies.

    PubMed

    Eblé, P L; Quak, S; Geurts, Y; Moonen-Leusen, H W M; Loeffen, W L A

    2014-11-01

    There is a need for live DIVA (differentiating infected from vaccinated animals) vaccines against classical swine fever (CSF). The aim of this study was to investigate whether vaccination with the chimeric pestivirus vaccine CP7_E2alf is efficacious to protect young piglets born from vaccinated sows, thus with maternally derived antibodies (MDAs). Groups of 10 piglets each, with or without MDAs, were vaccinated either intramuscularly (IM), at an age of 3 or 6 weeks, or orally (OR), at an age of 6 weeks. Five piglets of each group were challenged with CSFV strain Koslov and protection against clinical disease, virus shedding and transmission were studied. Vaccination with CP7_E2alf, both in the presence of MDA's and in piglets without MDA's, protected against severe clinical signs, but virus shedding from most inoculated piglets and transmission to contact pigs was observed. However, virus transmission in the vaccinated piglets was significantly reduced as compared to non-vaccinated piglets, although the reproduction ratio's R calculated from the results in the vaccinated pigs from our study were not yet significantly below 1. The efficacy of vaccination with CP7_E2alf in the presence of MDAs (R IMvac=0.8, R ORvac=0.4) seemed to be slightly less as compared to vaccination in the absence of MDAs (R IMvac=0.2, R ORvac=0). On a population level, the results suggest that the CP7_E2alf vaccine is an effective tool in the control and eradication of CSF and, moreover, can be applied for both IM and oral use for young age groups, with MDAs having a limited effect on the efficacy. PMID:25265929

  9. Silvaco ATLAS model of ESA's Gaia satellite e2v CCD91-72 pixels

    NASA Astrophysics Data System (ADS)

    Seabroke, George; Holland, Andrew; Burt, David; Robbins, Mark

    2010-07-01

    The Gaia satellite is a high-precision astrometry, photometry and spectroscopic ESA cornerstone mission, currently scheduled for launch in 2012. Its primary science drivers are the composition, formation and evolution of the Galaxy. Gaia will achieve its unprecedented accuracy requirements with detailed calibration and correction for CCD radiation damage and CCD geometric distortion. In this paper, the third of the series, we present our 3D Silvaco ATLAS model of the Gaia e2v CCD91-72 pixel. We publish e2v's design model predictions for the capacities of one of Gaia's pixel features, the supplementary buried channel (SBC), for the first time. Kohley et al. (2009) measured the SBC capacities of a Gaia CCD to be an order of magnitude smaller than e2v's design. We have found the SBC doping widths that yield these measured SBC capacities. The widths are systematically 2 μm offset to the nominal widths. These offsets appear to be uncalibrated systematic offsets in e2v photolithography, which could either be due to systematic stitch alignment offsets or lateral ABD shield doping diffusion. The range of SBC capacities were used to derive the worst-case random stitch error between two pixel features within a stitch block to be +/-0.25 μm, which cannot explain the systematic offsets. It is beyond the scope of our pixel model to provide the manufacturing reason for the range of SBC capacities, so it does not allow us to predict how representative the tested CCD is. This open question has implications for Gaia's radiation damage and geometric calibration models.

  10. E2-2 Dependent Plasmacytoid Dendritic Cells Control Autoimmune Diabetes

    PubMed Central

    Hansen, Lisbeth; Schmidt-Christensen, Anja; Gupta, Shashank; Fransén-Pettersson, Nina; Hannibal, Tine D.; Reizis, Boris; Santamaria, Pere; Holmberg, Dan

    2015-01-01

    Autoimmune diabetes is a consequence of immune-cell infiltration and destruction of pancreatic β-cells in the islets of Langerhans. We analyzed the cellular composition of the insulitic lesions in the autoimmune-prone non-obese diabetic (NOD) mouse and observed a peak in recruitment of plasmacytoid dendritic cells (pDCs) to NOD islets around 8–9 weeks of age. This peak coincides with increased spontaneous expression of type-1-IFN response genes and CpG1585 induced production of IFN-α from NOD islets. The transcription factor E2-2 is specifically required for the maturation of pDCs, and we show that knocking out E2-2 conditionally in CD11c+ cells leads to a reduced recruitment of pDCs to pancreatic islets and reduced CpG1585 induced production of IFN-α during insulitis. As a consequence, insulitis has a less aggressive expression profile of the Th1 cytokine IFN-γ and a markedly reduced diabetes incidence. Collectively, these observations demonstrate a disease-promoting role of E2-2 dependent pDCs in the pancreas during autoimmune diabetes in the NOD mouse. PMID:26624013

  11. A specific subset of E2 ubiquitin-conjugating enzymes regulate Parkin activation and mitophagy differently.

    PubMed

    Fiesel, Fabienne C; Moussaud-Lamodière, Elisabeth L; Ando, Maya; Springer, Wolfdieter

    2014-08-15

    Loss-of-function mutations in the genes encoding PINK1 and Parkin (also known as PARK2) are the most common causes of recessive Parkinson's disease. Both together mediate the selective degradation of mitochondrial proteins and whole organelles via the proteasome and the autophagy-lysosome pathway (mitophagy). The mitochondrial kinase PINK1 activates and recruits the E3 ubiquitin ligase Parkin to de-energized mitochondria. However, the cognate E2 co-enzymes of Parkin in this ubiquitin-dependent pathway have not been investigated. Here, we discovered a total of four E2s that either positively or negatively regulate the activation, translocation and enzymatic functions of Parkin during mitochondrial quality control. UBE2D family members and UBE2L3 redundantly charged the RING-HECT hybrid ligase Parkin with ubiquitin, resulting in its initial activation and translocation to mitochondria. UBE2N, however, primarily operated through a different mechanism in order to mediate the proper clustering of mitochondria, a prerequisite for degradation. Strikingly, in contrast to UBE2D, UBE2L3 and UBE2N, depletion of UBE2R1 resulted in enhanced Parkin translocation and clustering upon mitochondrial uncoupling. Our study uncovered redundant, cooperative or antagonistic functions of distinct E2 enzymes in the regulation of Parkin and mitophagy that might suggest a putative role in Parkinson's disease pathogenesis. PMID:24928900

  12. A specific subset of E2 ubiquitin-conjugating enzymes regulate Parkin activation and mitophagy differently

    PubMed Central

    Fiesel, Fabienne C.; Moussaud-Lamodière, Elisabeth L.; Ando, Maya; Springer, Wolfdieter

    2014-01-01

    ABSTRACT Loss-of-function mutations in the genes encoding PINK1 and Parkin (also known as PARK2) are the most common causes of recessive Parkinson's disease. Both together mediate the selective degradation of mitochondrial proteins and whole organelles via the proteasome and the autophagy-lysosome pathway (mitophagy). The mitochondrial kinase PINK1 activates and recruits the E3 ubiquitin ligase Parkin to de-energized mitochondria. However, the cognate E2 co-enzymes of Parkin in this ubiquitin-dependent pathway have not been investigated. Here, we discovered a total of four E2s that either positively or negatively regulate the activation, translocation and enzymatic functions of Parkin during mitochondrial quality control. UBE2D family members and UBE2L3 redundantly charged the RING-HECT hybrid ligase Parkin with ubiquitin, resulting in its initial activation and translocation to mitochondria. UBE2N, however, primarily operated through a different mechanism in order to mediate the proper clustering of mitochondria, a prerequisite for degradation. Strikingly, in contrast to UBE2D, UBE2L3 and UBE2N, depletion of UBE2R1 resulted in enhanced Parkin translocation and clustering upon mitochondrial uncoupling. Our study uncovered redundant, cooperative or antagonistic functions of distinct E2 enzymes in the regulation of Parkin and mitophagy that might suggest a putative role in Parkinson's disease pathogenesis. PMID:24928900

  13. Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

    SciTech Connect

    Kanginakudru, Sriramana; DeSmet, Marsha; Thomas, Yanique; Morgan, Iain M.; Androphy, Elliot J.

    2015-04-15

    The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication.

  14. CMIP5 historical simulations (1850-2012) with GISS ModelE2

    NASA Astrophysics Data System (ADS)

    Miller, Ron L.; Schmidt, Gavin A.; Nazarenko, Larissa S.; Tausnev, Nick; Bauer, Susanne E.; DelGenio, Anthony D.; Kelley, Max; Lo, Ken K.; Ruedy, Reto; Shindell, Drew T.; Aleinov, Igor; Bauer, Mike; Bleck, Rainer; Canuto, Vittorio; Chen, Yonghua; Cheng, Ye; Clune, Thomas L.; Faluvegi, Greg; Hansen, James E.; Healy, Richard J.; Kiang, Nancy Y.; Koch, Dorothy; Lacis, Andy A.; LeGrande, Allegra N.; Lerner, Jean; Menon, Surabi; Oinas, Valdar; Prez Garca-Pando, Carlos; Perlwitz, Jan P.; Puma, Michael J.; Rind, David; Romanou, Anastasia; Russell, Gary L.; Sato, Makiko; Sun, Shan; Tsigaridis, Kostas; Unger, Nadine; Voulgarakis, Apostolos; Yao, Mao-Sung; Zhang, Jinlun

    2014-06-01

    Observations of climate change during the CMIP5 extended historical period (1850-2012) are compared to trends simulated by six versions of the NASA Goddard Institute for Space Studies ModelE2 Earth System Model. The six models are constructed from three versions of the ModelE2 atmospheric general circulation model, distinguished by their treatment of atmospheric composition and the aerosol indirect effect, combined with two ocean general circulation models, HYCOM and Russell. Forcings that perturb the model climate during the historical period are described. Five-member ensemble averages from each of the six versions of ModelE2 simulate trends of surface air temperature, atmospheric temperature, sea ice and ocean heat content that are in general agreement with observed trends, although simulated warming is slightly excessive within the past decade. Only simulations that include increasing concentrations of long-lived greenhouse gases match the warming observed during the twentieth century. Differences in twentieth-century warming among the six model versions can be attributed to differences in climate sensitivity, aerosol and ozone forcing, and heat uptake by the deep ocean. Coupled models with HYCOM export less heat to the deep ocean, associated with reduced surface warming in regions of deepwater formation, but greater warming elsewhere at high latitudes along with reduced sea ice. All ensembles show twentieth-century annular trends toward reduced surface pressure at southern high latitudes and a poleward shift of the midlatitude westerlies, consistent with observations.

  15. Studies on orientation and rotation parameters of 4179 Toutatis from Chang'e-2 mission

    NASA Astrophysics Data System (ADS)

    Zhao, Yuhui; Ji, Jianghui; Hu, Shoucun

    The ginger-shaped near-Earth asteroid 4179 Toutatis is close to a 4:1 orbital resonance with the Earth and has made close Earth flybys approximately every four years in the recent 20 years. China’s lunar probe Chang’e-2 achieved a successful flyby the Toutatis on 13th Dec 2012 during its most recent flyby of Earth. During the mission, a series of image with high resolution has been obtained. Combined with the radar model of Toutatis, these figures show the attitude of the asteroid from the camera’s point of view and the orientation of it is then deduced based on the attitude of the camera and the relative position between 4179 Toutatis and Chang'e-2 in our works. According to the previous ground-based observations and works on the rotation parameters of Toutatis, this paper studies the rotating rate of the asteroid in accordance with the imaging result of Toutatis by Chang’e-2 and puts forward a correction to the spin rate parameters.

  16. CMIP5 Historical Simulations (1850-2012) with GISS ModelE2

    NASA Technical Reports Server (NTRS)

    Miller, Ronald Lindsay; Schmidt, Gavin A.; Nazarenko, Larissa S.; Tausnev, Nick; Bauer, Susanne E.; DelGenio, Anthony D.; Kelley, Max; Lo, Ken K.; Ruedy, Reto; Shindell, Drew T.; Aleinov, Igor; Bauer, Mike; Bleck, Rainer; Canuto, Vittorio; Chen, Yonghua; Cheng, Ye; Clune, Thomas L.; Faluvegi, Greg; Healy, Richard J.; Kiang, Nancy Y.; Lacis, Andy A.; LeGrande, Allegra N.; Lerner, Jean; Rind, David; Russell, Gary L.

    2014-01-01

    Observations of climate change during the CMIP5 extended historical period (1850-2012) are compared to trends simulated by six versions of the NASA Goddard Institute for Space Studies ModelE2 Earth System Model. The six models are constructed from three versions of the ModelE2 atmospheric general circulation model, distinguished by their treatment of atmospheric composition and the aerosol indirect effect, combined with two ocean general circulation models, HYCOM and Russell. Forcings that perturb the model climate during the historical period are described. Five-member ensemble averages from each of the six versions of ModelE2 simulate trends of surface air temperature, atmospheric temperature, sea ice and ocean heat content that are in general agreement with observed trends, although simulated warming is slightly excessive within the past decade. Only simulations that include increasing concentrations of long-lived greenhouse gases match the warming observed during the twentieth century. Differences in twentieth-century warming among the six model versions can be attributed to differences in climate sensitivity, aerosol and ozone forcing, and heat uptake by the deep ocean. Coupled models with HYCOM export less heat to the deep ocean, associated with reduced surface warming in regions of deepwater formation, but greater warming elsewhere at high latitudes along with reduced sea ice. All ensembles show twentieth-century annular trends toward reduced surface pressure at southern high latitudes and a poleward shift of the midlatitude westerlies, consistent with observations.

  17. E2F4 Promotes Neuronal Regeneration and Functional Recovery after Spinal Cord Injury in Zebrafish

    PubMed Central

    Sasagawa, Shota; Nishimura, Yuhei; Hayakawa, Yuka; Murakami, Soichiro; Ashikawa, Yoshifumi; Yuge, Mizuki; Okabe, Shiko; Kawaguchi, Koki; Kawase, Reiko; Tanaka, Toshio

    2016-01-01

    Mammals exhibit poor recovery after spinal cord injury (SCI), whereas non-mammalian vertebrates exhibit significant spontaneous recovery after SCI. The mechanisms underlying this difference have not been fully elucidated; therefore, the purpose of this study was to investigate these mechanisms. Using comparative transcriptome analysis, we demonstrated that genes related to cell cycle were significantly enriched in the genes specifically dysregulated in zebrafish SCI. Most of the cell cycle-related genes dysregulated in zebrafish SCI were down-regulated, possibly through activation of e2f4. Using a larval zebrafish model of SCI, we demonstrated that the recovery of locomotive function and neuronal regeneration after SCI were significantly inhibited in zebrafish treated with an E2F4 inhibitor. These results suggest that activation of e2f4 after SCI may be responsible, at least in part, for the significant recovery in zebrafish. This provides novel insight into the lack of recovery after SCI in mammals and informs potential therapeutic strategies.

  18. New observations of asteroid (4179) Toutatis as closely observed by Chang’e-2

    NASA Astrophysics Data System (ADS)

    Ji, Jianghui; Jiang, Yun; Zhao, Yuhui

    2015-08-01

    Toutatis, as an Apollo near-Earth asteroid in an eccentric orbit, was successfully visited by Chang'e-2 on December 13th 2012 after the spacecraft completed its lunar exploration and an extended mission of space-environment exploration at the Sun-Earth Lagrangian point. This flyby as close as a distance of 770 m away from the asteroid’s surface, reveals many geological features on Toutatis’ surface, like concavities, boulders, lineaments and regolith, particularly an ~ 800 m giant basin at the end of the large lobe as well as a sharply perpendicular silhouette near the neck region, indicating that Toutatis is probably a rubble-pile asteroid (Huang et al. 2013). We further identify more than 200 boulders over the imaged area of Toutatis, and infer that most boulders cannot solely be generated by impact cratering, but probably originate from the fragments of parent body of Toutatis. Incorporation with ground-based radar observations over the last two decades, we investigate the orientation and the rotational parameters of Toutatis based on the images captured by Chang'e-2. Hence, Chang'e-2's flyby enables us to have an in-depth understanding of the formation and evolution of this asteroid.

  19. Lysine Activation and Functional Analysis of E2-Mediated Conjugation in the SUMO Pathway

    SciTech Connect

    Yunus,A.; Lima, C.

    2006-01-01

    E2 conjugating proteins that transfer ubiquitin and ubiquitin-like modifiers to substrate lysine residues must first activate the lysine nucleophile for conjugation. Genetic complementation revealed three side chains of the E2 Ubc9 that were crucial for normal growth. Kinetic analysis revealed modest binding defects but substantially lowered catalytic rates for these mutant alleles with respect to wild-type Ubc9. X-ray structures for wild-type and mutant human Ubc9-RanGAP1 complexes showed partial loss of contacts to the substrate lysine in mutant complexes. Computational analysis predicted pK perturbations for the substrate lysine, and Ubc9 mutations weakened pK suppression through improper side chain coordination. Biochemical studies with p53, RanGAP1 and the Nup358/RanBP2 E3 were used to determine rate constants and pK values, confirming both structural and computational predictions. It seems that Ubc9 uses an indirect mechanism to activate lysine for conjugation that may be conserved among E2 family members.

  20. Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress.

    PubMed

    Fox, Julie M; Long, Feng; Edeling, Melissa A; Lin, Hueylie; van Duijl-Richter, Mareike K S; Fong, Rachel H; Kahle, Kristen M; Smit, Jolanda M; Jin, Jing; Simmons, Graham; Doranz, Benjamin J; Crowe, James E; Fremont, Daved H; Rossmann, Michael G; Diamond, Michael S

    2015-11-19

    We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern. PMID:26553503

  1. A molecular basis for phosphorylation-dependent SUMO conjugation by the E2 UBC9.

    PubMed

    Mohideen, Firaz; Capili, Allan D; Bilimoria, Parizad M; Yamada, Tomoko; Bonni, Azad; Lima, Christopher D

    2009-09-01

    Phosphorylation and small ubiquitin-like modifier (SUMO) conjugation contribute to the spatial and temporal regulation of substrates containing phosphorylation-dependent SUMO consensus motifs (PDSMs). Myocyte-enhancement factor 2 (MEF2) is a transcription factor and PDSM substrate whose modification by SUMO drives postsynaptic dendritic differentiation. NMR analysis revealed that the human SUMO E2 interacted with model substrates for phosphorylated and nonphosphorylated MEF2 in similar extended conformations. Mutational and biochemical analysis identified a basic E2 surface that enhanced SUMO conjugation to phosphorylated PDSM substrates MEF2 and heat-shock transcription factor 1 (HSF1), but not to nonphosphorylated MEF2 or HSF1, nor the non-PDSM substrate p53. Mutant ubiquitin-conjugating enzyme UBC9 isoforms defective in promoting SUMO conjugation to phosphorylated MEF2 in vitro and in vivo also impair postsynaptic differentiation in organotypic cerebellar slices. These data support an E2-dependent mechanism that underlies phosphorylation-dependent SUMO conjugation in pathways that range from the heat-shock response to nuclear hormone signaling to brain development. PMID:19684601

  2. The prostaglandin E2 receptor EP4 is integral to a positive feedback loop for prostaglandin E2 production in human macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Nishimura, Tomoyasu; Zhao, Xiaomin; Gan, Huixian; Koyasu, Shigeo; Remold, Heinz G

    2013-09-01

    Prostaglandin E2 (PGE2) is an important biological mediator involved in the defense against Mycobacterium tuberculosis (Mtb) infection. Previously, we reported that in macrophages (Mϕs), infection with avirulent Mtb H37Ra resulted in inhibition of necrosis by an inhibitory effect on mitochondrial permeability transition via the PGE2 receptor EP2. However, human Mϕs also express EP4, a PGE2 receptor functionally closely related to EP2 that also couples to stimulatory guanine nucleotide binding protein, but the functional differences between EP2 and EP4 in Mtb-infected Mϕs have been unclear. EP4 antagonist addition to H37Ra-infected Mϕs inhibited the expression of cyclooxygenase 2 (COX2) and microsomal prostaglandin E synthase-1 (mPGES-1), which are involved in PGE2 production. Moreover, H37Ra infection induced PGE2 production through the Toll-like receptor (TLR) 2/p38 mitogen-activated protein kinase (MAPK) signaling pathway. Induction of COX2 and mPGES-1 expression by TLR2 stimulation or Mtb infection was increased after additional stimulation with EP4 agonist. Hence, in Mtb-infected Mϕs, PGE2 production induced by pathogen recognition receptors/p38 MAPK signaling is up-regulated by EP4-triggered signaling to maintain an effective PGE2 concentration. PMID:23759445

  3. Downregulation of thymidylate synthase and E2F1 by arsenic trioxide in mesothelioma.

    PubMed

    Lam, Sze-Kwan; Li, Yuan-Yuan; Zheng, Chun-Yan; Ho, James Chung-Man

    2015-01-01

    Malignant pleural mesothelioma is a global health issue. Arsenic trioxide (ATO) has been shown to suppress thymidylate synthase (TYMS) in lung adenocarcinoma and colorectal cancer, and induce apoptosis in acute promyelocytic leukemia. With TYMS as a putative therapeutic target, the effect of ATO in mesothelioma was therefore studied. A panel of 5 mesothelioma cell lines was used to study the effect of ATO on cell viability, protein expression, mRNA expression and TYMS activity by MTT assay, western blot, qPCR and tritium-release assay, respectively. The knockdown of TYMS and E2F1 was performed with a specific siRNA. Phosphatidylserine externalization and mitochondrial membrane depolarization were measured by Annexin V and JC-1 staining respectively. The in vivo effect of ATO was studied using a nude mouse xenograft model. Application of ATO demonstrated anticancer effects in the cell line model with clinically achievable concentrations. Downregulation of TYMS protein (except H226 cells and 1.25 µM ATO in H2052 cells) and mRNA expression (H28 cells), pRB1 (H28 cells) and E2F1 and TYMS activity (except H226 cells) were also evident. E2F1 knockdown decreased cell viability more significantly than TYMS knockdown. In general, thymidine kinase 1, ribonucleotide reductase M1, c-myc and skp2 were downregulated by ATO. p-c-Jun was downregulated in H28 cells while upregulated in 211H cells. Phosphatidylserine externalization, mitochondrial membrane depolarization, downregulation of Bcl-2 and Bcl-xL, and upregulation of Bak and cleaved caspase-3 were observed. In the H226 xenograft model, the relative tumor growth was aborted, and E2F1 was downregulated while cleaved caspase-3 was elevated and localized to the nucleus in the ATO treatment group. ATO has potent antiproliferative and cytotoxic effects in mesothelioma in vitro and in vivo, partially mediated through E2F1 targeting (less effect through TYMS targeting). There is sound scientific evidence to support the clinical application of ATO in treatment of mesothelioma. PMID:25335113

  4. Future Climate Change Simulated By Giss ModelE2 Under Representative Concentration Pathways

    NASA Astrophysics Data System (ADS)

    Nazarenko, L.; Tausnev, N.; Schmidt, G. A.

    2014-12-01

    We examine the anthropogenically forced climate response for the twenty first century representative concentration pathway emission scenarios and their extensions for the period 2101-2500. The experiments were performed with ModelE2, a new version of the NASA Goddard Institute for Space Sciences coupled general circulation model that includes three different versions for the atmospheric composition components: a non-interactive version (NINT) with prescribed composition and a tuned aerosol indirect effect (AIE), the TCAD version with fully interactive aerosols, whole-atmosphere chemistry and the tuned AIE , and the TCADI version which further includes a parameterized first indirect aerosol effect on clouds. Each atmospheric version is coupled to two different ocean general circulation models: the Russell ocean model (GISS-E2-R) and HYCOM (GISS-E2-H). By 2100, global mean warming in the RCP scenarios ranges from 1.0ºC to 4.5ºC relative to 1850-1860 mean temperature in the historical simulations. In the RCP2.6 scenario, the surface warming in all simulations stays below a 2°C threshold at the end of the twenty-first century. For RCP8.5, the range is 3.5-4.5ºC at 2100. Decadally averaged sea ice area changes are highly correlated to global mean surface air temperature anomalies and show steep declines in both hemispheres, with a larger sensitivity during winter months. By the year 2500, there are complete recoveries of the globally averaged surface air temperature for all versions of the GISS climate model in the low forcing scenario RCP2.6. TCADI simulations show enhanced warming due to greater sensitivity to CO2, aerosol effects and greater methane feedbacks. Both coupled models have decreases in the Atlantic overturning streamfunction by 2100. In RCP2.6 there is a complete recovery of the Atlantic overturning stream function by the year 2500 while with scenario RCP8.5. E2-R climate model produces a complete shutdown of the Atlantic overturning in the RCP8.5 while it reaches the equilibrium state of 35-40% of the control simulation in the E2-H model by the end of 25th century.

  5. Structure Determination and Characterization of the Vitamin B[superscript 6] Degradative Enzyme (E)-2-(Acetamidomethylene)succinate Hydrolase

    SciTech Connect

    McCulloch, Kathryn M.; Mukherjee, Tathagata; Begley, Tadhg P.; Ealick, Steven E.

    2010-06-22

    The gene identification and kinetic characterization of (E)-2-(acetamidomethylene)succinate (E-2AMS) hydrolase has recently been described. This enzyme catalyzes the final reaction in the degradation of vitamin B{sub 6} and produces succinic semialdehyde, acetate, ammonia, and carbon dioxide from E-2AMS. The structure of E-2AMS hydrolase was determined to 2.3 {angstrom} using SAD phasing. E-2AMS hydrolase is a member of the {alpha}/{beta} hydrolase superfamily and utilizes a serine/histidine/aspartic acid catalytic triad. Mutation of either the nucleophilic serine or the aspartate resulted in inactive enzyme. Mutation of an additional serine residue in the active site causes the enzyme to be unstable and is likely structurally important. The structure also provides insight into the mechanism of hydrolysis of E-2AMS and identifies several potential catalytically important residues.

  6. Failure of estradiol to improve spontaneous or rehabilitation-facilitated recovery after hemorrhagic stroke in rats.

    PubMed

    Nguyen, Angela P; Arvanitidis, Anastasia P; Colbourne, Frederick

    2008-02-01

    Estrogen influences not only the incidence of stroke, but also the amount of injury sustained from a stroke including intracerebral hemorrhage (ICH). In this study we tested whether delayed 17beta-estradiol (E2) treatment affects recovery following striatal ICH. Female rats were trained and tested on several behavioral tests to assess skilled reaching, spontaneous forelimb usage and walking ability. Two weeks following ovariectomy, rats were subjected to a moderate-sized ICH via infusion of collagenase into the striatum. One week later they were implanted with either an E2 pellet (0.36 mg; 60-day release) or they underwent a sham procedure. They were further divided into groups that received either environmental enrichment (EE) rehabilitation therapy (group housing in a complex cage with ramps, tunnels, etc.) or a control condition (group housing in a standard cage). Rats were then behaviorally evaluated out to 8 weeks post-ICH and then euthanized. Neither EE nor E2 affected lesion size, which averaged 62.8 mm(3) across all groups. The EE therapy improved recovery on some tests (e.g., traversing a horizontal ladder) whereas E2 treatment did not notably affect either spontaneous or EE-facilitated recovery. Thus, E2 fails to improve recovery or protect against brain injury when given after a 1-week delay in contrast to its clear neuroprotective effects when given before or soon after ICH. PMID:18178174

  7. Functional Interactions between 17?-Estradiol and Progesterone Regulate Autophagy during Acini Formation by Bovine Mammary Epithelial Cells in 3D Cultures

    PubMed Central

    Zielniok, Katarzyna; Motyl, Tomasz

    2014-01-01

    Mammary gland epithelium forms a network of ducts and alveolar units under control of ovarian hormones: 17-beta-estradiol (E2) and progesterone (P4). Mammary epithelial cells (MECs) cultured on reconstituted basement membrane (rBM) form three-dimensional (3D) acini composed of polarized monolayers surrounding a lumen. Using the 3D culture of BME-UV1 bovine MECs we previously demonstrated that autophagy was induced in the centrally located cells of developing spheroids, and sex steroids increased this process. In the present study we showed that E2 and P4 enhanced the expression of ATG3, ATG5, and BECN1 genes during acini formation, and this effect was accelerated in the presence of both hormones together. The stimulatory action of E2 and P4 was also reflected by increased levels of Atg5, Atg3, and LC3-II proteins. Additionally, the activity of kinases involved in autophagy regulation, Akt, ERK, AMPK, and mTOR, was examined. E2 + P4 slightly increased the level of phosphorylated AMPK but diminished phosphorylated Akt and mTOR on day 9 of 3D culture. Thus, the synergistic actions of E2 and P4 accelerate the development of bovine mammary acini, which may be connected with stimulation of ATGs expression, as well as regulation of signaling pathways (PI3K/Akt/mTOR; AMPK/mTOR) involved in autophagy induction. PMID:24895572

  8. Mechanism of estrogen-mediated neuroprotection: regulation of mitochondrial calcium and Bcl-2 expression.

    PubMed

    Nilsen, Jon; Diaz Brinton, Roberta

    2003-03-01

    Estrogens are neuroprotective against glutamate excitotoxicity caused by an excessive rise in intracellular calcium ([Ca(2+)](i)). In this study, we demonstrate that 17 beta-estradiol (E(2)) treatment of hippocampal neurons attenuated the excitotoxic glutamate-induced rise in bulk-free [Ca(2+)](i) despite potentiating the influx of Ca(2+) induced by glutamate. E(2)-induced attenuation of bulk-free [Ca(2+)](i) depends on mitochondrial sequestration of Ca(2+), which is blocked in the presence of the combination of rotenone and oligomycin or in the presence of antimycin, which collapse the mitochondrial membrane potential, thereby preventing mitochondrial Ca(2+) transport. Release of mitochondrial Ca(2+) by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) after excitotoxic glutamate treatment resulted in a greater [Ca(2+)](i) in E(2)-treated cells, indicating an E(2)-induced increase in the mitochondrial calcium ([Ca(2+)](m)) load. The increased [Ca(2+)](m) load was accompanied by increased expression of Bcl-2, which can promote mitochondrial Ca(2+) load tolerance. These findings provide a mechanism of E(2)-induced neuronal survival by attenuation of excitotoxic glutamate [Ca(2+)](i) rise via increased mitochondrial sequestration of cytosolic Ca(2+) coupled with an increase in Bcl-2 expression to sustain mitochondrial Ca(2+) load tolerance and function. PMID:12604781

  9. Estrogen replacement therapy prevents airway dysfunction in a murine model of allergen-induced asthma.

    PubMed

    Dimitropoulou, Christiana; Drakopanagiotakis, Fotios; Chatterjee, Anuran; Snead, Connie; Catravas, John D

    2009-01-01

    We previously reported that 17beta-estradiol (E2) prevents hyperresponsiveness to carbachol of murine asthmatic tracheal rings in vitro. We now investigated whether E2 is similarly effective in reducing airway hyperreactivity in a murine model of allergic asthma in vivo. Female ovariectomized BALB/c mice were rendered asthmatic by a 25-day protocol of sensitization to ovalbumin. Under positive-pressure ventilation, anesthetized asthmatic mice exhibited a dramatic increase in airway responsiveness to increasing doses of inhaled methacholine compared to PBS-sensitized controls, as reflected in decreased dynamic compliance of the respiratory system and increased tissue damping, tissue elastance, and airway resistance. Furthermore, asthmatic mice exhibited hypercellularity and increased protein concentration in the bronchoalveolar lavage, strong signs of peribronchial cuffing with inflammatory cells and increased goblet cell activity. To test the effects of estrogen, three additional groups of mice were implanted subcutaneously with different amounts of slow-release E2 pellets at the time of ovariectomy and rendered asthmatic as before. E2 dose-dependently inhibited airway hyperresponsiveness to methacholine, reduced bronchoalveolar lavage hypercellularity, and virtually eliminated histologic signs of inflammation and goblet cell hyperactivity. The inflammation and airway hyperactivity in asthmatic mice was associated with an increase in bronchoalveolar lavage levels of TGFbeta1, which was completely abolished in E2-treated asthmatic mice. We conclude that estrogen replacement therapy effectively ameliorates the pathologic profile of murine allergic asthma. PMID:19083056

  10. Anaerobic biodegradation of estrogens--hard to digest.

    PubMed

    de Mes, T Z D; Kujawa-Roeleveld, K; Zeeman, G; Lettinga, G

    2008-01-01

    Although many publications are available on the fate of estrone (E1), 17beta-estradiol (E2) and 17alpha-ethynylestradiol (EE2) during aerobic wastewater treatment, little is published on their fate under strictly anaerobic conditions. Present research investigated the digestibility of E1 and EE2, using digested pig manure, granular UASB sludge, UASB-septic tank sludge and activated sludge as inocula. Besides, actual concentrations were measured in a UASB septic tank treating black water. Under anaerobic conditions E1 is reduced to E2 but the extent of this reduction depends on type of inoculum. No significant loss of the sum of E1 and E2 and of EE2 was observed. Adsorption was responsible for a 32-35% loss of E1 and E2 from the liquid phase in the UASB septic tank and the effluent still contained considerable concentrations of respectively 4.02 microg/l and 18.79 microg/l for E1 and E2 with a large fraction present in conjugated form. No EE2 was detected in the UASB effluent. PMID:18469388

  11. Characterization of a peptide domain within the GB virus C envelope glycoprotein (E2) that inhibits HIV replication.

    PubMed

    Xiang, Jinhua; McLinden, James H; Kaufman, Thomas M; Mohr, Emma L; Bhattarai, Nirjal; Chang, Qing; Stapleton, Jack T

    2012-08-15

    GB virus C (GBV-C) infection is associated with prolonged survival in HIV-infected cohorts, and GBV-C E2 protein inhibits HIV entry when added to CD4+ T cells. To further characterize E2 effects on HIV replication, stably transfected Jurkat cell lines expressing GBV-C E2 or control sequences were infected with HIV and replication was measured. HIV replication (all 6 isolates studied) was inhibited in all cell lines expressing a region of 17 amino acids of GBV-C E2, but not in cell lines expressing E2 without this region. In contrast, mumps and yellow fever virus replication was not inhibited by E2 protein expression. Synthetic GBV-C E2 17mer peptides did not inhibit HIV replication unless they were fused to a tat-protein-transduction-domain (TAT) for cellular uptake. These data identify the region of GBV-C E2 protein involved in HIV inhibition, and suggest that this GBV-C E2 peptide must gain entry into the cell to inhibit HIV. PMID:22608061

  12. Global identification of genes regulated by estrogen signaling and demethylation in MCF-7 breast cancer cells

    SciTech Connect

    Putnik, Milica; Zhao, Chunyan; Gustafsson, Jan-Ake; Department of Biology and Biochemistry, Science and Engineering Research Center Bldg, University of Houston, Houston, TX 77204-5056 ; Dahlman-Wright, Karin

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Estrogen signaling and demethylation can both control gene expression in breast cancers. Black-Right-Pointing-Pointer Cross-talk between these mechanisms is investigated in human MCF-7 breast cancer cells. Black-Right-Pointing-Pointer 137 genes are influenced by both 17{beta}-estradiol and demethylating agent 5-aza-2 Prime -deoxycytidine. Black-Right-Pointing-Pointer A set of genes is identified as targets of both estrogen signaling and demethylation. Black-Right-Pointing-Pointer There is no direct molecular interplay of mediators of estrogen and epigenetic signaling. -- Abstract: Estrogen signaling and epigenetic modifications, in particular DNA methylation, are involved in regulation of gene expression in breast cancers. Here we investigated a potential regulatory cross-talk between these two pathways by identifying their common target genes and exploring underlying molecular mechanisms in human MCF-7 breast cancer cells. Gene expression profiling revealed that the expression of approximately 140 genes was influenced by both 17{beta}-estradiol (E2) and a demethylating agent 5-aza-2 Prime -deoxycytidine (DAC). Gene ontology (GO) analysis suggests that these genes are involved in intracellular signaling cascades, regulation of cell proliferation and apoptosis. Based on previously reported association with breast cancer, estrogen signaling and/or DNA methylation, CpG island prediction and GO analysis, we selected six genes (BTG3, FHL2, PMAIP1, BTG2, CDKN1A and TGFB2) for further analysis. Tamoxifen reverses the effect of E2 on the expression of all selected genes, suggesting that they are direct targets of estrogen receptor. Furthermore, DAC treatment reactivates the expression of all selected genes in a dose-dependent manner. Promoter CpG island methylation status analysis revealed that only the promoters of BTG3 and FHL2 genes are methylated, with DAC inducing demethylation, suggesting DNA methylation directs repression of these genes in MCF-7 cells. In a further analysis of the potential interplay between estrogen signaling and DNA methylation, E2 treatment showed no effect on the methylation status of these promoters. Additionally, we show that the ER{alpha} recruitment occurs at the FHL2 promoter in an E2- and DAC-independent fashion. In conclusion, we identified a set of genes regulated by both estrogen signaling and DNA methylation. However, our data does not support a direct molecular interplay of mediators of estrogen and epigenetic signaling at promoters of regulated genes.

  13. Hyperforin, an Anti-Inflammatory Constituent from St. John's Wort, Inhibits Microsomal Prostaglandin E2 Synthase-1 and Suppresses Prostaglandin E2 Formation in vivo

    PubMed Central

    Koeberle, Andreas; Rossi, Antonietta; Bauer, Julia; Dehm, Friederike; Verotta, Luisella; Northoff, Hinnak; Sautebin, Lidia; Werz, Oliver

    2010-01-01

    The acylphloroglucinol hyperforin (Hyp) from St. John's wort possesses anti-inflammatory and anti-carcinogenic properties which were ascribed among others to the inhibition of 5-lipoxygenase. Here, we investigated whether Hyp also interferes with prostanoid generation in biological systems, particularly with key enzymes participating in prostaglandin (PG)E2 biosynthesis, i.e., cyclooxygenases (COX)-1/2 and microsomal PGE2 synthase (mPGES)-1 which play key roles in inflammation and tumorigenesis. Similar to the mPGES-1 inhibitors MK-886 and MD-52, Hyp significantly suppressed PGE2 formation in whole blood assays starting at 0.03–1 μM, whereas the concomitant generation of COX-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, thromboxane B2, and 6-keto PGF1α was not significantly suppressed up to 30 μM. In cell-free assays, Hyp efficiently blocked the conversion of PGH2 to PGE2 mediated by mPGES-1 (IC50 = 1 μM), and isolated COX enzymes were not (COX-2) or hardly (COX-1) suppressed. Intraperitoneal (i.p.) administration of Hyp (4 mg kg−1) to rats impaired exudate volume and leukocyte numbers in carrageenan-induced pleurisy associated with reduced PGE2 levels, and Hyp (given i.p.) inhibited carrageenan-induced mouse paw edema formation (ED50 = 1 mg kg−1) being superior over indomethacin (ED50 = 5 mg kg−1). We conclude that the suppression of PGE2 biosynthesis in vitro and in vivo by acting on mPGES-1 critically contributes to the anti-inflammatory efficiency of Hyp. PMID:21687502

  14. [Induced abortion using prostaglandin E2 and F2alpha gel].

    PubMed

    Lippert, T H; Modly, T

    1974-01-01

    In this study of 20 patients in the 13th-17th week of pregnancy abortion was induced with intrauterine, extraamniotic application of prostaglandins (PG) E2 or F2 in gel form. The gel composition was as follows: 4% tylose MH 300, 2% glycerine, 1% chlorhexidine digluconate, 83% sterile distilled water and 10% PG stock solution. Both PGE2 and PGF2 gels were used. Final concentration was 2.5 mg E2 or 2.5 mg F2 per g of gel. Gel was applied via transcervical, extraamniotic polyethylene catheter every 2-3 hours. Results: PGE2-gel was used in 14 cases. After 3-4 applications both fetus and placenta were expelled. Average dose used was 4.6 mg E2/patient. First contractions started in 30 minutes; induction to expulsion time was 11 hours 35 minutes. F2-gel given to 6 patients resulted in expulsion of the fetus in all cases but placenta needed removal by curettage in 4 patients. Average dose per patient was 17.7 mg of F2; first contractions in 30 minutes, average expulsion time 17 hours 38 minutes. With both PGs there were painful contractions which were controlled with a combination of pentazocine and Valium. PGE2 caused vomiting in 5 patients. No increased bleeding or postabortion infection occurred. Follow-up curettage was done in all patients to ensure removal of all tissues. Overall evaluation of the PG-gels was considered good. PG stability in gel form is good; during 8 months of preservation in sterile aluminum tubes at -25 degrees Celsius no decline in clinical effectiveness was noted. The gel application is less expensive than the slow-injection pump method. PMID:4473406

  15. Comparative analysis of the replicon regions of eleven ColE2-related plasmids.

    PubMed Central

    Hiraga, S; Sugiyama, T; Itoh, T

    1994-01-01

    The incA gene product of ColE2-P9 and ColE3-CA38 plasmids is an antisense RNA that regulates the production of the plasmid-coded Rep protein essential for replication. The Rep protein specifically binds to the origin and synthesizes a unique primer RNA at the origin. The IncB incompatibility is due to competition for the Rep protein among the origins of the same binding specificity. We localized the regions sufficient for autonomous replication of 15 ColE plasmids related to ColE2-P9 and ColE3-CA38 (ColE2-related plasmids), analyzed their incompatibility properties, and determined the nucleotide sequences of the replicon regions of 9 representative plasmids. The results suggest that all of these plasmids share common mechanisms for initiation of DNA replication and its control. Five IncA specificity types, 4 IncB specificity types, and 9 of the 20 possible combinations of the IncA and IncB types were found. The specificity of interaction of the Rep proteins and the origins might be determined by insertion or deletion of single nucleotides and substitution of several nucleotides at specific sites in the origins and by apparently corresponding insertion or deletion and substitution of amino acid sequences at specific regions in the C-terminal portions of the Rep proteins. For plasmids of four IncA specificity types, the nine-nucleotide sequences at the loop regions of the stem-loop structures of antisense RNAs are identical, suggesting an evolutionary significance of the sequence. The mosaic structures of the replicon regions with homologous and nonhomologous segments suggest that some of them were generated by exchanging functional parts through homologous recombination. PMID:7525540

  16. Electronic, magnetic, transport, and thermal properties of single-crystalline UF e2A l10

    NASA Astrophysics Data System (ADS)

    Troć, R.; Samsel-Czekała, M.; Talik, E.; Wawryk, R.; Gajek, Z.; Pasturel, M.

    2015-09-01

    The valence and core-level x-ray photoemission spectra (XPS), performed on an UF e2A l10 single crystal, were measured using the Al Kα radiation. The results of valence XPS show practically two separate regions of spectral intensity, one just at the Fermi level (EF) and the other one being a wide content with its maximum at about 0.8 eV below EF. These give rise to two electronic configurations of the 5 f states in the studied aluminide, itinerant and localized ones, i.e., their dual character. In such a situation the corresponding valence spectra, calculated within the local density approximation (LDA), well explain the former configuration, being responsible for a metallic behavior of the studied compound. Moreover, this behavior is confirmed clearly also by our results of magnetotransport measurements. On the other hand, the obtained magnetic susceptibility, specific heat, electrical resistivity, and thermoelectric power data support very well the local character of the 5 f2 -electron configuration of the U4 + ion in UF e2A l10 having the orthorhombic and cage-type crystal structure. Based on that configuration, the magnetic and thermal characteristics of the compound were modeled by the effective crystal field (CF) potential in the intermediate coupling scheme using initial parameters obtained by the angular overlap model (AOM). The obtained final CF parameters yielded the CF level scheme, composed of only singlets, proper for orthorhombic symmetry. Such a set of singlets reproduces in a satisfactory way both the strongly anisotropic temperature variations of the magnetic susceptibility, measured along the three main crystallographic directions, as well as the Schottky anomaly, evaluated using specific heat results of isomorphic ThF e2A l10 as a phonon reference. Also, the strongly anisotropic behavior of the Seebeck coefficient and its low temperature maxima observed for the compound studied here have been explained roughly by the CF effect.

  17. Evidence suggesting that HCV p7 protects E2 glycoprotein from premature degradation during virus production.

    PubMed

    Atoom, Ali M; Jones, Daniel M; Russell, Rodney S

    2013-09-01

    The hepatitis C virus (HCV) genome encodes a 63 amino acid (aa) protein, p7, which is located between the structural and non-structural proteins. p7 localizes to endoplasmic reticulum membranes and is composed of two transmembrane domains (TM1 and TM2) and a cytoplasmic loop. While its exact role is unknown, p7 is crucial for assembly and/or release of infectious virus production in cell culture, as well as infectivity in chimpanzees. The contribution of p7 to the HCV life cycle may result from at least two distinct roles. Firstly, several studies have shown that p7 acts as an ion channel, the functionality of which is critical for infection. Secondly, p7 interacts with NS2 in a manner that may regulate the targeting of other structural proteins during the assembly process. In this study, we observed that mutations in TM1 and the cytoplasmic loop of p7 decreased infectious virus production in a single-cycle virus production assay. Analysis of intra- and extracellular virus titers indicated that p7 functions at a stage prior to generation of infectious particles. These effects were not due to altered RNA replication since no effects on levels of NS3 or NS5A protein were observed, and were not a consequence of altered recruitment of core protein to lipid droplets. Similarly, these mutations seemingly did not prevent nucleocapsid oligomerization. Importantly, we found that an alanine triplet substitution including the two basic residues of the cytoplasmic loop, which is integral to p7 ion channel function, significantly reduced E2 glycoprotein levels. A time course experiment tracking E2 levels indicated that E2 was degraded over time, as opposed to being synthesized in reduced quantities. The results of this study provide strong evidence that one of the functions of p7 is to protect HCV glycoproteins from premature degradation during virion morphogenesis. PMID:23816605

  18. GPR30 FORMS AN INTEGRAL PART OF E2-PROTECTIVE PATHWAY IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

    PubMed Central

    Bodhankar, Sheetal; Offner, Halina

    2011-01-01

    A major focus of our laboratory has been an in-depth evaluation as to how estrogens exert a pronounced protective effect on clinical and histological disease in the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). An important issue regarding their therapeutic application has been the undesirable estrogenic side effects thought to be mediated primarily through 17β-estradiol (E2) binding to intracellular estrogen receptor alpha (ERα). With the discovery and characterization of GPR30 as the putative membrane estrogen receptor, we sought to study whether signaling through GPR30 was sufficient to mediate protection against EAE without engagement of ERα. Treatment of EAE in WT mice with G-1, a selective GPR30 agonist, retained estradiol’s ability to protect against clinical and histological EAE without estrogenic side effects. G-1 treatment deviated cytokine profiles and enhanced suppressive activity of CD4+Foxp3+ Treg cells through a GPR30- and programmed death 1 (PD-1)-dependent mechanism. This novel finding was indicative of the protective effect of GPR30 activation in EAE and provides a strong foundation for the clinical application of GPR30 agonists such as G-1 in MS. However, future studies are needed to elucidate cross-signaling and evaluate possible additive effects of combined signaling through both GPR30 and ER-α. Deciphering the possible mechanism of involvement of GPR30 in estrogen-mediated protection against EAE may result in lowering treatment doses of E2 and GPR30 agonists that could minimize risks and maximize immunoregulation and therapeutic effects in MS. Alternatively, one might envision using E2 derivatives with reduced estrogenic activity alone or in combination with GPR30 agonists as therapies for both male and female MS patients. PMID:22247749

  19. Secondary-electron emission mechanism of LiF film by (e,2e) spectroscopy

    NASA Astrophysics Data System (ADS)

    Samarin, S.; Berakdar, J.; Suvorova, A.; Artamonov, O. M.; Waterhouse, D. K.; Kirschner, J.; Williams, J. F.

    2004-01-01

    Secondary-electron emission (SEE) from LiF films deposited on Si(0 0 1) surface was studied using time-of-flight two-electron coincidence spectroscopy. A set of energy-distribution curves (EDCs) of secondary electrons excited from a LiF film by electrons with energies in the range of 20-50 eV exhibits emission features at about 7 and 11 eV. The energy positions of these maxima do not depend on the incident energy. To reveal the origin of these features, each of the EDCs was spanned in the second dimension E2 using two-electron coincidence spectroscopy. Two-dimensional mapping of the energy sharing between correlated electrons shows that above 25 eV incident energy, one electron of the pair is preferentially emitted with E1=7.2±0.3 eV energy and the second one with energy E2=( Ep-23.3)±0.5 eV, where Ep is the incident electron energy. At about 30 eV incident energy, a second favoured emission energy of 10.9 ± 0.3 eV is observed. The unique capability of (e,2e) spectroscopy established the links between electron energy loss process and emission features in the EDCs. It is suggested that the mechanism of SEE from LiF film includes the excitation of two collective excitations with subsequent decay via electron ejection. It was shown that the mechanism of secondary emission from LiF film depends on the film thickness and its structure.

  20. E6AP/UBE3A Ubiquitin Ligase Harbors Two E2?ubiquitin Binding Sites*

    PubMed Central

    Ronchi, Virginia P.; Klein, Jennifer M.; Haas, Arthur L.

    2013-01-01

    By exploiting 125I-polyubiquitin chain formation as a functional readout of enzyme activity, we have quantitatively examined the mechanism of human E6AP/UBE3A for the first time. Initial rate studies identify UbcH7 as the cognate E2 carrier protein for E6AP, although related Ubc5 isoforms and the ISG15-specific UbcH8 paralog also support E6AP with reduced efficacy due to impaired binding and catalytic competence. Initial rates of polyubiquitin chain formation displayed hyperbolic kinetics with respect to UbcH7 concentration (Km = 57.6 5.7 nm and kcat = 0.032 0.001 s?1) and substrate inhibition above 2 ?m. Competitive inhibition by an isosteric UbcH7C86S-ubiquitin oxyester substrate analog (Ki = 64 18 nm) demonstrates that Km reflects intrinsic substrate affinity. In contrast, noncompetitive inhibition by a UbcH7C86A product analog (Ki = 7 0.7 ?m) and substrate inhibition at high concentrations require two functionally distinct E2?ubiquitin substrate binding sites. The kinetics of polyubiquitin chain formation reflect binding at a cryptic Site 1 not previously recognized that catalyzes E6AP?ubiquitin thioester formation. Subsequent binding of E2?ubiquitin at the canonical Site 2 present in the extant crystal structure is responsible for polyubiquitin chain elongation. Other rate studies show that the conserved ?4 Phe849 residue is required for polyubiquitin chain formation rather than target protein conjugation as originally suggested. The present studies unambiguously preclude earlier models for the mechanism of Hect domain-catalyzed conjugation through the canonical binding site suggested by the crystal structure and define a novel two-step mechanism for formation of the polyubiquitin degradation signal. PMID:23439649

  1. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors.

    PubMed

    Qiu, Jiazhang; Sheedlo, Michael J; Yu, Kaiwen; Tan, Yunhao; Nakayasu, Ernesto S; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

    2016-05-01

    Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP. PMID:27049943

  2. Interferons and interleukin-6 suppress the DNA-binding activity of E2F in growth-sensitive hematopoietic cells.

    PubMed Central

    Melamed, D; Tiefenbrun, N; Yarden, A; Kimchi, A

    1993-01-01

    Transcription factor E2F binds to cellular promoters of certain growth- and cell cycle-controlling genes and forms distinct heteromeric complexes with other nuclear proteins. We show here that alpha and beta interferons (alpha, beta) and interleukin-6 abolished the E2F-containing DNA-binding complexes in Daudi Burkitt lymphoma cells and in M1 myeloblastic cells, which responded to the cytokines by suppression of c-myc transcription. Time kinetics studies showed that the abolishment of E2F complexes coincided with reduction of c-myc expression and that both molecular events preceded the cell cycle block in G0/G1 phase. In contrast, the pattern of E2F complexes remained unchanged in an interferon-treated growth-resistant Daudi cell mutant that displayed relaxed regulation of c-myc. All of the DNA-binding E2F complexes, including those containing the retinoblastoma protein (pRB), cyclin A-p33cdk2, and the free forms of E2F, were reduced by interferons or interleukin-6. Their abolishment was unperturbed by pharmacological treatments that alleviated the cyclin A and pRB responses to interferon. Thus, changes in cyclin A expression and pRB phosphorylation are not primary events that influence the pattern of E2F responses to cytokines. Addition of EDTA to cell extracts of interferon-treated Daudi cells restored the DNA-binding activity of E2F, resulting in the appearance of a single E2F complex that exclusively contained pRB. It is suggested that the regulation of E2F by growth-inhibitory cytokines that induce cell cycle exit takes place at the level of the DNA-binding activity, and by that mean it differs basically from the phase-specific regulation of E2F in cycling cells. Images PMID:7689148

  3. A new photon counting imaging detector for the Chinese ChangE-2 EUV explorer mission

    NASA Astrophysics Data System (ADS)

    Zhu, Xiangping; Zhao, Baosheng; Liu, Yongan; Zhang, Xinghua; Miao, Zhenhua; Wei, Yonglin

    2008-11-01

    This paper describes the preliminary design and performances of a new developed photon counting imaging detector for Chinese ChangE-2 EUV explorer mission. The detector consists of microchannel plate (MCP) stacks and wedge and strip anode and corresponding data read out electronics. The experimental results shows that the new developed detector has a spatial resolution of about 75μm, image distortions are small and dark noise count rate less than 0.4 counts cm-2s-1. The pulse height distribution vs MCP operating voltages and the flat field performances are also discussed.

  4. Dynamical (e, 2e) studies using tetrahydrofuran as a DNA analog.

    PubMed

    Colyer, C J; Bellm, S M; Lohmann, B; Hanne, G F; Al-Hagan, O; Madison, D H; Ning, C G

    2010-09-28

    Triple differential cross sections for the electron-impact ionization of the outer valence orbital of tetrahydrofuran have been measured using the (e, 2e) technique. The measurements have been performed with coplanar asymmetric kinematics, at an incident electron energy of 250 eV and at an ejected electron energy of 10 eV, over a range of momentum transfers. The experimental results are compared with theoretical calculations carried out using the molecular three-body distorted wave model. The results obtained are important for gaining an understanding of electron driven processes at a molecular level and for modeling energy deposition in living tissue. PMID:20886927

  5. X-ray spectroscopy of E2 and M3 transitions in Ni-like W

    SciTech Connect

    Clementson, J.; Beiersdorfer, P.; Gu, M. F.

    2010-01-15

    The electric quadrupole (E2) and magnetic octupole (M3) ground-state transitions in Ni-like W{sup 46+} have been measured using high-resolution crystal spectroscopy at the LLNL electron-beam ion trap facility. The lines fall in the soft x-ray region near 7.93 A and were originally observed as an unresolved feature in tokamak plasmas. Using flat ammonium dihydrogen phosphate and quartz crystals, the wavelengths, intensities, and polarizations of the two lines have been measured for various electron-beam energies and compared to intensity and polarization calculations performed using the Flexible Atomic Code (FAC).

  6. Future climate change under RCP emission scenarios with GISS ModelE2

    DOE PAGESBeta

    Nazarenko, L.; Schmidt, G. A.; Miller, R. L.; Tausnev, N.; Kelley, M.; Ruedy, R.; Russell, G. L.; Aleinov, I.; Bauer, M.; Bauer, S.; et al

    2015-02-24

    We examine the anthropogenically forced climate response for the 21st century representative concentration pathway (RCP) emission scenarios and their extensions for the period 2101–2500. The experiments were performed with ModelE2, a new version of the NASA Goddard Institute for Space Sciences (GISS) coupled general circulation model that includes three different versions for the atmospheric composition components: a noninteractive version (NINT) with prescribed composition and a tuned aerosol indirect effect (AIE), the TCAD version with fully interactive aerosols, whole-atmosphere chemistry, and the tuned AIE, and the TCADI version which further includes a parameterized first indirect aerosol effect on clouds. Each atmosphericmore » version is coupled to two different ocean general circulation models: the Russell ocean model (GISS-E2-R) and HYCOM (GISS-E2-H). By 2100, global mean warming in the RCP scenarios ranges from 1.0 to 4.5° C relative to 1850–1860 mean temperature in the historical simulations. In the RCP2.6 scenario, the surface warming in all simulations stays below a 2 °C threshold at the end of the 21st century. For RCP8.5, the range is 3.5–4.5° C at 2100. Decadally averaged sea ice area changes are highly correlated to global mean surface air temperature anomalies and show steep declines in both hemispheres, with a larger sensitivity during winter months. By the year 2500, there are complete recoveries of the globally averaged surface air temperature for all versions of the GISS climate model in the low-forcing scenario RCP2.6. TCADI simulations show enhanced warming due to greater sensitivity to CO₂, aerosol effects, and greater methane feedbacks, and recovery is much slower in RCP2.6 than with the NINT and TCAD versions. All coupled models have decreases in the Atlantic overturning stream function by 2100. In RCP2.6, there is a complete recovery of the Atlantic overturning stream function by the year 2500 while with scenario RCP8.5, the E2-R climate model produces a complete shutdown of deep water formation in the North Atlantic.« less

  7. Future climate change under RCP emission scenarios with GISS ModelE2

    SciTech Connect

    Nazarenko, L.; Schmidt, G. A.; Miller, R. L.; Tausnev, N.; Kelley, M.; Ruedy, R.; Russell, G. L.; Aleinov, I.; Bauer, M.; Bauer, S.; Bleck, R.; Canuto, V.; Cheng, Y.; Clune, T. L.; Del Genio, A. D.; Faluvegi, G.; Hansen, J. E.; Healy, R. J.; Kiang, N. Y.; Koch, D.; Lacis, A. A.; LeGrande, A. N.; Lerner, J.; Lo, K. K.; Menon, S.; Oinas, V.; Perlwitz, J.; Puma, M. J.; Rind, D.; Romanou, A.; Sato, M.; Shindell, D. T.; Sun, S.; Tsigaridis, K.; Unger, N.; Voulgarakis, A.; Yao, M. -S.; Zhang, Jinlun

    2015-02-24

    We examine the anthropogenically forced climate response for the 21st century representative concentration pathway (RCP) emission scenarios and their extensions for the period 2101–2500. The experiments were performed with ModelE2, a new version of the NASA Goddard Institute for Space Sciences (GISS) coupled general circulation model that includes three different versions for the atmospheric composition components: a noninteractive version (NINT) with prescribed composition and a tuned aerosol indirect effect (AIE), the TCAD version with fully interactive aerosols, whole-atmosphere chemistry, and the tuned AIE, and the TCADI version which further includes a parameterized first indirect aerosol effect on clouds. Each atmospheric version is coupled to two different ocean general circulation models: the Russell ocean model (GISS-E2-R) and HYCOM (GISS-E2-H). By 2100, global mean warming in the RCP scenarios ranges from 1.0 to 4.5° C relative to 1850–1860 mean temperature in the historical simulations. In the RCP2.6 scenario, the surface warming in all simulations stays below a 2 °C threshold at the end of the 21st century. For RCP8.5, the range is 3.5–4.5° C at 2100. Decadally averaged sea ice area changes are highly correlated to global mean surface air temperature anomalies and show steep declines in both hemispheres, with a larger sensitivity during winter months. By the year 2500, there are complete recoveries of the globally averaged surface air temperature for all versions of the GISS climate model in the low-forcing scenario RCP2.6. TCADI simulations show enhanced warming due to greater sensitivity to CO₂, aerosol effects, and greater methane feedbacks, and recovery is much slower in RCP2.6 than with the NINT and TCAD versions. All coupled models have decreases in the Atlantic overturning stream function by 2100. In RCP2.6, there is a complete recovery of the Atlantic overturning stream function by the year 2500 while with scenario RCP8.5, the E2-R climate model produces a complete shutdown of deep water formation in the North Atlantic.

  8. Development of an (e,2e) electron momentum spectroscopy apparatus using an ultrashort pulsed electron gun

    SciTech Connect

    Yamazaki, M.; Kasai, Y.; Oishi, K.; Nakazawa, H.; Takahashi, M.

    2013-06-15

    An (e,2e) apparatus for electron momentum spectroscopy (EMS) has been developed, which employs an ultrashort-pulsed incident electron beam with a repetition rate of 5 kHz and a pulse duration in the order of a picosecond. Its instrumental design and technical details are reported, involving demonstration of a new method for finding time-zero. Furthermore, EMS data for the neutral Ne atom in the ground state measured by using the pulsed electron beam are presented to illustrate the potential abilities of the apparatus for ultrafast molecular dynamics, such as by combining EMS with the pump-and-probe technique.

  9. Future climate change under RCP emission scenarios with GISS ModelE2

    NASA Astrophysics Data System (ADS)

    Nazarenko, L.; Schmidt, G. A.; Miller, R. L.; Tausnev, N.; Kelley, M.; Ruedy, R.; Russell, G. L.; Aleinov, I.; Bauer, M.; Bauer, S.; Bleck, R.; Canuto, V.; Cheng, Y.; Clune, T. L.; Del Genio, A. D.; Faluvegi, G.; Hansen, J. E.; Healy, R. J.; Kiang, N. Y.; Koch, D.; Lacis, A. A.; LeGrande, A. N.; Lerner, J.; Lo, K. K.; Menon, S.; Oinas, V.; Perlwitz, J.; Puma, M. J.; Rind, D.; Romanou, A.; Sato, M.; Shindell, D. T.; Sun, S.; Tsigaridis, K.; Unger, N.; Voulgarakis, A.; Yao, M.-S.; Zhang, Jinlun

    2015-03-01

    We examine the anthropogenically forced climate response for the 21st century representative concentration pathway (RCP) emission scenarios and their extensions for the period 2101-2500. The experiments were performed with ModelE2, a new version of the NASA Goddard Institute for Space Sciences (GISS) coupled general circulation model that includes three different versions for the atmospheric composition components: a noninteractive version (NINT) with prescribed composition and a tuned aerosol indirect effect (AIE), the TCAD version with fully interactive aerosols, whole-atmosphere chemistry, and the tuned AIE, and the TCADI version which further includes a parameterized first indirect aerosol effect on clouds. Each atmospheric version is coupled to two different ocean general circulation models: the Russell ocean model (GISS-E2-R) and HYCOM (GISS-E2-H). By 2100, global mean warming in the RCP scenarios ranges from 1.0 to 4.5°C relative to 1850-1860 mean temperature in the historical simulations. In the RCP2.6 scenario, the surface warming in all simulations stays below a 2°C threshold at the end of the 21st century. For RCP8.5, the range is 3.5-4.5°C at 2100. Decadally averaged sea ice area changes are highly correlated to global mean surface air temperature anomalies and show steep declines in both hemispheres, with a larger sensitivity during winter months. By the year 2500, there are complete recoveries of the globally averaged surface air temperature for all versions of the GISS climate model in the low-forcing scenario RCP2.6. TCADI simulations show enhanced warming due to greater sensitivity to CO2, aerosol effects, and greater methane feedbacks, and recovery is much slower in RCP2.6 than with the NINT and TCAD versions. All coupled models have decreases in the Atlantic overturning stream function by 2100. In RCP2.6, there is a complete recovery of the Atlantic overturning stream function by the year 2500 while with scenario RCP8.5, the E2-R climate model produces a complete shutdown of deep water formation in the North Atlantic.

  10. Magnetoelectric, photovoltaic, and magnetophotovoltaic effects in KBiF e2O5

    NASA Astrophysics Data System (ADS)

    Mettout, B.; Tolédano, P.; Sombra, A. S. B.; Furtado Filho, A. F. G.; do Nascimento, J. P. C.; Santos da Silva, M. A.; Gisse, P.; Vasseur, H.

    2016-05-01

    Multiferroic materials are intensively investigated as potential candidates for a new generation of solar cells, due to the coexistence in their phases of ferroelectricity and magnetic order with low band gap. However, symmetry considerations, which determine the interplay between the magnetoelectric and photovoltaic properties of a light irradiated multiferroic crystal, are not fully taken into account in the current theory of the photovoltaic effect. Here the remarkable photovoltaic and magnetophotovoltaic effects occurring in the multiferroic phase of KBiF e2O5 are described using a theoretical approach that takes directly into account the crystal and light beam wave symmetries.

  11. X-ray Spectroscopy of E2 and M3 Transitions in Ni-like W

    SciTech Connect

    Clementson, J; Beiersdorfer, P; Gu, M F

    2009-11-09

    The electric quadrupole (E2) and magnetic octupole (M3) ground state transitions in Ni-like W{sup 46+} have been measured using high-resolution crystal spectroscopy at the Livermore electron beam ion trap facility. The lines fall in the soft x-ray region near 7.93 {angstrom} and were originally observed as an unresolved feature in tokamak plasmas. Using flat ADP and quartz crystals the wavelengths, intensities, and polarizations of the two lines have been measured for various electron beam energies and compared to intensity and polarization calculations performed using the Flexible Atomic Code (FAC).

  12. Bis[(E)-2-(2,4-dichloro-5-nitrostyryl)-1,3-benzothiazole] hydrate.

    PubMed

    Cox, O; Cordero, M; Piñeiro, S; Huang, S D

    1997-03-15

    The title compound, bis[(E)-2-(2,4-dichloro-5-nitrostyryl)-1,3-benzothiazole] hydrate, 2C15H8Cl2N2O2S.-H2O, was obtained from the condensation of 2-methyl-benzothiazole with 2,4-dichloro-5-nitrobenzaldehyde. Single-crystal X-ray analysis showed that the asymmetric unit contains two crystallographically unique but structurally similar molecules. The dihedral angle between the benzothiazole fragment and the phenyl ring is 6.1 (4) degrees in molecule A and 19.5 (4) degrees in molecule B. PMID:9094194

  13. Prostaglandin I2 and prostaglandin E2 modulate human intrarenal artery contractility through prostaglandin E2-EP4, prostacyclin-IP, and thromboxane A2-TP receptors.

    PubMed

    Eskildsen, Morten P; Hansen, Pernille B L; Stubbe, Jane; Toft, Anja; Walter, Steen; Marcussen, Niels; Rasmussen, Lars M; Vanhoutte, Paul M; Jensen, Boye L

    2014-09-01

    Cyclooxygenase inhibitors decrease renal blood flow in settings with decreased effective circulating volume. The present study examined the hypothesis that prostaglandins, prostaglandin E2 (PGE2) and prostacyclin (PGI2), induce relaxation of human intrarenal arteries through PGE2-EP and PGI2-IP receptors. Intrarenal arteries were microdissected from human nephrectomy samples (n=53, median diameter ≈362 μm, 88% viable, 76% relaxed in response to acetylcholine). Rings were suspended in myographs to record force development. In vessels with K(+)-induced tension (EC70: -log [mol/L]=1.36±0.03), PGE2 and PGI2 induced concentration-dependent relaxation (-log EC50: PGE2=7.1±0.3 and PGI2=7.7). The response to PGE2 displayed endothelium dependence and desensitization. Relaxation by PGE2 was mimicked by an EP4 receptor agonist (CAY10598, EC50=6.7±0.2). The relaxation after PGI2 was abolished by an IP receptor antagonist (BR5064, 10(-8) mol/L). Pretreatment of quiescent arteries with PGE2 for 5 minutes (10(-6) mol/L) led to a significant right shift of the concentration-response to norepinephrine (EC50 from 6.6±0.1-5.9±0.1). In intrarenal arteries with K(+)-induced tone, PGE2 and PGI2 at 10(-5) mol/L elicited increased tension. This was abolished by thromboxane receptor (TP) antagonist (S18886, 10(-6) mol/L). A TP agonist (U46619, n=6) evoked tension (EC50=8.1±0.2) that was inhibited by S18886. Polymerase chain reaction and immunoblotting showed EP4, IP, and TP receptors in intrarenal arteries. In conclusion, PGE2 and PGI2 may protect renal perfusion by activating cognate IP and EP4 receptors associated with smooth muscle cells and endothelium in human intrarenal arteries and contribute to increased renal vascular resistance at high pathological concentrations mediated by noncognate TP receptor. PMID:24914192

  14. N-LINKED GLYCOSYLATION STATUS OF CLASSICAL SWINE FEVER VIRUS STRAIN BRECIA E2 GLYCOPROTEIN INFLUENCES VIRULENCE IN SWINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Although E2 have been involved in virus attachment to target cells, the induction of a protective immune response as well in the process of viral pathogenesis, the role of glycosylation in the functionality of the p...

  15. 37 CFR 1.760 - Interim extension of patent term under 35 U.S.C. 156(e)(2).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... term under 35 U.S.C. 156(e)(2). 1.760 Section 1.760 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE GENERAL RULES OF PRACTICE IN PATENT CASES Adjustment... extension of patent term under 35 U.S.C. 156(e)(2). An applicant who has filed a formal application...

  16. N-Linked Glycosylation Status Of Classical Swine Fever Virus Strain Brescia E2 Glycoprotein Influences Virulence In Swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Previous studies indicate that E2 is involved in several functions including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Her...

  17. Two distinct regions of the BPV1 E1 replication protein interact with the activation domain of E2.

    PubMed

    Moscufo, N; Sverdrup, F; Breiding, D E; Androphy, E J

    1999-12-15

    Papillomavirus E1 and E2 proteins co-operation in viral DNA replication is mediated by protein-protein interactions that lead to formation of an E1-E2 complex. To identify the domains involved, portions of the two proteins were expressed as fusions to the DNA-binding protein LexA or the transactivation domain of VP16 and analyzed by the yeast two-hybrid system. The C-terminal 266 amino acids of BPV1 E1 (E1C266) interacted strongly with E2 in the yeast system and in a mammalian two-hybrid assay. VP16-E1C266 interacted with a region encompassing amino acids 1-200 of the transactivation domain of E2 that was fused to LexA. The interaction between E1 full length and E2 was clearly observed only when E1 was expressed as LexA-E1 chimera. In addition, we found that in the LexA context also the N-terminal region encompassing the first 340 amino acids of E1 (E1N340) interacted with E2 full length. The interactions of E1N340 and E1C266 with E2 were confirmed also by in vitro binding studies. These observations demonstrate that two distinct regions of E1 mediate the interaction with E2 in vivo. PMID:10581387

  18. 37 CFR 1.760 - Interim extension of patent term under 35 U.S.C. 156(e)(2).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... term under 35 U.S.C. 156(e)(2). 1.760 Section 1.760 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE GENERAL RULES OF PRACTICE IN PATENT CASES Adjustment... extension of patent term under 35 U.S.C. 156(e)(2). An applicant who has filed a formal application...

  19. 37 CFR 1.760 - Interim extension of patent term under 35 U.S.C. 156(e)(2).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... term under 35 U.S.C. 156(e)(2). 1.760 Section 1.760 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE GENERAL RULES OF PRACTICE IN PATENT CASES Adjustment... extension of patent term under 35 U.S.C. 156(e)(2). An applicant who has filed a formal application...

  20. 37 CFR 1.760 - Interim extension of patent term under 35 U.S.C. 156(e)(2).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... term under 35 U.S.C. 156(e)(2). 1.760 Section 1.760 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE GENERAL RULES OF PRACTICE IN PATENT CASES Adjustment... extension of patent term under 35 U.S.C. 156(e)(2). An applicant who has filed a formal application...

  1. Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties

    PubMed Central

    Beaumont, Elodie; Roch, Emmanuelle; Chopin, Lucie; Roingeard, Philippe

    2016-01-01

    Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system. PMID:26966906

  2. THE E2/FRB PATHWAY REGULATION OF DNA REPLICATION AND PROTEIN BIOSYNTHESIS

    EPA Science Inventory

    The E2F/Rb pathway plays a pivotal role in the control of cell cycle progression and regulates the expression of genes required for Gl/S transition. Our study examines the genomic response in Drosophila embryos after overexpression and mutation of E2F/Rb pathway molecules. Hierar...

  3. HPV-18 E2circumflexE4 chimera: 2 new spliced transcripts and proteins induced by keratinocyte differentiation

    SciTech Connect

    Tan, Chye Ling; Gunaratne, Jayantha; Lai, Deborah; Carthagena, Laetitia; Wang, Qian; Xue, Yue Zhen; Quek, Ling Shih; Doorbar, John; Bachelerie, Francoise; Thierry, Francoise; Bellanger, Sophie

    2012-07-20

    The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1circumflexE4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2circumflexE4 transcripts resulting from 2 splice donor sites in the 5 Prime part of E2, while the splice acceptor site is the one used for E1circumflexE4. Both E2circumflexE4 transcripts are up-regulated by keratinocyte differentiation in vitro and can be detected in clinical samples containing low-grade HPV-18-positive cells from Pap smears. They give rise to two fusion proteins in vitro, E2circumflexE4-S and E2circumflexE4-L. Whereas we could not differentiate E2circumflexE4-S from E1circumflexE4 in vivo, E2circumflexE4-L could be formally identified as a 23 kDa protein in raft cultures in which the corresponding transcript was also found, and in a biopsy from a patient with cervical intraepithelial neoplasia stage I-II (CINI-II) associated with HPV-18, demonstrating the physiological relevance of E2circumflexE4 products.

  4. Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA

    PubMed Central

    Kim, Sanghyun; Zbaida, David; Elbaum, Michael; Leh, Hervé; Nogues, Claude; Buckle, Malcolm

    2015-01-01

    VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein–protein interactions. In order to isolate the protein–DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1–VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1–VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1. PMID:26044711

  5. A comprehensive framework of E2–RING E3 interactions of the human ubiquitin–proteasome system

    PubMed Central

    van Wijk, Sjoerd J L; de Vries, Sjoerd J; Kemmeren, Patrick; Huang, Anding; Boelens, Rolf; Bonvin, Alexandre M J J; Timmers, H Th Marc

    2009-01-01

    Covalent attachment of ubiquitin to substrates is crucial to protein degradation, transcription regulation and cell signalling. Highly specific interactions between ubiquitin-conjugating enzymes (E2) and ubiquitin protein E3 ligases fulfil essential roles in this process. We performed a global yeast-two hybrid screen to study the specificity of interactions between catalytic domains of the 35 human E2s with 250 RING-type E3s. Our analysis showed over 300 high-quality interactions, uncovering a large fraction of new E2–E3 pairs. Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others. We also found that the physical interactions of our screen compare well with reported functional E2–E3 pairs in in vitro ubiquitination experiments. For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B). Our data provide a detailed, genome-wide overview of binary E2–E3 interactions of the human ubiquitination system. PMID:19690564

  6. E2F1 promotes tumor cell invasion and migration through regulating CD147 in prostate cancer.

    PubMed

    Liang, Yu-Xiang; Lu, Jian-Ming; Mo, Ru-Jun; He, Hui-Chan; Xie, Jian; Jiang, Fu-Neng; Lin, Zhuo-Yuan; Chen, Yan-Ru; Wu, Yong-Ding; Luo, Hong-Wei; Luo, Zheng; Zhong, Wei-De

    2016-04-01

    Increased expression of E2F1 has been reported to be associated with tumor growth and cell survival of prostate cancer (PCa). However, its roles and mechanisms on PCa have not been fully elucidated. The present study found that E2F1 overexpression in PCa tissues was significantly associated with high Gleason score (P=0.01) and advanced pathological stage (P=0.02). In addition, PCa patients with high E2F1 expression more frequently had shorter biochemical recurrence-free survival (P=0.047) than those with low E2F1 expression. Then, we confirmed that the knock-down of E2F1 expression was able to inhibit cell cycle progression, invasion and migration of PCa cell lines in vitro, along with tumor xenograft growth and epithelial-to-mesenchymal transition (EMT) in vivo. Moreover, we identified CD147 as a novel interaction partner for E2F1 through bio-informatic binding site prediction, combined with chromatin immunoprecipitation-PCR (ChIP-PCR) and western blot analysis. Taken together, our data delineate an as yet unrecognized function of E2F1 as enhancer of tumor invasion and migration of PCa via regulating the expression of CD147 in PCa. Importantly, E2F1 may function as a biomarker that can differentiate patients with biochemical recurrent and non-biochemical recurrent disease following radical prostatectomy, highlighting its potential as a therapeutic target. PMID:26891801

  7. The retinoblastoma protein binds E2F residues required for activation in vivo and TBP binding in vitro.

    PubMed Central

    Hagemeier, C; Cook, A; Kouzarides, T

    1993-01-01

    The retinoblastoma (RB) tumour suppressor protein is capable of repressing the activity of promoters containing DNA binding sites for the transcription factor E2F. Recently a protein which binds RB and possesses the DNA binding characteristics of E2F has been cloned. Here we show that the E2F activation domain is the target for RB-induced repression. RB can silence the 57 residue E2F activation domain but cannot effectively repress an E2F mutant which has reduced RB binding capacity. Extensive mutagenesis of E2F shows residues involved in RB binding are required for transcription activation. Mutations which affect both functions most dramatically lie within the minimal RB binding region. A further subset of sensitive residues lies within a new repeat motif E/DF XX L X P which flanks the minimum RB binding site. These data show that RB can mask E2F residues involved in the activation process, possibly by mimicking a component of the transcriptional machinery. Consistent with this model, we find that the TATA box binding protein TBP can bind to the E2F activation domain in vitro in a manner indistinguishable from that of RB. Images PMID:8255752

  8. 40 CFR Table E-2 to Subpart E of... - Spectral Energy Distribution and Permitted Tolerance for Conducting Radiative Tests

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 5 2011-07-01 2011-07-01 false Spectral Energy Distribution and... REFERENCE AND EQUIVALENT METHODS Procedures for Testing Physical (Design) and Performance Characteristics of... E-2 Table E-2 to Subpart E of Part 53—Spectral Energy Distribution and Permitted Tolerance...

  9. 40 CFR Table E-2 to Subpart E of... - Spectral Energy Distribution and Permitted Tolerance for Conducting Radiative Tests

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 6 2014-07-01 2014-07-01 false Spectral Energy Distribution and... REFERENCE AND EQUIVALENT METHODS Procedures for Testing Physical (Design) and Performance Characteristics of..., Table E-2 Table E-2 to Subpart E of Part 53—Spectral Energy Distribution and Permitted Tolerance...

  10. 40 CFR Table E-2 to Subpart E of... - Spectral Energy Distribution and Permitted Tolerance for Conducting Radiative Tests

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 6 2012-07-01 2012-07-01 false Spectral Energy Distribution and... REFERENCE AND EQUIVALENT METHODS Procedures for Testing Physical (Design) and Performance Characteristics of... E-2 Table E-2 to Subpart E of Part 53—Spectral Energy Distribution and Permitted Tolerance...

  11. 40 CFR Table E-2 to Subpart E of... - Spectral Energy Distribution and Permitted Tolerance for Conducting Radiative Tests

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 6 2013-07-01 2013-07-01 false Spectral Energy Distribution and... REFERENCE AND EQUIVALENT METHODS Procedures for Testing Physical (Design) and Performance Characteristics of..., Table E-2 Table E-2 to Subpart E of Part 53—Spectral Energy Distribution and Permitted Tolerance...

  12. Structure of an E3:E2~Ub Complex Reveals an Allosteric Mechanism Shared among RING/U-box Ligases

    SciTech Connect

    Pruneda, Jonathan N.; Littlefield, Peter J.; Soss, Sarah E.; Nordquist, Kyle A.; Chazin, Walter J.; Brzovic, Peter S.; Klevit, Rachel E.

    2012-09-28

    Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanismby which this class of enzymes facilitates Ub transfer remains enigmatic. Here, we present a structural model for a RING/U-box E3:E2~Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2~Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 side chain and an E2 backbone carbonyl, observed in all structures of active RING/ U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2~Ub. The conformational biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/ U-box E3:E2~Ub ligase complex and identify the long sought-after source of allostery for RING/UBox activation of E2~Ub conjugates.

  13. Interaction of CSFV E2 protein with swine host factors as detected by yeast two-hybrid system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV). E2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. Howev...

  14. [The enhancement of DNA binding ability of a mutated E2 (A338V) protein of HPV-2].

    PubMed

    Pan, Ming-ming; Gao, Chen; Li, Xiao-li; Gong, Han-shi; Shi, Qi; Yuan, Yu-kang; Fan, Gui-xiang; Dong, Xiao-ping

    2010-05-01

    HPV-2 is a very common type of HPV which causes common warts. The E2 protein of virus can repress the activity of the viral early promoter through binding to the specific binding sites in viral LCR. Previously we reported that the repression of a mutated E2 protein of HPV-2 isolated from a patient with huge common wart on the viral early promoter was obviously decreased, and A338V mutation located at the C terminal DNA binding region of E2 protein. In this study, we expressed and purified the recombinant mutated and prototype E2 fusion proteins, both in the contexts of the C terminal and the full length, by prokaryotic expression system. The electrophoretic mobility shift assay showed E2 protein could bind to double-stranded DNA oligos labeled with biotin that covered two E2 binding sites. The DNA binding abilities of both C terminal and full-length mutated E2 proteins were stronger than the prototype analogs. This result indicates that the enhancement of the mutated E2 DNA binding ability may be the molecular mechanism for its impact on the activity of viral promoter, which correlates with the phenotype of extensive common wart. PMID:20572344

  15. Temporal variation in the estrogenicity of a sewage treatment plant effluent and its biological significance.

    PubMed

    Martinović, Dalma; Denny, Jeffrey S; Schmieder, Patricia K; Ankley, Gerald T; Sorensen, Peter W

    2008-05-01

    Daily variation in the estrogenic activity of effluent released by a modern sewage treatment plant (STP) was measured and its effects on the physiology, behavior, and reproductive success of male fish were evaluated. As measured by an estrogen receptor binding assay, the daily estrogenic activity of this effluent was both high and extremely variable (42 +/- 25.4 [mean +/- SD] ng 17beta-estradiol (E2) equivalents/L; n = 18). Liver VTG mRNA expression in male fathead minnows (FHM) covaried with the binding assay estimates, suggesting that these fluctuations are biologically relevant. Tests which exposed male FHMs to either fluctuating levels of E2, a constant concentration of E2 (time-weighted to reflect average concentrations), or control (no E2) demonstrated that while the estrogenic activity of this effluent was detrimental to male spawning success, the fact that its concentration varied in a daily manner was without additional influence. The variability of the effluent's estrogenicity suggests that studies concerned with the effects of STP effluents should collect multiple daily samples and then test them on an appropriate time-weighted basis. PMID:18522128

  16. 17β-Estradiol and Inflammation: Implications for Ischemic Stroke

    PubMed Central

    Petrone, Ashley B.; Simpkins, James W.; Barr, Taura L.

    2014-01-01

    Although typically associated with maintenance of female reproductive function, estrogens mediate physiological processes in nearly every body tissue, including the central nervous system. Numerous pre-clinical studies have shown that estrogen, specifically 17-beta-estradiol (17β-E2), protects the brain from ischemic injury following stroke. There are multiple mechanisms of 17β-E2’s neuroprotection, including activation of several neuroprotective pathways in the brain, but 17β-E2 also mediates the local and systemic immune response to ischemic stroke. This review summarizes the immune response to stroke, sex differences in stroke pathophysiology, and the role of estrogen as an immunomodulator. This review will focus almost entirely on the role of 17β-E2; however, there will be a brief review and comparison to other forms of estrogen. Understanding the immunomodulatory action of estrogens may provide an opportunity for the use of estrogens in treatment of stroke and other inflammatory disease. PMID:25276492

  17. Development and validation of an analytical method for detection of estrogens in water.

    PubMed

    Noppe, Herlinde; De Wasch, Katia; Poelmans, Sofie; Van Hoof, Nathalie; Verslycke, Tim; Janssen, Colin R; De Brabander, Hubert F

    2005-05-01

    An analytical procedure enabling routine analysis of four environmental estrogens at concentrations below 1 ng L(-1) in estuarine water samples has been developed and validated. The method includes extraction of water samples using solid-phase extraction discs and detection by gas chromatography (GC) with tandem mass spectrometry (MS-MS) in electron-impact (EI) mode. The targeted estrogens included 17alpha- and 17beta-estradiol (aE2, bE2), estrone (E1), and 17alpha-ethinylestradiol (EE2), all known environmental endocrine disruptors. Method performance characteristics, for example trueness, recovery, calibration, precision, accuracy, limit of quantification (LOQ), and the stability of the compounds are presented for each of the selected estrogens. Application of the procedure to water samples from the Scheldt estuary (Belgium - The Netherlands), a polluted estuary with reported incidences of environmental endocrine disruption, revealed that E1 was detected most frequently at concentrations up to 7 ng L(-1). aE2 was detected once only and concentrations of bE2 and EE2 were below the LOQ. PMID:15900457

  18. Modulation of the cytosolic androgen receptor in striated muscle by sex steroids

    NASA Technical Reports Server (NTRS)

    Rance, N. E.; Max, S. R.

    1984-01-01

    The effects of orchiectomy (GDX) and of subsequent administration of testosterone propionate (TP) or 17(beta)-estradiol (E2) on the maximum binding (Bmax) and apparent Kd of the cytosolic androgen receptor in levator ani (LA) and skeletal muscles of adult male Sprague-Dawley rats are investigated experimentally. The results are presented in graphs and discussed. In LA, BMAX is found to rise from a control level of 2.5 fmol/mg protein to 280, 600, 478, and 133 percent of control at 12 h, 14 d, 30 d, and 44 d after GDX, respectively, while Kd increased only insignificantly (from 680 to 960 fM); Bmax is held at control levels for 6 h by cycloheximide given at GDX, is unaffected by TP given at 30 d, and is further increased (by 480 percent at 44 d) by administration of E2 at 30 d. Bmax in skeletal muscles is found to increase to 139, 212, 220, and 158 percent of control at 12 h, 14 d, 30 d, and 44 d, respectively; Bmax is returned to control at 44 d by TP at 30 d but is not affected by E2. The effect of E2 in LA is attributed to either induction of the cytosolic receptor or a decreased rate of receptor degradation.

  19. Gonadotropin and testosterone measurements after estrogen administration to adult men, prepubertal and pubertal boys, and men with hypogonadotropism: evidence for maturation of positive feedback in the male.

    PubMed

    Kulin, H E; Reiter, E O

    1976-01-01

    Nineteen male subjects were fiven five daily injections of 17beta-estradiol and circulating levels of estradiol (E2), testerone (T), and gonadotropins were determined by radioimmunoassay before, during, and after the steroid course. Peak levels of E2 attained during the 5 days of treatment ranged from 173-577 pg/ml. Four of seven normal adult men and one castrate man demonstrated suppression of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) with a subsequent rise in LH (positive feedback) while E2 levels remained elevated. A rise in T was associated with the LH increment in the four normal men. Nine pre-, early, or midpubertal boys and two men with hypogonadotropic hypogonadism displayed only gonadotropin suppression after E2 administration. The difference in LH response to estrogen (i.e., positive feedback) between the adult men with normal or elevated gonadotropin levels as compared with the endocrinologically normal boys is significant (P less than 0.01). PMID:1107952

  20. Detection of ethylene glycol - toward W51/e2 and G34.3+0.02

    NASA Astrophysics Data System (ADS)

    Lykke, Julie M.; Favre, Cécile

    2014-07-01

    Ethylene glycol (HOCH2CH2OH), also commenly known as antifreeze, is the reduced alcohol version of glycolaldehyde (CH2OHCHO). Glycoladehyde - the simplest possible aldehyde sugar (Marstokk and Møllendal 1973) - is the first intermediate step in the path toward forming more complex and biologically relevant molecules through the the formose reaction, which begins with formaldehyde (H2CO) and ends with the formation of sugars and ultimately ribose, the backbone of RNA (e.g., Larralde et al. 1995). The presence of glycolaldehyde is therefore an important indication that processes leading to biologically relevant molecules are taking place. It is however, still unclear as to how glycolaldehyde and ethylene glycol are formed in the ISM. It has been proposed that they share a common formation pathway through UV-irradiation of methanol (CH3OH) ices mixed with CO (Öberg et al. 2009). So far, ethylene glycol, in its lower energy con-former (g’Ga(CH2OH)2), has been detected toward SgrB2 (N) by Hollis et al. (2002), tentatively toward IRAS 16293-2422 (Jørgensen et al. 2012) and marginally by Kalenskii and Johansson (2010) toward W51 e1/e2. Here we present a firm detection of ethylene glycol toward W51/e2 as well as a first detection toward G34.3+0.02 at 1mm and 3mm using the IRAM 30m telescope.

  1. A dynamic Asp-Arg interaction is essential for catalysis in microsomal prostaglandin E2 synthase.

    PubMed

    Brock, Joseph S; Hamberg, Mats; Balagunaseelan, Navisraj; Goodman, Michael; Morgenstern, Ralf; Strandback, Emilia; Samuelsson, Bengt; Rinaldo-Matthis, Agnes; Haeggström, Jesper Z

    2016-01-26

    Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of hum