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Sample records for 17beta-estradiol e2 plasmatic

  1. 17 beta-estradiol-BSA conjugates and 17 beta-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction.

    PubMed

    Sylvia, V L; Walton, J; Lopez, D; Dean, D D; Boyan, B D; Schwartz, Z

    2001-01-01

    Nuclear receptors for 17 beta-estradiol (E(2)) are present in growth plate chondrocytes from both male and female rats and regulation of chondrocytes through these receptors has been studied for many years; however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the cell response. E(2) was found to directly affect the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E(2) activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E(2)-dependent alkaline phosphatase activity and proteoglycan sulfation in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of the present study were: (1) to examine the effect of a cell membrane-impermeable 17 beta-estradiol-bovine serum albumin conjugate (E(2)-BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; (2) to determine the pathway that mediates the membrane effect of E(2)-BSA on PKC; and (3) to compare the action of E(2)-BSA to that of E(2). Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10(-9) to 10(-7) M E(2) or E(2)-BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [(3)H]-thymidine incorporation measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E(2)-BSA in the presence or absence of GDP beta S (inhibitor of G-proteins), GTP gamma S (activator of G-proteins), U73122 or D609 (inhibitors of phospholipase C [PLC]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3-kinase). E(2)-BSA mimicked the effects of E(2) on alkaline phosphatase specific activity and proteoglycan sulfation, causing dose-dependent increases in both RC and GC cell cultures. Both forms of estradiol inhibited [(3)H

  2. 17 beta-estradiol modulates GABAergic synaptic transmission and tonic currents during development in vitro.

    PubMed

    Pytel, Maria; Wójtowicz, Tomasz; Mercik, Katarzyna; Sarto-Jackson, Isabella; Sieghart, Werner; Ikonomidou, Chrysanthy; Mozrzymas, Jerzy W

    2007-05-01

    Estrogens exert a variety of modulatory effects on the structure and function of the nervous system. In particular, 17 beta-estradiol was found to affect GABAergic inhibition in adult animals but its action on GABAergic currents during development has not been elucidated. In the present study, we investigated the effect of 17 beta-estradiol on hippocampal neurons developing in vitro. In this model, mIPSC kinetics showed acceleration with age along with increased alpha1 subunit expression, similarly as in vivo. Long-term treatment with 17 beta-estradiol increased mIPSC amplitudes in neurons cultured for 6-8 and 9-11DIV and prolonged the mIPSC decaying phase only in the 9-11DIV group. The time needed for the onset of 17 beta-estradiol effect on mIPSC amplitude was approximately 48 h. In the period of 9-11DIV, treatment with 17 beta-estradiol strongly reduced the tonic conductance activated by low GABA concentrations. The effects of 17 beta-estradiol on mIPSCs and tonic conductance were not correlated with any change in expression of considered GABAAR subunits (alpha1-3, alpha5-6, gamma2) while alpha4 and delta subunits were at the detection limit. In conclusion, we provide evidence that 17 beta-estradiol differentially affects the phasic and tonic components of GABAergic currents in neurons developing in vitro.

  3. 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.

  4. Fate of nonylphenol and 17beta-estradiol contained in composted sewage sludge after land application.

    PubMed

    Minamiyama, M; Ochi, S; Suzuki, Y

    2008-01-01

    Many environmental problems caused by endocrine disrupters (EDs) have been reported. Because little is known about the fate of EDs accumulated in sewage sludge, we carried out a study to clarify the fate of EDs in composted sludge after its application to soil. Nonylphenol (NP) and 17beta-estradiol (E2) were measured for leachate and soil. High concentrations of NP and E2 were detected in the leachate at the early stage, but they decreased rapidly. Also, the high contents of NP and E2 in soil decreased significantly within 300 days. Because the decrease of NP and E2 in the soil was much larger than that of NP and E2 in the leachate, there must have been a physicochemical or biological decomposition mechanism in the soil layer. We also tried to clarify the transfer of NPs to plants from compost. In the experimental conditions of this study, the transfer of NPs to plants from compost was not observed. PMID:18235167

  5. Genistein potentiates the effect of 17-beta estradiol on human hepatocellular carcinoma cell line

    PubMed Central

    Kavoosi, Fraidoon; Dastjerdi, Mehdi Nikbakht; Valiani, Ali; Esfandiari, Ebrahim; Sanaei, Masumeh; Hakemi, Mazdak Ganjalikhani

    2016-01-01

    Background: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. This cancer may be due to a multistep process with an accumulation of epigenetic alterations in tumor suppressor genes (TSGs), leading to hypermethylation of the genes. Hypermethylation of TSGs is associated with silencing and inactivation of them. It is well-known that DNA hypomethylation is the initial epigenetic abnormality recognized in human tumors. Estrogen receptor alpha (ERα) is one of the TSGs which modulates gene transcription and its hypermethylation is because of overactivity of DNA methyltransferases. Fortunately, epigenetic changes especially hypermethylation can be reversed by pharmacological compounds such as genistein (GE) and 17-beta estradiol (E2) which involve in preventing the development of certain cancers by maintaining a protective DNA methylation. The aim of the present study was to analyze the effects of GE on ERα and DNMT1 genes expression and also apoptotic and antiproliferative effects of GE and E2 on HCC. Materials and Methods: Cells were treated with various concentrations of GE and E2 and the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay was used. Furthermore, cells were treated with single dose of GE and E2 (25 μM) and flow cytometry assay was performed. The expression level of the genes was determined by quantitative real-time reverse transcription polymerase chain reaction. Results: GE increased ERα and decreased DNMT1 genes expression, GE and E2 inhibited cell viability and induced apoptosis significantly. Conclusion: GE can epigenetically increase ERα expression by inhibition of DNMT1 expression which in turn increases apoptotic effect of E2. Furthermore, a combination of GE and E2 can induce apoptosis more significantly. PMID:27656602

  6. Genistein potentiates the effect of 17-beta estradiol on human hepatocellular carcinoma cell line

    PubMed Central

    Kavoosi, Fraidoon; Dastjerdi, Mehdi Nikbakht; Valiani, Ali; Esfandiari, Ebrahim; Sanaei, Masumeh; Hakemi, Mazdak Ganjalikhani

    2016-01-01

    Background: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. This cancer may be due to a multistep process with an accumulation of epigenetic alterations in tumor suppressor genes (TSGs), leading to hypermethylation of the genes. Hypermethylation of TSGs is associated with silencing and inactivation of them. It is well-known that DNA hypomethylation is the initial epigenetic abnormality recognized in human tumors. Estrogen receptor alpha (ERα) is one of the TSGs which modulates gene transcription and its hypermethylation is because of overactivity of DNA methyltransferases. Fortunately, epigenetic changes especially hypermethylation can be reversed by pharmacological compounds such as genistein (GE) and 17-beta estradiol (E2) which involve in preventing the development of certain cancers by maintaining a protective DNA methylation. The aim of the present study was to analyze the effects of GE on ERα and DNMT1 genes expression and also apoptotic and antiproliferative effects of GE and E2 on HCC. Materials and Methods: Cells were treated with various concentrations of GE and E2 and the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay was used. Furthermore, cells were treated with single dose of GE and E2 (25 μM) and flow cytometry assay was performed. The expression level of the genes was determined by quantitative real-time reverse transcription polymerase chain reaction. Results: GE increased ERα and decreased DNMT1 genes expression, GE and E2 inhibited cell viability and induced apoptosis significantly. Conclusion: GE can epigenetically increase ERα expression by inhibition of DNMT1 expression which in turn increases apoptotic effect of E2. Furthermore, a combination of GE and E2 can induce apoptosis more significantly.

  7. Effects of 17 beta-estradiol exposure on Xenopus laevis gonadal histopathology.

    PubMed

    Wolf, Jeffrey C; Lutz, Ilka; Kloas, Werner; Springer, Timothy A; Holden, Larry R; Krueger, Henry O; Hosmer, Alan J

    2010-05-01

    The natural estrogen 17 beta-estradiol (E2) is a potential environmental contaminant commonly employed as a positive control substance in bioassays involving estrogenic effects. The aquatic anuran Xenopus laevis is a frequent subject of reproductive endocrine disruptor research; however, histopathological investigations have tended to be less than comprehensive. Consequently, a study was designed to characterize gross and microscopic changes in the gonads of X. laevis as a result of E2 exposure. Additional goals of this study, which consisted of three separate experiments, included the standardization of diagnostic terminology and criteria, the validation of statistical methodology, and the establishment of a half maximal effective concentration (EC50) for E2 as defined by an approximately 50% conversion of presumptive genotypic males to phenotypic females. In the first experiment, frogs were exposed to nominal concentrations of 0, 0.2, 1.5, or 6.0 microg/L E2. From these experimental results and those of a subsequent range finding trial, the EC50 for E2 was determined to be approximately 0.2 microg/L. This E2 concentration was utilized in the other two experiments, which were performed at different facilities to confirm the reproducibility of results. Experiments were conducted according to Good Laboratory Practice guidelines, and the histopathologic evaluations were peer reviewed by an independent pathologist. Among the three trials, the histopathological findings that were strongly associated with E2-exposure (p<0.001 to 0.0001) included an increase in the proportion of phenotypic females, mixed sex, dilated testis tubules, dividing gonocytes in the testis, and dilated ovarian cavities in phenotypic ovaries. A comparison of the gross and microscopic evaluations suggested that some morphologic changes in the gonads may potentially be missed if studies rely entirely on macroscopic assessment.

  8. Comparative responses in rare minnow exposed to 17beta-estradiol during different life stages.

    PubMed

    Liao, T; Guo, Q L; Jin, S W; Cheng, W; Xu, Y

    2009-08-01

    Present in the excrement of humans and animals, 17beta-estradiol (E(2)) has been detected in the aquatic environment in a range from several nanograms to several hundred nanograms per liter. In this study, the sensitivities of rare minnows during different life stages to E(2) at environmentally relevant (5, 25, and 100 ng l(-1)) and high (1000 ng l(-1)) concentrations were compared using vitellogenin (VTG) and gonad development as biomarkers under semistatic conditions. After 21 days of exposure, VTG concentrations in whole-body homogenates were analyzed; the results indicated that the lowest observed effective concentration for VTG induction was 25 ng l(-1) E(2) in the adult stage, but 100 ng l(-1) E(2) in the larval and juvenile stages. After exposure in the early life stage, the larval and juvenile fish were transferred to clean water until gonad maturation. No significant difference in VTG induction was found between the exposure and control groups in the adults. However, a markedly increased proportion of females and appearance of hermaphrodism were observed in the juvenile-stage group exposed to 25 ng l(-1) E(2). These results showed that VTG induction in the adult stage is more sensitive than in larval and juvenile stages following exposure to E(2). The juvenile stage may be the critical period of gonad development. Sex ratio could be a sensitive biomarker indicating exposure to xenoestrogens in early-life-stage subchronic exposure tests. The results of this study provide useful information for selecting sensitive biomarkers properly in aquatic toxicology testing.

  9. In vivo occupancy of female rat brain estrogen receptors by 17beta-estradiol and tamoxifen.

    PubMed

    Pareto, D; Alvarado, M; Hanrahan, S M; Biegon, A

    2004-11-01

    Estrogens or antiestrogens are currently used by millions of women, but the interaction of these hormonal agents with brain estrogen receptors (ER) in vivo has not been characterized to date. Our goal was to assess, in vivo, the extent and regional distribution of brain ER occupancy in rats chronically exposed to 17beta-estradiol (E(2)) or tamoxifen (TAM). For that purpose, female ovariectomized Sprague-Dawley rats were implanted with subcutaneous pellets containing either placebo (OVX), E(2), or TAM for 3 weeks. ER occupancy in grossly dissected regions was quantified with 16alpha-[(18)F]fluoroestradiol ([(18)F]FES). Both E(2) and TAM produced significant decreases in radioligand uptake in the brain although the effect of E(2) was larger and more widespread than the effect of TAM. Detailed regional analysis of the interaction was then undertaken using a radioiodinated ligand, 11beta-methoxy-16alpha-[(125)I]iodo-estradiol ([(125)I]MIE(2)), and quantitative ex vivo autoradiography. E(2) treatment resulted in near-complete (86.6 +/- 17.5%) inhibition of radioligand accumulation throughout the brain, while ER occupancy in the TAM group showed a marked regional distribution such that percentage inhibition ranged from 40.5 +/- 15.6 in the ventrolateral part of the ventromedial hypothalamic nucleus to 84.6 +/- 4.5 in the cortical amygdala. These results show that exposure to pharmacologically relevant levels of TAM produces a variable, region-specific pattern of brain ER occupancy, which may be influenced by the regional proportion of ER receptor subtypes. These findings may partially explain the highly variable and region-specific effects observed in neurochemical, metabolic, and functional studies of the effects of TAM in the brain of experimental animals as well as human subjects.

  10. 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.

  11. 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.

  12. Effect of 17beta-estradiol on the expression of somatostatin genes in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Holloway, A C; Melroe, G T; Ehrman, M M; Reddy, P K; Leatherland, J F; Sheridan, M A

    2000-08-01

    In the present study, the effects of 17beta-estradiol (E(2)) treatment on the expression of preprosomatostatin (PPSS) I, PPSS II', and PPSS II" mRNA in the hypothalamus and endocrine pancreas (Brockmann body), as well as the effects of E(2) treatment on plasma somatostatin (SS)-14 and -25 concentrations in sexually immature rainbow trout (Oncorhynchus mykiss), were investigated. E(2) treatment significantly (P < 0.001) depressed both plasma SS-14 and SS-25. In the hypothalamus, E(2) treatment significantly (P < 0.001) decreased the levels of PPSS I and PPSS II" mRNA. However, there was no effect of E(2) treatment on PPSS II' mRNA levels. In the pancreas, E(2) treatment had no significant effect on the levels of either PPSS II' mRNA or PPSS II" mRNA. However, E(2) treatment significantly (P < 0.005) decreased levels of PPSS I mRNA. These data suggest that E(2) acts, in part, to increase plasma growth hormone levels in rainbow trout by decreasing the endogenous inhibitory somatostatinergic tone by inhibiting plasma levels of both SS-14 and SS-25 and hypothalamic levels of mRNA encoding these proteins. PMID:10938224

  13. 17beta-estradiol and tamoxifen stimulate rapid and transient ERK activationin MCF-7 cells via distinct signaling mechanisms.

    PubMed

    Visram, Hasina; Greer, Peter A

    2006-12-01

    Traditionally, estrogen signaling was thought to be mediated strictly through genomic pathways. Recently, however, it has been demonstrated that estrogen stimulation of cells leads to rapid nongenomic effects including ERK activation. While the precise mechanism of this action is still under investigation, it is known that activation of the epidermal growth factor receptor, the Src tyrosine kinase, and metalloproteinases are involved in this process. More recently, tamoxifen, an anti-hormonal agent used to treat breast cancer, has been shown to also activate ERK. The pathways by which it does so, however, are not known. Using the MCF-7 human breast carcinoma cell line as a model system, we show that ERK is rapidly and transiently activated in cells challenged with epidermal growth factor (EGF), 17beta-estradiol (E2) or tamoxifen. The ERK activation response to E2 and tamoxifen was kinetically similar, although the response to tamoxifen was delayed relative to that of E2 stimulation. The effect of the EGFR inhibitor AG1517 revealed that E2 and tamoxifen were both equally dependent on EGFR for activation of ERK. In contrast, inhibition of Src or metalloproteinases caused distinct effects on ERK activation by E2 and tamoxifen. Thus, while both E2 and tamoxifen induced activation of ERK, the differences in the effects of inhibitors of Src or metalloproteinases on ERK activation indicated that E2 and tamoxifen do so via distinct molecular mechanisms.

  14. 17beta-estradiol suppresses cytotoxicity and proliferative capacity of murine splenic NK1.1+ cells.

    PubMed

    Hao, Sha; Li, Pengfei; Zhao, Junli; Hu, Yali; Hou, Yayi

    2008-10-01

    In order to clarify the effects of 17beta-estradiol (E2) on natural killer (NK) cells and the possibly regulatory mechanisms, we obtained highly purified and viable NK cells from C57BL/6J mouse spleen by a magnetic cell sorter (MACS). These cells were treated with E2 and then their cytotoxicity and proliferative capacity were examined. To further investigate the mechanisms on the effect of E2 on NK cells, expressions of activation-associated markers (CD69, CD122) and inhibitory receptors (CD94, Ly49), and intracellular cytokine production were analyzed. At last, we performed the cDNA microarray to explore the possible involved genes. We found that E2 could suppress NK cell cytotoxicity and proliferative capacity in vitro. E2 reduced NK cell cytotoxicity and proliferative capacity, which may be through influencing the phenotypes and cytokine expression of NK cells, mainly involving CD94 and IFN-gamma. Furthermore, regulation of Stat4, Fyn, Sh2d1a, Eat2, Cd244, Irf1, Runx1, Irf7, Irf5, Esrra and Nr5a1 genes may be related to the cytotoxicity, proliferation and cytokine production of E2-mediated purified NK cells.

  15. Residual of 17beta-estradiol in digestion liquid generated from a biogas plant using livestock waste.

    PubMed

    Suzuki, Y; Kubota, A; Furukawa, T; Sugamoto, K; Asano, Y; Takahashi, H; Sekito, T; Dote, Y; Sugimoto, Y

    2009-06-15

    A biogas plant using livestock waste in which a methane fermentation process is applied is a useful facility for generating energy. The digestion liquid generated from the biogas plant as a residue has high potential for use as a crop fertilizer. However, high-density estrogens such as 17beta-estradiol (E2) are included in livestock waste, and there is little information on the behavior of E2 in the digestion liquid. In this study, a survey of E2 concentration at each process in a biogas plant using livestock waste was carried out. In addition, the efficiencies of E2 removal from the digestion liquid by activated carbon adsorption and soil infiltration were examined. The total concentration of E2 in raw livestock waste was reduced to 2 microg/l after treatment, and the removal efficiency of E2 was about 80% for the plant. The methane fermentation process is important not only for the generation of methane but also for the removal of E2. The proportion of E2 conjugates comprising the total E2 concentration was 10% or less in all treated samples. In the plant, there is no likelihood of an increase in estrogen activity by the cleaving of E2 conjugates. By carrying out activated carbon adsorption to remove E2 from the digestion liquid, a large portion of E2 was removed from the digestion liquid, but an E2 concentration of 0.5 microg/l still remained in the treated digestion liquid. In contrast, it was possible to purify the digestion liquid to an E2 concentration of less than 0.002-0.011 microg/l by soil infiltration. It is thus possible to utilize the digestion liquid as a fertilizer without causing aquatic environmental pollution, but factors such as application rate, soil characteristics, and the E2 concentration of digestion liquid should be considered first.

  16. A possible alternative to 17beta-estradiol in a viviparous lizard, Tiliqua nigrolutea.

    PubMed

    Edwards, Ashley; Jones, Susan M; Davies, Noel W

    2002-11-01

    We have detected an unusual polar steroid as a major end product of gonadal steroid biosynthesis in male and female blotched blue-tongued lizards, Tiliqua nigrolutea. In an investigation of the steroidogenic pathways in blue-tongued lizards, we incubated gonads of both male and female lizards with tritiated pregnenolone and identified the products of steroid biosynthesis using thin layer chromatography and high performance liquid chromatography with radiometric detection: gonads from both reproductively active and quiescent lizards were examined. The proportion of total radioactive substrate converted to the unknown polar steroid was greater in individuals with hypertrophied gonads (males (N=4) 28.1%; females (N=4) 45.3%) than in those with regressed gonads (males (N=4) 5.9%; females (N=4) 33.3%). Properties of the unknown steroid were identical across all incubations. Incubation time-course information, thin layer and high performance liquid chromatography properties of the polar steroid are presented. The steroid is more polar than 17beta-estradiol, which was not detected, but less polar than all tri-hydroxylated estrogens to which it was compared. The possibility of an alternative estrogen to 17beta-estradiol in some reptiles is discussed.

  17. Upregulation of steroidogenic enzymes and ovarian 17beta-estradiol in human granulosa-lutein cells by Cordyceps sinensis mycelium.

    PubMed

    Huang, Bu-Miin; Hsiao, Kuei-Yang; Chuang, Pei-Chin; Wu, Meng-Hsing; Pan, Hsien-An; Tsai, Shaw-Jenq

    2004-05-01

    There is increasing evidence that 17beta-estradiol (E2) directly influences the quality of maturing oocytes and thus the outcome of assisted reproduction treatment. Although Cordyceps sinensis (CS) mycelium, a Chinese herbal medicine, is believed to enhance libido and fertility in both sexes, the mechanism of its effect in women has not been determined. The aim of the present study was to evaluate the effects of CS on steroidogenic enzyme expression and E2 biosynthesis in human granulosa-lutein cells (GLC). We found that CS induced E2 production by GLC in a dose- and time-dependent manner and that a 3-h treatment with CS induced increased levels of mRNAs coding for the P450 side chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), and aromatase. Western blot analysis demonstrated that, after treatment with CS for 3 h, protein levels of steroidogenic acute regulatory protein (StAR) and aromatase were upregulated while P450scc and 3beta-HSD levels showed no substantial change. New protein synthesis was required for CS-induced E2 production because it was abrogated by cycloheximide pretreatment. Addition of 22(R)-hydroxycholesterol, thus bypassing the need for StAR protein, did not induce as much E2 production as CS treatment, indicating that upregulation of StAR protein was not the only factor contributing to CS-induced steroidogenesis. Cotreatment of GLCs with CS and aminoglutethimide, an aromatase inhibitor, completely abolished CS-induced E2 production. In conclusion, treatment of GLCs with CS results in increased E2 production due, at least in part, to increased StAR and aromatase expression. These data may help in the development of treatment regimens to improve the success rate of in vitro fertilization. PMID:14711788

  18. 17beta-estradiol attenuates hyperoxia-induced apoptosis in mouse C8-D1A cell line.

    PubMed

    Huppmann, Stephanie; Römer, Susanne; Altmann, Rodica; Obladen, Michael; Berns, Monika

    2008-11-15

    In premature infants, oxygen free radicals generated following neonatal resuscitation are associated with subsequent diseases such as retinopathy of prematurity and bronchopulmonary dysplasia. Recent studies in brain tissue samples have shown that nonphysiologic oxygen levels play a key role in induction of apoptosis in the developing brain. Estrogen is a well-established agent in neuroprotection and, therefore, is thought to be neuroprotective even in the premature brain. Astrocytes appear to have a critical role in protection and survival of neurons in the brain. As one of the glial cell types, they have a great potential for possible involvement in the mediation of estrogen neuroprotective effects. The aim of our study was to analyze whether astrocytes in cell cultures are damaged by hyperoxia and whether 17beta-estradiol (E2) can protect them against apoptosis. Additionally, we investigated the mechanism of the protection by E2, hypothesizing that it is mediated through extracellular signal-regulated kinase (ERK1/2). Cells underwent eightfold more apoptosis when cultivated in hyperoxia compared with normoxia. Addition of E2 reduced apoptosis in hyperoxia by more than 50%. Levels of ERK1/2 and phosphorylated ERK1/2 were increased after hyperoxia compared with normoxia. Preincubation with E2 prior to exposure to hyperoxia resulted in decreased levels of ERK1/2 and pERK1/2. Hyperoxia induces apoptosis in C8-D1A cells, and E2 seems to be a protecting factor for astrocytes in hyperoxia. This effect is not mediated through up-regulation of pERK1/2.

  19. Effects of 17-beta estradiol and 4-nonylphenol on phase II electrophilic detoxification pathways in largemouth bass (Micropterus salmoides) liver.

    PubMed

    Hughes, Erin M; Gallagher, Evan P

    2004-03-01

    The effects of in vivo exposure to a natural and synthetic estrogen upon three hepatic phase II enzyme pathways involved in cellular protection against reactive intermediates were investigated in the largemouth bass (Micropterus salmoides). The pathways analyzed included glutathione S-transferases (GST), glutathione (GSH) biosynthesis and NAD(P)H-dependent quinone reductase (QR). Following exposure to 17-beta estradiol (E2, a model natural estrogen; 2 mg/kg, i.p.) or 4-nonylphenol (NP, a model synthetic estrogen; 5 mg/kg and 50 mg/kg, i.p.), serum vitellogenin concentrations in male fish were markedly increased. Exposure to E2 did not affect steady-state GST-A mRNA expression, although GST catalytic activity toward 1-chloro 2,4-dinitrobenzene (CDNB) was elevated at 48 h post-injection. In addition, the rates of bass liver GST-4-hydroxy-2-nonenal (GST-4HNE) conjugation were elevated by E2 exposure at all timepoints. In contrast, exposure to NP decreased steady-state GST-A mRNA levels, but did not alter GST catalytic activities. Hepatic GSH levels were not significantly affected by exposure to either compound, although a trend towards increased GSH biosynthesis was observed with both compounds. Although bass liver quinone reductase catalyzed 2,6-dichloroindophenol (DCP) reduction, unlike in rodents, these catalytic activities were not inhibited by dicoumarol. Exposure to 5 mg/kg NP significantly increased hepatic QR activities. Collectively, our data suggest that exposure to E2 or NP alters the ability of largemouth bass to biotransform environmental chemicals through glutathione S-transferase and quinone reductase catalytic pathways.

  20. Fate of sulfamethoxazole, 4-nonylphenol, and 17beta-estradiol in groundwater contaminated by wastewater treatment plant effluent.

    PubMed

    Barber, Larry B; Keefe, Steffanie H; Leblanc, Denis R; Bradley, Paul M; Chapelle, Francis H; Meyer, Michael T; Loftin, Keith A; Kolpin, Dana W; Rubio, Fernando

    2009-07-01

    Organic wastewater contaminants (OWCs) were measured in samples collected from monitoring wells located along a 4.5-km transect of a plume of groundwater contaminated by 60 years of continuous rapid infiltration disposal of wastewater treatment plant effluent. Fifteen percent of the 212 OWCs analyzed were detected, including the antibiotic sulfamethoxazole (SX), the nonionic surfactant degradation product 4-nonylphenol (NP), the solvent tetrachloroethene (PCE), and the disinfectant 1,4-dichlorobenzene (DCB). Comparison of the 2005 sampling results to data collected from the same wells in 1985 indicates that PCE and DCB are transported more rapidly in the aquiferthan NP, consistent with predictions based on compound hydrophobicity. Natural gradient in situ tracer experiments were conducted to evaluate the subsurface behavior of SX, NP, and the female sex hormone 17beta-estradiol (E2) in two oxic zones in the aquifer: (1) a downgradient transition zone at the interface between the contamination plume and the overlying uncontaminated groundwater and (2) a contaminated zone located beneath the infiltration beds, which have not been loaded for 10 years. In both zones, breakthrough curves for the conservative tracer bromide (Br-) and SX were nearly coincident, whereas NP and E2 were retarded relative to Br- and showed mass loss. Retardation was greater in the contaminated zone than in the transition zone. Attenuation of NP and E2 in the aquifer was attributed to biotransformation, and oxic laboratory microcosm experiments using sediments from the transition and contaminated zones show that uniform-ring-labeled 14C 4-normal-NP was biodegraded more rapidly 130-60% recovered as 14CO2 in 13 days) than 4-14C E2 (20-90% recovered as 14CO2 in 54 days). There was little difference in mineralization potential between sites.

  1. Gene expression responses of European flounder (Platichthys flesus) to 17-beta estradiol.

    PubMed

    Williams, Tim D; Diab, Amer M; George, Stephen G; Sabine, Victoria; Chipman, James K

    2007-02-01

    Male European flounder (Platichthys flesus) were intraperitoneally injected with 10mg/kg 17-beta estradiol and tissues taken from individuals over a timecourse of 16 days. The GENIPOL P. flesus cDNA microarray was employed to detect hepatic gene expression differences between fish treated with estradiol and saline controls. Known biomarkers of estrogen exposure, choriogenin L and vitellogenins, showed sustained induction over the time-course. Among 175 identified clones showing sustained statistically significant induction or repression, those associated with the Gene Ontology terms mitochondria, amino acid synthesis, ubiquitination and apoptosis were included amongst those induced while those associated with immune function, electron transport, cell signalling and protein phosphorylation were repressed. Thus, we show the gene expression response of an environmentally relevant fish species to a high dose of an estrogenic endocrine disruptor and also report the sequencing of a further 2121 flounder ESTs.

  2. Treatment of oophorectomized guinea pigs with intrauterine 17 beta-estradiol pellets may modulate myometrial beta-adrenergic receptor binding properties.

    PubMed

    Hatjis, C G; Grogan, D M; Koritnik, D R

    1989-12-01

    Intrauterine 17 beta-estradiol pellets can induce an up-regulation of guinea pig myometrial beta-adrenergic receptor density and l-isoproterenol-dependent adenylate cyclase activity. Does 17 beta-estradiol influence the ability of beta-adrenergic receptors to form a "high affinity" state with l-isoproterenol, which is a necessary step for adenylate cyclase activation? Nonpregnant, oophorectomized guinea pigs received intrauterine pellets of either placebo, 17 beta-estradiol, progesterone, or 17 beta-estradiol plus progesterone for 1 week. 17 beta-Estradiol resulted in pharmacologic, whereas progesterone resulted in physiologic plasma 17 beta-estradiol and progesterone concentrations, respectively. The affinity of myometrial beta-adrenergic receptors for l-isoproterenol was measured by percentage of inhibition of -[125I]cyanopindolol binding. In all groups, the competition curves in the presence of magnesium chloride could be resolved into two affinity states of the beta-adrenergic receptor, "high" and "low," respectively. The ratio of their dissociation constants was not influenced by hormonal treatment. However, the relative concentration of beta-adrenergic receptors in the high affinity state was significantly higher in the 17 beta-estradiol-treated group than that in the control group. This correlates with the up-regulation in myometrial adenylate cyclase activity and suggests that myometrial beta-adrenergic receptor-adenylate cyclase function may be modulated by 17 beta-estradiol.

  3. 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.

  4. 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

  5. 90-day feeding and one-generation reproduction study in Crl:CD BR rats with 17 beta-estradiol.

    PubMed

    Biegel, L B; Flaws, J A; Hirshfield, A N; O'Connor, J C; Elliott, G S; Ladics, G S; Silbergeld, E K; Van Pelt, C S; Hurtt, M E; Cook, J C; Frame, S R

    1998-08-01

    Over the past several years, there has been increasing concern that chemicals and pesticides found in the environment may mimic endogenous estrogens, potentially producing adverse effects in wildlife and human populations. Because estrogenicity is one of the primary concerns, a 90-day/one-generation reproduction study with 17 beta-estradiol was designed to set dose levels for future multigenerational reproduction and combined chronic toxicity/oncogenicity studies. The purpose of these studies is to evaluate the significance of a range of responses as well as to provide benchmark data for a risk assessment for chemicals with estrogen-like activities. This 90-day/one-generation reproduction study was conducted in male and female Crl:CD BR rats using dietary concentrations of 0, 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol. Endpoints were chosen in order to evaluate both subchronic and reproductive toxicity. In addition, several mechanistic/biochemical endpoints were evaluated for their usefulness in follow-up studies. In the P1 generation, dietary administration of 2.5, 10, and 50 ppm 17 beta-estradiol produced dose-dependent decreases in body weight, body weight gain, food consumption, and food efficiency. At 10 and 50 ppm 17 beta-estradiol, minimal to mild nonregenerative anemia, lymphopenia, decreased serum cholesterol (50 ppm only), and altered splenic lymphocyte subtypes were also observed in the P1 generation. Additionally, at these concentrations, there were changes in the weights of several organs. Evidence of ovarian malfunction, characterized by reduced numbers of corpora lutea and large antral follicles, was observed at 2.5 ppm 17 beta-estradiol and above. Other pathologic changes in males and females fed 10 and 50 ppm 17 beta-estradiol included centrilobular hepatocellular hypertrophy; diffuse hyperplasia of the pituitary gland; feminization of the male mammary glands; mammary gland hyperplasia in females; increased number of cystic follicles in the ovary

  6. Assessment of Neuroprotective Effects of Local Administration of 17- Beta- Estradiol on Peripheral Nerve Regeneration in Ovariectomized Female Rats

    PubMed Central

    Nobakhti-Afshar, Ahmadreza; Najafpour, Alireza; Mohammadi, Rahim; Zarei, Leila

    2016-01-01

    Objective: To assess the neuroprotective effects of local administration of 17- beta- estradiol on nerve regeneration. Methods: Sixty female Wistar rats were overiectomized and divided into four experimental groups (n = 15), randomly: In autograft group a segment of sciatic nerve was transected and re-implanted reversely. In sham-surgery group sciatic nerve was exposed and manipulated. In transected group left sciatic nerve was transected and stumps were fixed in adjacent muscle. In treatment group defect was bridged using a silicon conduit filled with 10 µL (0.1 mg/mL) 17- beta- estradiol. Each group was subdivided into four subgroups of five animals each and nerve fibers were studied in a 12-week period. Results: Behavioral, functional, biomechanical, electrophysiological and gastrocnemius muscle mass findings and morphometric indices confirmed faster recovery of regenerated axons in treatment group than in other groups (p<0.05). Immunohistochemical reactions to S-100 in treatment group were more positive than that in other groups. Conclusion: Local administration of 17-beta-estradiol improved functional recovery and morphometric indices of sciatic nerve. It could have clinical implications for the surgical management of patients after facial nerve transection. PMID:27540548

  7. Modulation of ovine fetal adrenocorticotropin secretion by androstenedione and 17beta-estradiol.

    PubMed

    Saoud, C J; Wood, C E

    1997-04-01

    Parturition in sheep is initiated by increases in activity of the fetal hypothalamic-pituitary-adrenal axis. We have previously reported that cortisol negative feedback efficacy is decreased at the end of gestation. The present study was designed to test the hypothesis that increasing plasma estrogen and/or androgen concentrations in the fetus might increase plasma adrenocorticotropic hormone (ACTH) concentration, either by stimulating ACTH secretion or by altering the negative feedback effect of cortisol on ACTH. Fetal sheep were chronically catheterized and treated with no steroid (control), 17beta-estradiol, or androstenedione (each approximately 0.24 mg/day). After catheterization and implantation of steroid pellet, fetuses were subjected to two short (10 min) periods of sodium nitroprusside-induced hypotension with or without pretreatment with intravenous infusion of hydrocortisone sodium succinate (0.5 microg/min) to test fetal ACTH responsiveness to stress and cortisol negative feedback efficacy. Estradiol treatment significantly increased basal plasma ACTH and cortisol concentrations relative to control fetuses but did not interfere with the inhibition of ACTH secretion by cortisol. Fetal plasma ACTH responses to hypotension were significantly suppressed approximately 60% in both control and estradiol-treated groups. Androstenedione treatment significantly increased basal fetal plasma ACTH and decreased basal fetal plasma cortisol concentration. Androstenedione did not alter stimulated levels of fetal ACTH but did block the inhibition of stimulated ACTH by cortisol. We conclude that increased fetal cortisol and ACTH secretion at the end of gestation may be due to the combined effects of the gonadal steroids in that estradiol increases basal plasma ACTH secretion while androstenedione reduces cortisol negative feedback efficacy.

  8. 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.

  9. Altered sexual characteristics in guppies (Poecilia reticulata) exposed to 17beta-estradiol and 4-tert-octylphenol during sexual development.

    PubMed

    Toft, Gunnar; Baatrup, Erik

    2003-10-01

    The effects of estrogenic compounds on the development of sexual characteristics in juvenile guppies (Poecilia reticulata) were examined. After exposure for 3 months, the sex ratio was female biased in guppies exposed to 0.5 microg/L 17beta-estradiol (E2) or higher but unaffected by exposure to 4-tert-octylphenol (OP). When exposed to 100 microg/L OP, the male guppy sexual behavior and sperm count were increased. In contrast, the male sperm count was markedly reduced after exposure to a high concentration of E2. The length of the gonopodium (copulatory organ) was increased when exposed to 100 microg/L OP and at all the tested concentrations of E2. Similar concentrations of OP and E2 reduced the gonad weight of the females. The results demonstrate that the development of sexual characteristics in guppies can be disturbed by environmentally realistic concentrations of E2 but higher concentrations of OP are needed to induce similar alterations.

  10. 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.

  11. A computational model of the hypothalamic-pituitary-gonadal axis in male fathead minnows exposed to 17alpha-ethinylestradiol and 17beta-estradiol.

    PubMed

    Watanabe, Karen H; Li, Zhenhong; Kroll, Kevin J; Villeneuve, Daniel L; Garcia-Reyero, Natàlia; Orlando, Edward F; Sepúlveda, Maria S; Collette, Timothy W; Ekman, Drew R; Ankley, Gerald T; Denslow, Nancy D

    2009-06-01

    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, 17alpha-ethinylestradiol (EE(2)) and 17beta-estradiol (E(2)), have been measured in wastewater treatment effluents and have been shown to cause adverse effects in fish. To further our understanding of how estrogen exposure affects reproductive endpoints in the male fathead minnow (FHM, Pimephales promelas), a physiologically based computational model was developed of the hypothalamic-pituitary-gonadal (HPG) axis. Apical reproductive endpoints in the model include plasma steroid hormone and vitellogenin concentrations. Using Markov chain Monte Carlo simulation, the model was calibrated with data from unexposed FHM, and FHM exposed to EE(2) and E(2). Independent experimental data sets were used to evaluate model predictions. We found good agreement between our model predictions and a variety of measured reproductive endpoints, although the model underpredicts unexposed FHM reproductive endpoint variances, and overpredicts variances in estrogen-exposed FHM. We conclude that this model provides a robust representation of the HPG axis in male FHM.

  12. Gamma irradiation for terminal sterilization of 17beta-estradiol loaded poly-(D,L-lactide-co-glycolide) microparticles.

    PubMed

    Mohr, D; Wolff, M; Kissel, T

    1999-08-27

    17beta-Estradiol-loaded microparticles using poly-(D, L-lactide-co-glycolide) polymer (PLG) were prepared by a modified spray-drying method and the effects of gamma-irradiation on drug substance, polymer and microparticles were investigated. Irradiation doses ranging from 5.1 to 26.6 kGy were applied using a 60Co-radiation source. 17beta-Estradiol drug substance showed excellent stability against gamma-irradiation in the investigated dose range, whereas microencapsulated estradiol seems to be converted to conjugation products with PLG, and to a lesser extent to the degradation product 9,11-dehydroestradiol. The weight-average molecular weight of the PLG polymers decreased with increasing irradiation dose while polydispersity indices (M(w)/M(n)) remained nearly unchanged, compatible with a random chain scission mechanism in lactide/glycolide-copolymer degradation. In vitro drug release studies showed accelerated kinetics with increasing irradiation doses due to dose dependent polymer degradation. Microbiological process monitoring showed decreasing bioburden with increasing spraying time, which was successfully further reduced by applying irradiation sterilization. Microencapsulated test spore suspensions of Bacillus pumilus ATCC 27142, the official test specimen for the gamma-sterilization process, revealed effective reduction of bioburden, confirming its published D(10) value. In conclusion, our studies demonstrated efficacy of gamma-irradiation as terminal sterilization method for poly-(D,L-lactide-co-glycolide) polymer-based drug delivery systems. The sterilization conditions need to be carefully adjusted for the final dosage form. PMID:10469916

  13. Effects of garlic oil on postmenopausal osteoporosis using ovariectomized rats: comparison with the effects of lovastatin and 17beta-estradiol.

    PubMed

    Mukherjee, M; Das, A S; Das, D; Mukherjee, S; Mitra, S; Mitra, C

    2006-01-01

    The purpose of this study was to examine the antiosteoporosis effects of garlic oil in an ovariectomized (Ovx) rat model of osteoporosis and to compare its efficacy with lovastatin (a synthetic hypocholesterolemic drug) and 17beta-estradiol (a potent antiosteoporotic agent). Animals were divided into five groups: sham-operated control, ovariectomized, ovariectomized supplemented with lovastatin, ovariectomized supplemented with garlic oil and ovariectomized supplemented with 17beta-estradiol. In our study, the development of a high rate of bone turnover and osteoporosis in the ovariectomized animals were confirmed by significant alterations of serum alkaline phosphatase activity, serum tartrate-resistant acid phosphatase activity, urinary excretion of calcium, phosphate, hydroxyproline and urinary calcium to creatinine ratio, when compared with the sham-operated control group. Supplementation of these animals with either garlic oil or lovastatin or 17beta-estradiol, in addition to their hypocholesterolemic effect, could counterbalance all these changes. The results revealed that all three compounds significantly protected the hypogonadal bone loss as reflected by higher bone densities and higher bone mineral contents than the ovariectomized group of animals. The results emphasize that, like 17beta-estradiol, the hypocholesterolemic compounds garlic oil and lovastatin are also effective in suppressing bone loss owing to estrogen deficiency and their efficacy in the order of lower to higher is garlic < lovastatin < 17beta-estradiol.

  14. 17beta-estradiol attenuates programmed cell death in cortical pericontusional zone following traumatic brain injury via upregulation of ERalpha and inhibition of caspase-3 activation.

    PubMed

    Li, Li-Zhuo; Bao, Yi-Jun; Zhao, Min

    2011-01-01

    Pericontusional zone (PCZ) of traumatic cerebral contusion is a target of pharmacological intervention. It is well studied that 17beta-estradiol has a protective role in ischemic brain injury, but its role in brain protection of traumatic brain damage deserves further investigation, especially in pericontusional zone. Here we show that 17beta-estradiol enhances the protein expression and mRNA induction of estrogen alpha receptor (ERalpha) and prevents from programmed cell death in cortical pericontusional zone. ERalpha specific antagonist blocks this protective effect of 17beta-estradiol. Caspase-3 activation occurs in cortical pericontusional zone of the oil-treated injured rat brain and its activation is inhibited by 17beta-estradiol treatment. Additionally, ERalpha specific antagonist reverses this inhibition. Pan-caspase inhibitor also protect cortical pericontusional zone from programmed cell death. Our present study indicates 17beta-estradiol protects from programmed cell death in cortical pericontusional zone via enhancement of ERalpha and decrease of caspase-3 activation.

  15. Signaling responses after exposure to 5 alpha-dihydrotestosterone or 17 beta-estradiol in norepinephrine-induced hypertrophy of neonatal rat ventricular myocytes.

    PubMed

    Koshman, Yevgeniya E; Piano, Mariann R; Russell, Brenda; Schwertz, Dorie W

    2010-03-01

    Androgens appear to enhance, whereas estrogens mitigate, cardiac hypertrophy. However, signaling pathways in cells for short (3 min) and longer term (48 h) treatment with 17beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT) are understudied. We compared the effect of adrenergic stimulation by norepinephrine (NE; 1 microM) alone or in combination with DHT (10 nM) or E2 (10 nM) treatment in neonatal rat ventricular myocytes (NRVMs) by cell area, protein synthesis, sarcomeric structure, gene expression, phosphorylation of extracellular signal-regulated (ERK), and focal adhesion kinases (FAK), and phospho-FAK nuclear localization. NE alone elicited the expected hypertrophy and strong sarcomeric organization, and DHT alone gave a similar but more modest response, whereas E2 did not alter cell size. Effects of NE dominated when used with either E2 or DHT with all combinations. Both sex hormones alone rapidly activated FAK but not ERK. Long-term or brief exposure to E2 attenuated NE-induced FAK phosphorylation, whereas DHT had no effect. Neither hormone altered NE-elicited ERK activation. Longer term exposure to E2 alone reduced FAK phosphorylation and reduced nuclear phospho-FAK, whereas its elevation was seen in the presence of NE with both sex hormones. The mitigating effects of E2 on the NE-elicited increase in cell size and the hypertrophic effect of DHT in NRVMs are in accordance with results observed in whole animal models. This is the first report of rapid, nongenomic sex hormone signaling via FAK activation and altered FAK trafficking to the nucleus in heart cells. PMID:20044473

  16. Aqueous exposure to 4-nonylphenol and 17beta-estradiol increases stress sensitivity and disrupts ion regulatory ability of juvenile Atlantic salmon.

    PubMed

    Lerner, Darren T; Björnsson, Björn Thrandur; McCormick, Stephen D

    2007-07-01

    Population declines of wild Atlantic salmon have been attributed to an array of anthropogenic disturbances, including dams, commercial and recreational fishing, habitat loss, and pollution. Environmental contaminants in particular, can act as environmental stressors on fish, typically causing disruption of ion homeostasis due to their close association with the aquatic environment. To examine the effects of the xenoestrogen 4-nonylphenol (NP) or 17beta-estradiol (E2) on stress sensitivity and ion regulation, we exposed juvenile Atlantic salmon continuously for 21 d to either 10 or 100 microg/L NP (NP-L or NP-H), 2 microg/L E2 (positive control), or vehicle control during the parr-smolt transformation in April. After treatment, fish were sampled in freshwater (FW), transferred to 30 per thousand seawater (SW) for 24 h, or subjected to a handling stress. Estradiol and NP-H increased plasma vitellogenin in males and females, and E2 increased gonadosomatic index only in males. In FW, E2 reduced sodium potassium-activated adenosine triphosphatase activity as well as plasma levels of growth hormone, insulin-like growth factor I, and triiodothyronine. Both E2 and NP-H reduced plasma sodium in FW and increased plasma chloride in SW. Plasma cortisol levels pre- and poststressor were significantly elevated by all treatments relative to controls, but only E2 increased plasma glucose before and after the stressor. These results indicate that exposure of anadromous salmonids to environmental estrogens heightens sensitivity to external stressors, impairs ion regulation in both FW and SW, and disrupts endocrine pathways critical for smolt development. PMID:17665683

  17. 17beta-estradiol-dependent activation of signal transducer and activator of transcription-1 in human fetal osteoblasts is dependent on Src kinase activity.

    PubMed

    Kennedy, Angela M; Shogren, Kristen L; Zhang, Minzhi; Turner, Russell T; Spelsberg, Thomas C; Maran, Avudaiappan

    2005-01-01

    Estrogen is essential for normal growth and remodeling of bone. Although the mechanism of estrogen action on bone cells has been widely investigated, the full spectrum of signal transduction pathways activated by estrogen is unknown. In this report, we investigate the effects of the gonadal hormone 17beta-estradiol on the regulation of signal transducer and activator of transcription-1 (Stat1) protein in cultured human fetal osteoblast cells, devoid of the classical estrogen receptors (ERs). 17beta-estradiol (10 nM) led to rapid (within 15 min) activation of Stat1 protein as indicated by increases in tyrosine phosphorylation and DNA binding activity. Also, 17beta-estradiol increased gamma-activated sequence-dependent transcription in transient transfection assays, suggesting an increase in Stat protein-dependent transcription. Estrogen-dependent Stat1 activation was blocked in cells that transiently express dominant-negative Stat1 mutant protein. Activation of Stat1 by 17beta-estradiol was not inhibited by ER antagonist ICI 182,780, providing further evidence that it is not dependent on classical ERs. 17beta-Estradiol induced rapid (within 15 min) Stat1 phosphorylation and stimulated gamma-activated sequence-dependent transcription in ER-negative breast cancer cells, indicating that these results are not unique to bone cells. The rapid estrogenic effect involving the phosphorylation and activation of Stat1 was blocked in the presence of Src family kinase inhibitor PP2; activated Stat1 was associated with Src protein in estrogen-treated cells. These findings indicate the requirement for Src kinase pathways in estrogen-mediated Stat1 activation. Thus, the ER-independent activation of Stat1 in 17beta-estradiol-treated osteoblast and breast cancer cells may partially mediate the actions of estrogen on target cells.

  18. Effect of plasma lipoproteins in gonadotropin stimulation of 17 beta-estradiol production in the ovarian follicle of rainbow trout (Salmo gairdneri).

    PubMed

    Babin, P J

    1986-12-01

    The effect of trout plasma lipoproteins on the production of 17 beta-estradiol by trout ovarian follicles is investigated in vitro. 17 beta-Estradiol secretion into the medium was assayed as a function of follicular diameter in the presence of lipoproteins with and without salmonid gonadotropin (SGA-GTH). The presence of very low-density lipoproteins (VLDL) + chylomicrons (Chy), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) amplified the SGA-GTH effect at the lowest concentrations tested (less than 50 micrograms protein/ml). HDL is the most effective for increasing hormone accumulation on a microgram lipoprotein sterol basis. Autoradiography of 125I-labeled LDL showed that they were preferentially bound by thecal cells. Kinetics of 17 beta-estradiol release indicated that lipoprotein amplification occurred especially after 15 hr and subsequent metabolism of 17 beta-estradiol by follicular layers also led to an equilibrium. At the end of vitellogenesis apoprotein B lipoproteins (VLDL + Chy, LDL) apparently inhibited SGA-GTH stimulation. N',O'-Dibutyryl cAMP (10 mM) considerably stimulated 17 beta-estradiol production but lipoprotein amplification did not occur. Chloroquine (30 microM) inhibition of LDL and HDL amplification indicates that this process requires lysosomal degradation. Plasma lipoproteins in trout modulate SGA-GTH stimulation of 17 beta-estradiol production during exogenous vitellogenesis. Due to the ease and frequency with which the experiments can be carried out, the ovarian follicle of salmonids is an excellent model for the study of the role of lipoproteins in the regulation of ovarian steroids biosynthesis. PMID:3026884

  19. Larval exposure to 4-nonylphenol and 17beta-estradiol affects physiological and behavioral development of seawater adaptation in Atlantic salmon smolts.

    PubMed

    Lerner, Darren T; Björnsson, Björn Thrandur; McCormick, Stephen D

    2007-06-15

    Population declines of anadromous salmonids are attributed to anthropogenic disturbances including dams, commercial and recreational fisheries, and pollutants, such as estrogenic compounds. Nonylphenol (NP), a xenoestrogen, is widespread in the aquatic environment due to its use in agricultural, industrial, and household products. We exposed Atlantic salmon yolk-sac larvae to waterborne 10 or 100 microg L(-1) NP (NP-L or NP-H, respectively), 2 microg L(-1) 17beta-estradiol (E2), or vehicle, for 21 days to investigate their effects on smolt physiology and behavior 1 year later. NP-H caused approximately 50% mortality during exposure, 30 days after exposure, and 60 days after exposure. Mortality rates of NP-L and E2 fish were not affected until 60 days after treatment, when they were 4-fold greater than those of controls. Treatment with NP-L or E2 as yolk-sac larvae decreased gill sodium-potassium-activated adenosine triphosphatase (Na+,K(+)-ATPase) activity and seawater (SW) tolerance during smolt development, 1 year after exposure. Exposure to NP-L and E2 resulted in a latency to enter SW and reduced preference for SW approximately 2- and 5-fold, respectively. NP-L-exposed fish had 20% lower plasma insulin-like growth factor I (IGF-I) levels and 35% lower plasma triiodothyronine (T3). Plasma growth hormone and thyroxine (T4) were unaffected. Exposure to E2 did not affect plasma levels of IGF-I, GH, T3, or T4. Both treatment groups exhibited increased plasma cortisol and decreased osmoregulatory capacity in response to a handling stressor. These results suggest that early exposure to environmentally relevant concentrations of NP, and other estrogenic compounds, can cause direct and delayed mortalities and that this exposure can have long-term, "organizational" effects on life-history events in salmonids. PMID:17626455

  20. Stimulation of RNA polymerase I and II activities by 17 beta -estradiol receptor on chick liver chromatin.

    PubMed Central

    Dierks-Ventling, C; Bieri-Bonniot, F

    1977-01-01

    The endogenous transcriptional capacity (RNA polymerase I and II activity) of liver chromatin from chicks treated with 17 beta-estradiol for 24 h (E 24) was double that of the controls. E 24 chromatin contained estradiol receptor activity while control chromatin did not. Its presence suggested an implication in the enhanced activities of RNA polymerases of E 24 chromatin. When semi-purified estradiol receptor was added to control chromatin, the endogenous transcriptional capacity of this chromatin was greatly increased. Studies with alpha-amanitin showed that both RNA polymerase I and II were stimulated by the estradiol receptor. This stimulation was observed as long as homology of the system was maintained. Solubilized homologous RNA polymerases were stimulated much less by the hormone complex in the presence of heterologous DNA than with homologous chromatin. Prokaryotic RNA polymerase could not be stimulated by chick liver estradiol receptor in the presence of heterologous DNA. PMID:840645

  1. Stimulation of MCF-7 tumor progression in athymic nude mice by 17beta-estradiol induces WISP-2/CCN5 expression in xenografts: a novel signaling molecule in hormonal carcinogenesis.

    PubMed

    Ray, Gibanananda; Banerjee, Snigdha; Saxena, Neela K; Campbell, Donald R; Van Veldhuizen, Peter; Banerjee, Sushanta K

    2005-03-01

    There was 100% solid tumor formation following inoculation of MCF-7 cells. However, MCF-7 tumor progression was significantly greater in the mice exposed to 17beta-estradiol (17beta-E2) compared to unexposed mice. WISP-2/CCN5 mRNA expression was correspondingly increased in 17beta-E2 exposed MCF-7 tumors compared to unexposed xenografts. Moreover, estrogen exposure followed by anti-estrogen tamoxifen treatment drastically inhibited the tumor growth and WISP-2 expression in nude mice. Therefore, the study suggests that higher WISP-2/CCN5 expression by estrogen may be associated with the estrogen-induced growth of MCF-7 tumors in vivo. Finally, overexpression of WISP-2/CCN5 may be considered as a prognostic marker of estrogen-sensitive tumor growth.

  2. 17Beta-estradiol differentially protects cortical pericontusional zone from programmed cell death after traumatic cerebral contusion at distinct stages via non-genomic and genomic pathways.

    PubMed

    Bao, Yi-Jun; Li, Li-Zhuo; Li, Xin-Guo; Wang, Yun-Jie

    2011-11-01

    Pericontusional zone (PCZ) of traumatic cerebral contusion is a target of pharmacological intervention. Our previous study indicated that 17beta-estradiol has a protective role in PCZ after traumatic cerebral contusion via the upregulation of estrogen receptor (ER) alpha mRNA induction and protein expression as well as inhibition of caspase-3 activation, suggesting that genomic signaling pathway is implicated in the protective effect of 17beta-estrodiol. Recent findings demonstrated that 17beta-estradiol also acts on the extranuclear/membrane ER to activate non-genomic signaling pathway to regulate cellular functions and exert the protective effect in the brain. It is still unclear how and whether genomic and non-genomic pathways of 17beta-estradiol are involved in the neuroprotection in PCZ. Our current study demonstrates that 17beta-estradiol activates ERK1/2 and Akt at the early stage and induces ERalpha and survivin mRNA at the late stage to modulate its protection via the suppression of caspase-3 activation in PCZ. These findings suggest that 17beta-estrodiol differentially plays its protective roles via genomic and non-genomic signaling pathways in PCZ after traumatic cerebral contusion.

  3. 17beta-estradiol-mediated neuroprotection and ERK activation require a pertussis toxin-sensitive mechanism involving GRK2 and beta-arrestin-1.

    PubMed

    Dominguez, Reymundo; Hu, Eric; Zhou, Miou; Baudry, Michel

    2009-04-01

    17-beta-Estradiol (E2) is a steroid hormone involved in numerous bodily functions, including several brain functions. In particular, E2 is neuroprotective against excitotoxicity and other forms of brain injuries, a property that requires the extracellular signal-regulated kinase (ERK) pathway and possibly that of other signaling molecules. The mechanism and identity of the receptor(s) involved remain unclear, although it has been suggested that E2 receptor alpha (ERalpha) and G proteins are involved. We, therefore, investigated whether E2-mediated neuroprotection and ERK activation were linked to pertussis toxin (PTX)-sensitive G-protein-coupled effector systems. Biochemical and image analysis of organotypic hippocampal slices and cortical neuronal cultures showed that E2-mediated neuroprotection as well as E2-induced ERK activation were sensitive to PTX. The sensitivity to PTX suggested a possible role of G-protein- and beta-arrestin-mediated mechanisms. Western immunoblots from E2-treated cortical neuronal cultures revealed an increase in phosphorylation of both G-protein-coupled receptor-kinase 2 and beta-arrestin-1, a G-protein-coupled receptor adaptor protein. Transfection of neurons with beta-arrestin-1 small interfering RNA prevented E2-induced ERK activation. Coimmunoprecipitation experiments indicated that E2 increased the recruitment of beta-arrestin-1 and c-Src to ERalpha. These findings suggested that ERalpha is regulated by a mechanism associated with receptor desensitization and downregulation. In support of this idea, we found that E2 treatment of cortical synaptoneurosomes resulted in internalization of ERalpha, whereas treatment of cortical neurons with the ER agonists E-6-BSA-FITC [beta-estradiol-6-(O-carboxymethyl)oxime-bovine serum albumin conjugated with fluorescein isothiocyanate] and E-6-biotin [1,3,5(10)-estratrien-3,17beta-diol-6-one-6-carboxymethloxime-NH-propyl-biotin] resulted in agonist internalization. These results demonstrate that E2

  4. Role of 17 beta-estradiol on type IV collagen fibers volumetric density in the basement membrane of bladder wall.

    PubMed

    de Fraga, Rogerio; Dambros, Miriam; Miyaoka, Ricardo; Riccetto, Cássio Luís Zanettini; Palma, Paulo César Rodrigues

    2007-10-01

    The authors quantified the type IV collagen fibers volumetric density in the basement membrane of bladder wall of ovariectomized rats with and without estradiol replacement. This study was conducted on 40 Wistar rats (3 months old) randomly divided in 4 groups: group 1, remained intact (control); group 2, submitted to bilateral oophorectomy and daily replacement 4 weeks later of 17 beta-estradiol for 12 weeks; group 3, sham operated and daily replacement 4 weeks later of sesame oil for 12 weeks; and group 4, submitted to bilateral oophorectomy and killed after 12 weeks. It was used in immunohistochemistry evaluation using type IV collagen polyclonal antibody to stain the fibers on paraffin rat bladder sections. The M-42 stereological grid system was used to analyze the fibers. Ovariectomy had an increase effect on the volumetric density of the type IV collagen fibers in the basement membrane of rat bladder wall. Estradiol replacement in castrated animals demonstrated a significative difference in the stereological parameters when compared to the castrated group without hormonal replacement. Surgical castration performed on rats induced an increasing volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall and the estradiol treatment had a significant effect in keeping a low volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall.

  5. Stimulation of catecholamine synthesis through unique estrogen receptors in the bovine adrenomedullary plasma membrane by 17{beta}-estradiol

    SciTech Connect

    Yanagihara, Nobuyuki . E-mail: yanagin@med.uoeh-u.ac.jp; Liu, Minhui; Toyohira, Yumiko; Tsutsui, Masato; Ueno, Susumu; Shinohara, Yuko; Takahashi, Kojiro; Tanaka, Kazumi

    2006-01-13

    Incubation of cultured bovine adrenal medullary cells with 17{beta}-estradiol (E{sub 2}) (0.3-100 nM) or membrane-impermeable E{sub 2}-bovine serum albumin (100 nM) acutely increased {sup 14}C-catecholamine synthesis from [{sup 14}C]tyrosine. The stimulatory effect of E{sub 2} was not inhibited by ICI182,780, a nuclear estrogen receptor inhibitor. E{sub 2} also increased tyrosine hydroxylase activity and p44/42MAPK phosphorylation, the former of which was attenuated by U0126, an inhibitor of p44/42MAPK kinase. The plasma membrane isolated from the gland showed two classes of specific binding sites of [{sup 3}H]E{sub 2} with apparent K {sub d}s of 3.2 and 106 nM, and B {sub max}s of 0.44 and 8.5 pmol/mg protein, respectively. The high-affinity binding of [{sup 3}H]E{sub 2} was most strongly inhibited by E{sub 2} and phytoestrogens, and to lesser extents by other steroid hormones, while it was enhanced by ICI182,780 and environmental estrogenic pollutants. These findings suggest that E{sub 2} acutely stimulates catecholamine synthesis via activation of p44/42MAPK through unique estrogen receptors in the plasma membrane of bovine adrenal medulla.

  6. [The effect of repeated administration of carbetocin (Depotocin, Spofa) in the first days postpartum on levels of thyroxine, triiodothyronine, 17 beta-estradiol, progesterone and on fertilization].

    PubMed

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

    1992-07-01

    Application of new procedures in the sphere of the control of sexual functions requires an extension of present knowledge of postparturient endocrinium or endogenic factors comprised in postparturient physiology of sexual activity. According to recent data, oxytocin, besides its uterotonic and luteolytic activity, acts as an ovarian factor in the local intrafollicular regulation of stereidogenesis and as a modulator of uterine secretion of prostaglandines. Based on present knowledge of oxytocin effects, this study was aimed at investigation of the influence of repeated carbetocin (Depotocin inj. Spofa) administration on the dynamics of changes in thyroxine (T4), triiodothyronine (T3), 17 beta-estradiol (E2), progesterone (P4) concentrations and their mutual correlations from the 36th hour till the 51st day after parturition. Simultaneous study of a possible delayed influence of applied carbetocin on conception of ewes after oestrus evocation on day 51 after lambing was carried out. Nineteen ewes of the Slovak Merino breed, lambed in the first decade of February, were assigned to the experimental (n = 9) and to the control group (n = 10). Experimental ewes were subjected to repeated postparturient carbetocin treatment at the dose 0.07 mg per animal. The first dose was applied i. m. in 24 hours, and the second in 72 hours after parturition. On day 51 oestrus was induced in nine ewes of each group by combined treatment with chlorsuperlutin (Agelin, vaginal pessaries, Spofa) and PMSG (500 I.U./animal). On the day of PMSG application ewes were housed together with rams for the period of the next six days. Samples of blood were taken 24 hours before parturition (-1st day), up to 36 h after parturition and on days 4, 7, 14, 17, 21, 25, 34, 42 and 51 after parturition. Concentrations of T4, T3, E2 and P4 were determined by commercial kits RIA-test-T4; RIA-test-T3; RIA-test-ESTRA and RIA-test-PROG (URVJT Kosice). Animal of the control group showed variations of T4

  7. Characterization of atrazine-induced gonadal malformations in African clawed frogs (Xenopus laevis) and comparisons with effects of an androgen antagonist (cyproterone acetate) and exogenous estrogen (17beta-estradiol): Support for the demasculinization/feminization hypothesis.

    PubMed

    Hayes, Tyrone B; Stuart, A Ali; Mendoza, Magdalena; Collins, Atif; Noriega, Nigel; Vonk, Aaron; Johnston, Gwynne; Liu, Roger; Kpodzo, Dzifa

    2006-04-01

    Atrazine is a potent endocrine disruptor that both chemically castrates and feminizes male amphibians. It depletes androgens in adult frogs and reduces androgen-dependent growth of the larynx in developing male larvae. It also disrupts normal gonadal development and feminizes the gonads of developing males. Gonadal malformations induced by atrazine include hermaphrodites and males with multiple testes [single sex polygonadism (SSP)], and effects occur at concentrations as low as 0.1 ppb (microg/L). Here, we describe the frequencies at which these malformations occur and compare them with morphologies induced by the estrogen, 17beta-estradiol (E2) , and the antiandrogen cyproterone acetate, as a first step in testing the hypothesis that the effects of atrazine are a combination of demasculinization and feminization. The various forms of hermaphroditism did not occur in controls. Nonpigmented ovaries, which occurred at relatively high frequencies in atrazine-treated larvae, were found in four individuals out of more than 400 controls examined (1%). Further, we show that several types of gonadal malformations (SSP and three forms of hermaphroditism) are produced by E2 exposure during gonadal differentiation, whereas a final morphology (nonpigmented ovaries) appears to be the result of chemical castration (disruption of androgen synthesis and/or activity) by atrazine. These experimental findings suggest that atrazine-induced gonadal malformations result from the depletion of androgens and production of estrogens, perhaps subsequent to the induction of aromatase by atrazine, a mechanism established in fish, amphibians, reptiles, and mammals (rodents and humans).

  8. 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

  9. 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.

  10. 17-Beta-estradiol inhibits transforming growth factor-beta signaling and function in breast cancer cells via activation of extracellular signal-regulated kinase through the G protein-coupled receptor 30.

    PubMed

    Kleuser, Burkhard; Malek, Daniela; Gust, Ronald; Pertz, Heinz H; Potteck, Henrik

    2008-12-01

    Breast cancer development and breast cancer progression involves the deregulation of growth factors leading to uncontrolled cellular proliferation, invasion and metastasis. Transforming growth factor (TGF)-beta plays a crucial role in breast cancer because it has the potential to act as either a tumor suppressor or a pro-oncogenic chemokine. A cross-communication between the TGF-beta signaling network and estrogens has been postulated, which is important for breast tumorigenesis. Here, we provide evidence that inhibition of TGF-beta signaling is associated with a rapid estrogen-dependent nongenomic action. Moreover, we were able to demonstrate that estrogens disrupt the TGF-beta signaling network as well as TGF-beta functions in breast cancer cells via the G protein-coupled receptor 30 (GPR30). Silencing of GPR30 in MCF-7 cells completely reduced the ability of 17-beta-estradiol (E2) to inhibit the TGF-beta pathway. Likewise, in GPR30-deficient MDA-MB-231 breast cancer cells, E2 achieved the ability to suppress TGF-beta signaling only after transfection with GPR30-encoding plasmids. It is most interesting that the antiestrogen fulvestrant (ICI 182,780), which possesses agonistic activity at the GPR30, also diminished TGF-beta signaling. Further experiments attempted to characterize the molecular mechanism by which activated GPR30 inhibits the TGF-beta pathway. Our results indicate that GPR30 induces the stimulation of the mitogen-activated protein kinases (MAPKs), which interferes with the activation of Smad proteins. Inhibition of MAPK activity prevented the ability of E2 from suppressing TGF-beta signaling. These findings are of great clinical relevance, because down-regulation of TGF-beta signaling is associated with the development of breast cancer resistance in response to antiestrogens.

  11. Increase of carcinogenic risk via enhancement of cyclooxygenase-2 expression and hydroxyestradiol accumulation in human lung cells as a result of interaction between BaP and 17-beta estradiol.

    PubMed

    Chang, Louis W; Chang, Yun-Ching; Ho, Chia-Chi; Tsai, Ming-Hsien; Lin, Pinpin

    2007-07-01

    Animal studies demonstrated that females are more susceptible than males to benzo[a]pyrene (BaP)-induced toxicities, including lung carcinogenesis. Elevation of cyclooxygenase-2 (COX-2) expression has been shown to increase the risk of cancer development. BaP induces COX-2 expression, and an interaction between BaP and estrogen in relation to COX-2 expression is suspected. In the present study, 10 muM BaP alone only slightly increased COX-2 mRNA expression and 10 nM 17-beta estradiol (E(2)) alone slightly increased prostaglandin E2 (PGE2) secretion in human bronchial epithelial cells. However, co-treatment with BaP and E(2) potentiated COX-2 mRNA expression and significantly elevated PGE2 secretion. Utilizing specific inhibitors and reporter assays, we further investigated the potentiation mechanisms of E(2) on BaP-induced COX-2 expression. First, E(2) activated estrogen receptor to increase PGE2 secretion, which directly increased COX-2 expression. Second, E(2) potentiated BaP-induced nuclear factor-kappaB (NF-kappaB) activation, which regulates COX-2 expression. Third, although the aryl hydrocarbon receptor (AhR) did not play a role in BaP-induced COX-2 expression, the potentiation effect of E(2) itself was AhR dependent. We further demonstrated that BaP induced the production of genotoxic E(2) metabolites (2- and 4-hydroxyestradiols) via AhR-up-regulated cytochromes P450 1A1 and 1B1. These metabolites could directly activate NF-kappaB to further promote COX-2 mRNA expression in human lung epithelial cells. These findings were further supported by increased PGE2 secretion in rat lung slice cultures. Our findings that the BaP-E(2) interaction enhanced COX-2 expression and hydroxyestradiol accumulation in the media of cultivated lung cells and tissues provide the needed scientific basis for higher risk of BaP-associated lung cancer in females.

  12. 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.

  13. 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...

  14. Identification of centrarchid hepcidins and evidence that 17beta-estradiol disrupts constitutive expression of hepcidin-1 and inducible expression of hepcidin-2 in largemouth bass (Micropterus salmoides).

    PubMed

    Robertson, Laura S; Iwanowicz, Luke R; Marranca, Jamie Marie

    2009-06-01

    Hepcidin is a highly conserved antimicrobial peptide and iron-regulatory hormone. Here, we identify two hepcidin genes (hep-1 and hep-2) in largemouth bass (Micropterus salmoides) and smallmouth bass (Micropterus dolomieu). Hepcidin-1 contains a putative ATCUN metal-binding site in the amino-terminus that is missing in hepcidin-2, suggesting that hepcidin-1 may function as an iron-regulatory hormone. Both hepcidins are predominately expressed in the liver of largemouth bass, similar to other fish and mammals. Experimental exposure of pond-raised largemouth bass to 17beta-estradiol and/or the bacteria Edwardsiella ictaluri led to distinct changes in expression of hep-1 and hep-2. Estradiol reduced the constitutive expression of hep-1 in the liver. Bacterial exposure induced expression of hep-2, suggesting that hepcidin-2 may have an antimicrobial function, and this induction was abolished by estradiol. To our knowledge, this is the first report of the regulation of hepcidin expression by estradiol in either fish or mammals.

  15. DNA arrays to monitor gene expression in rat blood and uterus following 17beta-estradiol exposure: biomonitoring environmental effects using surrogate tissues.

    PubMed

    Rockett, John C; Kavlock, Robert J; Lambright, Christy R; Parks, Louise G; Schmid, Judith E; Wilson, Vickie S; Wood, Carmen; Dix, David J

    2002-09-01

    We propose that gene expression changes in accessible tissues such as blood often reflect those in inaccessible tissues, thus offering a convenient biomonitoring method to provide insight into the effects of environmental toxicants on such tissues. In this pilot study, gene expression changes in peripheral blood leukocytes (PBL) were compared to those in the uteri of adult rats to identify genes that were altered in both tissues following estradiol treatment. Ovariectomized rats were treated with either 17beta-estradiol or vehicle control (corn oil) for 3 days. PBL and uterine RNAs were hybridized to arrays containing 1185 genes. One hundred and ninety three genes were expressed in common between the PBL and uterus. Eighteen were changed significantly in both tissues, 9 of which were treatment- but not tissue-specific (e.g., jun-D, phospholipase A2, thymidine kinase). These results demonstrate that many genes are coexpressed between PBL and uterus, and that some are coregulated by estradiol. Given the limited number of genes examined in this study and the estimated size of other mammalian genomes, we conclude that many more genes will also be coregulated and suggest that accessible tissues such as PBL can serve as surrogate tissues for observing gene expression changes in inaccessible target tissues.

  16. 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.

  17. Rainfall and tillage effects on transport of fecal bacteria and sex hormones 17beta-estradiol and testosterone from broiler litter applications to a Georgia Piedmont Ultisol.

    PubMed

    Jenkins, Michael B; Truman, Clint C; Siragusa, Gregory; Line, Eric; Bailey, J Stan; Frye, Jonathan; Endale, Dinku M; Franklin, Dorcas H; Schomberg, Harry H; Fisher, Dwight S; Sharpe, Ronald R

    2008-09-15

    Poultry litter provides nutrients for crop and pasture production; however, it also contains fecal bacteria, sex hormones (17beta-estradiol and testosterone) and antibiotic residues that may contaminate surface waters. Our objective was to quantify transport of fecal bacteria, estradiol, testosterone and antibiotic residues from a Cecil sandy loam managed since 1991 under no-till (NT) and conventional tillage (CT) to which either poultry litter (PL) or conventional fertilizer (CF) was applied based on the nitrogen needs of corn (Zea mays L) in the Southern Piedmont of NE Georgia. Simulated rainfall was applied for 60 min to 2 by 3-m field plots at a constant rate in 2004 and variable rate in 2005. Runoff was continuously measured and subsamples taken for determining flow-weighted concentrations of fecal bacteria, hormones, and antibiotic residues. Neither Salmonella, nor Campylobacter, nor antimicrobial residues were detected in litter, soil, or runoff. Differences in soil concentrations of fecal bacteria before and after rainfall simulations were observed only for Escherichia coli in the constant rainfall intensity experiment. Differences in flow-weighted concentrations were observed only for testosterone in both constant and variable intensity rainfall experiments, and were greatest for treatments that received poultry litter. Total loads of E. coli and fecal enterococci, were largest for both tillage treatments receiving poultry litter for the variable rainfall intensity. Load of testosterone was greatest for no-till plots receiving poultry litter under variable rainfall intensity. Poultry litter application rates commensurate for corn appeared to enhance only soil concentrations of E. coli, and runoff concentrations of testosterone above background levels.

  18. Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation

    PubMed Central

    2011-01-01

    Background Melatonin is associated with direct or indirect actions upon female reproductive function. However, its effects on sex hormones and steroid receptors during ovulation are not clearly defined. This study aimed to verify whether exposure to long-term melatonin is able to cause reproductive hormonal disturbances as well as their role on sex steroid receptors in the rat ovary, oviduct and uterus during ovulation. Methods Twenty-four adult Wistar rats, 60 days old (+/- 250 g) were randomly divided into two groups. Control group (Co): received 0.9% NaCl 0.3 mL + 95% ethanol 0.04 mL as vehicle; Melatonin-treated group (MEL): received vehicle + melatonin [100 μg/100 g BW/day] both intraperitoneally during 60 days. All animals were euthanized by decapitation during the morning estrus at 4 a.m. Results Melatonin significantly reduced the plasma levels of LH and 17 beta-estradiol, while urinary 6-sulfatoximelatonin (STM) was increased at the morning estrus. In addition, melatonin promoted differential regulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and melatonin receptor (MTR) along the reproductive tissues. In ovary, melatonin induced a down-regulation of ER-alpha and PRB levels. Conversely, it was observed that PRA and MT1R were up-regulated. In oviduct, AR and ER-alpha levels were down-regulated, in contrast to high expression of both PRA and PRB. Finally, the ER-beta and PRB levels were down-regulated in uterus tissue and only MT1R was up-regulated. Conclusions We suggest that melatonin partially suppress the hypothalamus-pituitary-ovarian axis, in addition, it induces differential regulation of sex steroid receptors in the ovary, oviduct and uterus during ovulation. PMID:21810236

  19. Development of Simultaneous Derivative Spectrophotometric and HPLC Methods for Determination of 17-Beta-Estradiol and Drospirenone in Combined Dosage Form

    PubMed Central

    Aydoğmuş, Zeynep; Yılmaz, Ece Merve; Yörüsün, Sevgi; Akpınar, Samet

    2015-01-01

    Simple, rapid spectrophotometric, and reverse-phase high performance liquid chromatographic methods were developed for the concurrent analysis of 17-beta-estradiol (ESR) and drospirenone (DRS). The spectrophotometric method was based on the determination of first derivative spectra and determined ESR and DRS using the zero-crossing technique at 208 and 282 nm, respectively, in methanol. The linear range was 0.5–32.0 µg·mL−1 for DRS and 0.5–8.0 µg·mL−1 for EST. The limit of detection (LOD) values were 0.14 µg·mL−1 and 0.10 µg·mL−1 and limit of quantification (LOQ) values were 0.42 µg·mL−1 and 0.29 µg·mL−1 for ESR and DRS, respectively. The chromatographic method was based on the separation of both analytes on a C18 column with a mobile phase containing acetonitrile and water (70 : 30, v/v). Detection was performed with a UV-photodiode array detector at 279 nm. The linear range was 0.08–2.5 µg·mL−1 for DRS and 0.23–7.5 µg·mL−1 for EST. LOD values were 0.05 µg·mL−1 and 0.02 µg·mL−1 and LOQ values were 0.15 µg·mL−1 and 0.05 µg·mL−1 for ESR and DRS, respectively. These recommended methods have been applied for the simultaneous determination of ESR and DRS in their tablets. PMID:27347530

  20. Effect of alpha-interferon, 17 beta-estradiol, and tamoxifen on estrogen receptor concentration and cell cycle kinetics of MCF 7 cells.

    PubMed

    Bezwoda, W R; Meyer, K

    1990-09-01

    The interaction of alpha-interferon, 17 beta-estradiol, and tamoxifen on estrogen receptor content, growth fraction, proliferative rate, and total protein synthesis of MCF 7 cells was investigated under culture conditions (minus phenol red and at low concentrations of "stripped" fetal calf serum) allowing for direct stimulation of proliferation by estrogens. Exposure to estradiol alone resulted in a decrease of estrogen receptor content as measured by immunoassay, an increase of the proportion of cells in S phase, and increases in cell proliferation as well as total protein synthesis. alpha-Interferon treatment resulted in cell cycle arrest with reduced proliferation, an increase of estrogen receptor content, but a decrease in the rate of total protein synthesis. Pretreatment with alpha-interferon inhibited the estrogen induced stimulation of cell growth as well as the associated decrease of estrogen receptor content. Tamoxifen treatment resulted in decreased cell proliferation and decrease of estrogen receptor content and of total protein synthesis. These results suggest that the estrogen receptor concentration of MCF 7 cells is growth fraction related. Pretreatment with alpha-interferon enhanced the inhibitory effect of tamoxifen on cell proliferation while preventing the tamoxifen induced reduction of estrogen receptor content. The synergistic effect of alpha-interferon and tamoxifen are most marked following 72 h pretreatment with interferon, when the maximum interferon induced increase of estrogen receptor concentration is evident. The mechanism is thus due probably to an increase of cellular receptor as a ligand for tamoxifen binding and suggests a possible role for the clinical use of interferons combined with tamoxifen.

  1. 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.

  2. Development of Simultaneous Derivative Spectrophotometric and HPLC Methods for Determination of 17-Beta-Estradiol and Drospirenone in Combined Dosage Form.

    PubMed

    Aydoğmuş, Zeynep; Yılmaz, Ece Merve; Yörüsün, Sevgi; Akpınar, Samet

    2015-01-01

    Simple, rapid spectrophotometric, and reverse-phase high performance liquid chromatographic methods were developed for the concurrent analysis of 17-beta-estradiol (ESR) and drospirenone (DRS). The spectrophotometric method was based on the determination of first derivative spectra and determined ESR and DRS using the zero-crossing technique at 208 and 282 nm, respectively, in methanol. The linear range was 0.5-32.0 µg·mL(-1) for DRS and 0.5-8.0 µg·mL(-1) for EST. The limit of detection (LOD) values were 0.14 µg·mL(-1) and 0.10 µg·mL(-1) and limit of quantification (LOQ) values were 0.42 µg·mL(-1) and 0.29 µg·mL(-1) for ESR and DRS, respectively. The chromatographic method was based on the separation of both analytes on a C18 column with a mobile phase containing acetonitrile and water (70 : 30, v/v). Detection was performed with a UV-photodiode array detector at 279 nm. The linear range was 0.08-2.5 µg·mL(-1) for DRS and 0.23-7.5 µg·mL(-1) for EST. LOD values were 0.05 µg·mL(-1) and 0.02 µg·mL(-1) and LOQ values were 0.15 µg·mL(-1) and 0.05 µg·mL(-1) for ESR and DRS, respectively. These recommended methods have been applied for the simultaneous determination of ESR and DRS in their tablets. PMID:27347530

  3. 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

  4. In vitro and in vivo analysis of fatty acid effects on metabolism of 17beta-estradiol and progesterone in dairy cows.

    PubMed

    Piccinato, C A; Sartori, R; Sangsritavong, S; Souza, A H; Grummer, R R; Luchini, D; Wiltbank, M C

    2010-05-01

    Some studies have reported improved reproductive performance with dietary fat supplementation. This study examined effects of fatty acids with different lengths, or desaturation, or both, on metabolism of estradiol (E2) and progesterone (P4) in bovine liver slice incubations (experiments 1 and 2) and in vivo (experiment 3). In experiment 1, effects of fatty acids C16:0 (palmitic acid), C16:1 (palmitoleic acid), C18:1 (oleic acid), and C18:3 (linolenic acid) were evaluated at 30, 100, and 300 microM on P4 and E2 metabolism in vitro. In experiment 2, stearic acid (C18:0) and C18:3 were evaluated in the same incubation conditions. In experiment 1, all of the fatty acids had some significant inhibitory effect on metabolism of P4, E2, or both (300 microM C16:0 on E2; 100 microM C16:1 on E2; 300 microM C16:1 on both P4 and E2; 300 microM C18:1 on P4; and 100 and 300 microM C18:3 on both P4 and E2). In experiment 2, C18:3 (100 and 300 microM) but not C18:0 decreased P4 and E2 metabolism. Overall, the most profound increase (approximately 60%) in half-life of P4 and E2 was observed with incubations of 300 microM C18:3 in both in vitro experiments. Based on these in vitro results, in experiment 3 linseed oil (rich in C18:3) was supplemented into the abomasum and acute effects on metabolism of E2 and P4 were evaluated. Cows (n=4) had endogenous E2 and P4 minimized (corpus luteum regressed, follicles aspirated) before receiving continuous intravenous infusion of E2 and P4 to analyze metabolic clearance rate for these hormones during abomasal infusion of saline (control) or 70 mL of linseed oil every 4h for 28h. Linseed oil infusion increased C18:3 in plasma by 46%; however, metabolic clearance rate for E2 and P4 were similar for control cows compared with linseed-treated cows. Thus, in vitro experiments indicated that E2 and P4 metabolism can be inhibited by high concentrations of C18:3. Nevertheless, in vivo, linseed oil did not acutely inhibit E2 and P4 metabolism, perhaps

  5. 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; Riches, Kirsten; Mughal, Romana S.; Viriyavejakul, Parnpen; Chaisri, Urai; Maneerat, Yaowapa; Turner, Neil A.; 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

  6. 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...

  7. 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

  8. 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...

  9. In vitro and immunological assessment of the estrogenic activity and concentrations of 17beta-estradiol, estrone, and ethinyl estradiol in treated effluent from 45 wastewater treatment plants in Victoria, Australia.

    PubMed

    Allinson, M; Shiraishi, F; Salzman, S A; Allinson, G

    2010-04-01

    The project was conducted between May 2006 and September 2007, and involved the collection of effluent samples from 45 wastewater treatment plants (WWTPs). The 45 WWTPs included 16 lagoon-based plants and 29 with activated sludge-based processes. Permission was obtained from all the relevant water authorities to collect samples of final effluent at point of discharge to the environment, whether that was to a creek, a river, the ocean, or the land. Samples were collected on two occasions, namely, in August 2006 (winter) and late February-early March 2007 (summer), and subjected to a number of biological and chemical analyses, including toxicity tests, measurement of hormonal (estrogenic) activity using yeast-based bioassays, and measurement of specific hormonal concentrations using enzyme-linked immunosorbent assays (ELISAs). Almost all of the effluents examined showed estrogenic activity: in winter, no activity to 73 ng/l 17beta-estradiol equivalents (EEQ); and in summer, no activity to 20 ng/l EEQ. On the whole, the levels of estrogenic activity observed were comparable with the range recently reported in Australia and New Zealand using human estrogen receptor-based assays ("not detected" to approximately 10 ng/l EEQ). The low/no bioassay response was confirmed by the chemical assessment of estradiol, estrone, and ethinyl estradiol concentrations by ELISA, which returned concentrations of these compounds for the most part below 10 ng/l. PMID:20130850

  10. 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

  11. 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

  12. 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

  13. [Night workers and plasmatic cortisol].

    PubMed

    Palermo, P; Rosati, M V; Ciarrocca, M; Nicassio, P; Piccoli, F; Cerratti, D; Anzani, M F; Tomei, G; Perugi, F; Monti, C; Palitti, T; Tomao, E; Caciari, T; Tomei, F

    2007-01-01

    The aim of the study is to evaluate whether occupational exposure to night work could cause alterations in the levels of plasmatic cortisol. The interest toward this argument arises form several studies in scientific literature referring the presence of an alteration in the synthesis and release of cortisol in workers exposed to night work. We studied a population of workers employed in night security service and monitoring service of alarm systems in different museums compared to a control group not performing shift-work and/or night work. The exposed and control subjects were compared by age, length of service, smoking habit (n. cigarettes per day), habitual consumption of alcoholic drinks (n. glass of wine/beer per day). We evaluated the levels of plasmatic cortisol on 50 workers exposed to night work, all males of whom 30 smokers and 20 non-smokers and on 50 controls of whom 30 smokers and 20 non-smokers.

  14. 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.

  15. 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...

  16. 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...

  17. 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...

  18. A metabolomics study of the inhibitory effect of 17-beta-estradiol on osteoclast proliferation and differentiation.

    PubMed

    Liu, Xiaoyan; Liu, Yanqiu; Cheng, Mengchun; Zhang, Xiaozhe; Xiao, Hongbin

    2015-02-01

    Estradiol is a major drug used clinically to alleviate osteoporosis, partly through inhibition of the activity of osteoclasts, which play a crucial role in bone resorption. So far, little is known about the effects of estradiol on osteoclast metabolism. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC/MS)-based metabolomics strategy was used to investigate the metabolite response to 17β-estradiol in mouse osteoclast RAW264.7, a commonly used cell model for studying osteoporosis. Our results showed that the application of estradiol altered the levels of 27 intracellular metabolites, including lysophosphatidylcholines (LysoPCs), other lipids and amino acid derivants. The changes of all the 27 metabolites were observed in the study of estradiol induced osteoclast proliferation inhibition (1 μM estradiol applied), while the changes of only 18 metabolites were observed in the study of differentiation inhibition (0.1 μM estradiol applied). Further pathway impact analysis determined glycerophospholipid metabolism as the main potential target pathway of estradiol, which was further confirmed by LCAT (phosphatidylcholine-sterol acyltransferase) activity changes and lipid peroxidative product (MDA, methane dicarboxylic aldehyde) changes caused by estradiol. Additionally, we found that estradiol significantly decreased intracellular oxidative stress during cell proliferation but not during cell differentiation. Our study suggested that estradiol generated a highly condition-dependent influence on osteoclast metabolism.

  19. 17beta-Estradiol inhibits Ca2+-dependent homeostasis of airway surface liquid volume in human cystic fibrosis airway epithelia.

    PubMed

    Coakley, Ray D; Sun, Hengrui; Clunes, Lucy A; Rasmussen, Julia E; Stackhouse, James R; Okada, Seiko F; Fricks, Ingrid; Young, Steven L; Tarran, Robert

    2008-12-01

    Normal airways homeostatically regulate the volume of airway surface liquid (ASL) through both cAMP- and Ca2+-dependent regulation of ion and water transport. In cystic fibrosis (CF), a genetic defect causes a lack of cAMP-regulated CFTR activity, leading to diminished Cl- and water secretion from airway epithelial cells and subsequent mucus plugging, which serves as the focus for infections. Females with CF exhibit reduced survival compared with males with CF, although the mechanisms underlying this sex-related disadvantage are unknown. Despite the lack of CFTR, CF airways retain a limited capability to regulate ASL volume, as breathing-induced ATP release activates salvage purinergic pathways that raise intracellular Ca2+ concentration to stimulate an alternate pathway to Cl- secretion. We hypothesized that estrogen might affect this pathway by reducing the ability of airway epithelia to respond appropriately to nucleotides. We found that uridine triphosphate-mediated (UTP-mediated) Cl- secretion was reduced during the periovulatory estrogen maxima in both women with CF and normal, healthy women. Estrogen also inhibited Ca2+ signaling and ASL volume homeostasis in non-CF and CF airway epithelia by attenuating Ca2+ influx. This inhibition of Ca2+ signaling was prevented and even potentiated by estrogen antagonists such as tamoxifen, suggesting that antiestrogens may be beneficial in the treatment of CF lung disease because they increase Cl- secretion in the airways. PMID:19033671

  20. 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

  1. 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...

  2. A metabolomics study of the inhibitory effect of 17-beta-estradiol on osteoclast proliferation and differentiation.

    PubMed

    Liu, Xiaoyan; Liu, Yanqiu; Cheng, Mengchun; Zhang, Xiaozhe; Xiao, Hongbin

    2015-02-01

    Estradiol is a major drug used clinically to alleviate osteoporosis, partly through inhibition of the activity of osteoclasts, which play a crucial role in bone resorption. So far, little is known about the effects of estradiol on osteoclast metabolism. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC/MS)-based metabolomics strategy was used to investigate the metabolite response to 17β-estradiol in mouse osteoclast RAW264.7, a commonly used cell model for studying osteoporosis. Our results showed that the application of estradiol altered the levels of 27 intracellular metabolites, including lysophosphatidylcholines (LysoPCs), other lipids and amino acid derivants. The changes of all the 27 metabolites were observed in the study of estradiol induced osteoclast proliferation inhibition (1 μM estradiol applied), while the changes of only 18 metabolites were observed in the study of differentiation inhibition (0.1 μM estradiol applied). Further pathway impact analysis determined glycerophospholipid metabolism as the main potential target pathway of estradiol, which was further confirmed by LCAT (phosphatidylcholine-sterol acyltransferase) activity changes and lipid peroxidative product (MDA, methane dicarboxylic aldehyde) changes caused by estradiol. Additionally, we found that estradiol significantly decreased intracellular oxidative stress during cell proliferation but not during cell differentiation. Our study suggested that estradiol generated a highly condition-dependent influence on osteoclast metabolism. PMID:25474166

  3. 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 ...

  4. Carbon monoxide and iron modulate plasmatic coagulation in Alzheimer's disease.

    PubMed

    Nielsen, Vance G; Pretorius, Etheresia; Bester, Janette; Jacobsen, Wayne K; Boyle, Patrick K; Reinhard, Joao P

    2015-01-01

    Alzheimer's disease (AD) is a significant source of morbidity and mortality for millions of people worldwide, and multiple potential etiologies have been postulated to contribute to AD. Among these, spontaneous cerebral emboli and increased cerebral and circulating heme oxygenase (Hmox) activity in AD patients are of particular interest, as two of the products of Hmox activity, carbon monoxide (CO) and iron enhance plasmatic coagulation and modify the ultrastructure of thrombi. We hypothesized that patients afflicted with AD would have coagulation kinetics modulated by CO and iron. Using viscoelastic assessments of coagulation, it was determined with a small cohort (n=11) of AD patients that all had enhancement of coagulation by CO, iron, or both. In a complementary fashion, it was determined that a separate cohort (n=12) of AD patients had thrombi with ultrastructural features consistent with iron and CO exposure as assessed with scanning electron microscopy. Further, when stratified by normal or abnormally increased serum ferritin concentrations (which can be increased by Hmox), the AD patients with abnormal ferritin concentrations had significantly thinner fibrin fiber diameters, not unlike that noted when normal plasma is mixed with iron or CO. In sum, AD patients were noted to have plasmatic coagulation kinetic and thrombus ultrastructural changes consistent with exposure to CO and iron. Future investigation of CO and iron in the pathogenesis of Alzheimer's disease is warranted.

  5. 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...

  6. 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...

  7. 17beta-Estradiol and testosterone in drainage and runoff from poultry litter applications to tilled and no-till crop land under irrigation.

    PubMed

    Jenkins, Michael B; Endale, Dinku M; Schomberg, Harry H; Hartel, Peter G; Cabrera, Miguel L

    2009-06-01

    Thirteen million [corrected] metric tons of poultry litter are produced annually by poultry producers in the U.S. Poultry litter contains the sex hormones estradiol and testosterone, endocrine disruptors that have been detected in surface waters. The objective of this study was to evaluate the potential impact of poultry litter applications on estradiol and testosterone concentrations in subsurface drainage and surface runoff in irrigated crop land under no-till and conventional-till management. We conducted an irrigation study in fall of 2001 and spring of 2002. Four treatments, no-till plus poultry litter, conventional-till plus poultry litter, no-till plus conventional fertilizer, and conventional-till plus conventional fertilizer, were evaluated. Flow-weighted concentration and load ha(-1) of the two hormones were measured in drainage and runoff. Soil concentrations of estradiol and testosterone were measured. Based on comparisons to the conventional fertilizer (and control) treatments, poultry litter did not add to the flow-weighted concentration or load ha(-1) of either estradiol or testosterone in subsurface drainage or surface runoff. Significant differences were, however, observed between tillage treatments: flow-weighted concentrations of estradiol were greater for no-till than conventional-till plots of the June irrigation; and runoff loads of both estradiol and testosterone were less from no-till than conventional-till plots for the November irrigation. Although the differences between no-till and conventional-tillage appeared to affect the hydrologic transport of both hormones, the differences appeared to have inconsequential environmental impact.

  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. 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...

  10. 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...

  11. 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.

  12. Residual plasmatic activity of ADAMTS13 is correlated with phenotype severity in congenital thrombotic thrombocytopenic purpura.

    PubMed

    Lotta, Luca A; Wu, Haifeng M; Mackie, Ian J; Noris, Marina; Veyradier, Agnes; Scully, Marie A; Remuzzi, Giuseppe; Coppo, Paul; Liesner, Ri; Donadelli, Roberta; Loirat, Chantal; Gibbs, Richard A; Horne, April; Yang, Shangbin; Garagiola, Isabella; Musallam, Khaled M; Peyvandi, Flora

    2012-07-12

    The quantification of residual plasmatic ADAMTS13 activity in congenital thrombotic thrombocytopenic purpura (TTP) patients is constrained by limitations in sensitivity and reproducibility of commonly used assays at low levels of ADAMTS13 activity, blunting efforts to establish genotype-phenotype correlations. In the present study, the residual plasmatic activity of ADAMTS13 was measured centrally by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (limit of detection = 0.5%) in 29 congenital TTP patients. The results were used to study correlations among ADAMTS13 genotype, residual plasmatic activity, and clinical phenotype severity. An ADAMTS13 activity above 0.5% was measured in 26 (90%) patients and lower levels of activity were associated with earlier age at first TTP episode requiring plasma infusion, more frequent recurrences, and prescription of fresh-frozen plasma prophylaxis. Receiver operating characteristic curve analysis showed that activity levels of less than 2.74% and 1.61% were discriminative of age at first TTP episode requiring plasma infusion < 18 years, annual rate of TTP episodes > 1, and use of prophylaxis. Mutations affecting the highly conserved N-terminal domains of the protein were associated with lower residual ADAMTS13 activity and a more severe phenotype in an allelic-dose dependent manner. The results of the present study show that residual ADAMTS13 activity is associated with the severity of clinical phenotype in congenital TTP and provide insights into genotype-phenotype correlations.

  13. Oxidative stress and labile plasmatic iron in anemic patients following blood therapy

    PubMed Central

    Fernandes, Marília Sabo; Rissi, Tatiana Tamborena; Zuravski, Luisa; Mezzomo, Juliana; Vargas, Carmen Regla; Folmer, Vanderlei; Soares, Félix Alexandre Antunes; Manfredini, Vanusa; Ahmed, Mushtaq; Puntel, Robson Luiz

    2014-01-01

    AIM: To determine the plasmatic iron content and evaluate the oxidative stress (OS) markers in subjects receiving blood therapy. METHODS: Thirty-nine individuals with unspecified anemia receiving blood transfusions and 15 healthy subjects were included in the study. Anemic subjects were divided into three subgrouP: (1) those that received up to five blood transfusions (n = 14); (2) those that received from five to ten transfusions (n = 11); and (3) those that received more than ten transfusions (n = 14). Blood samples were collected by venous arm puncture and stored in tubes containing heparin. The plasma and cells were separated by centrifugation and subsequently used for analyses. Statistical analyses were performed using Kruskal-Wallis analysis of variance followed by Dunn’s multiple comparison tests when appropriate. RESULTS: The eletrophoretic hemoglobin profiles of the subjects included in this study indicated that no patients presented with hemoglobinopathy. Labile plasmatic iron, ferritin, protein carbonyl, thiobarbituric acid-reactive substances (TBARS) and dichlorofluorescein diacetate oxidation were significantly higher (P < 0.05), whereas total thiol levels were significantly lower (P < 0.05) in transfused subjects compared to controls. Additionally, the activity of catalase, superoxide dismutase and glutathione peroxidase were significantly lower in the transfused subjects (P < 0.05). Antioxidant enzyme activities and total thiol levels were positively correlated (P < 0.05), and negatively correlated with the levels of protein carbonyl and TBARS (P < 0.05). In contrast, protein carbonyl and TBARS were positively correlated (P < 0.05). Altogether, these data confirm the involvement of OS in patients following therapy with repeated blood transfusions. CONCLUSION: Our data reveal that changes in OS markers are correlated with levels of labile plasmatic iron and ferritin and the number of transfusions. PMID:25254188

  14. Erythrocyte membrane fluidity and indices of plasmatic oxidative damage after acute physical exercise in humans.

    PubMed

    Berzosa, C; Gómez-Trullén, E M; Piedrafita, E; Cebrián, I; Martínez-Ballarín, E; Miana-Mena, F J; Fuentes-Broto, L; García, J J

    2011-06-01

    Optimal levels of membrane fluidity are essential for numerous cell functions including cell growth, solute transport and signal transduction. Since exercise enhances free radical production, our aim was to evaluate in healthy male subjects the effects of an acute bout of maximal and submaximal exercise on the erythrocyte membrane fluidity and its possible relation to the oxidative damage overproduction due to exercise. Subjects (n = 34) performed three cycloergometric tests: a continuous progressive exercise, a strenuous exercise until exhaustion and an acute bout of exercise at an intensity corresponding to 70% of maximal work capacity for 30 min. Venous blood samples were collected before and immediately after these exercises. Erythrocyte membrane fluidity was assessed by fluorescence spectroscopy. Plasma malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations and carbonyl content of plasmatic proteins were used as an index of lipid and protein oxidation, respectively. Exercise produced a dramatic drop in the erythrocyte membrane fluidity as compared to resting time, but this was not accompanied by significant changes in the plasmatic MDA and 4-HDA concentrations. The highest erythrocyte membrane rigidity was detected immediately after strenuous exercise until exhaustion was performed. Protein carbonyl levels were higher after exhaustive exercises than at rest. Continuous progressive and strenuous exercises until exhaustion, but not submaximal workload, resulted in a significant enhanced accumulation of carbonylated proteins in the plasma. These findings are consistent with the idea that exercise exaggerates oxidative damage, which may contribute, at least partially, to explain the rigidity in the membrane of the erythrocytes due to acute exercise.

  15. Hemodialysis patients have plasmatic hypercoagulability and decreased fibrinolytic vulnerability: role of carbon monoxide.

    PubMed

    Matika, Ryan W; Nielsen, Vance G; Steinbrenner, Evangelina B; Sussman, Amy N; Madhrira, Machaiah

    2014-01-01

    Chronic hemodialysis is associated with significant thrombophilia. Of interest, hemodialysis patients have increased carboxyhemoglobin (COHb) and exhaled carbon monoxide (CO), signs of upregulated heme oxygenase (Hmox) activity. Given that CO enhances plasmatic coagulation, we determined whether patients requiring chronic hemodialysis had an increase in endogenous CO, plasmatic hypercoagulability and decreased fibrinolytic vulnerability. Carbon monoxide was determined by noninvasive pulse oximetry measurement of COHb. Blood samples were obtained just before hemodialysis. Thrombelastographic methods to assess plasma coagulation kinetics, fibrinolytic kinetics, and formation of carboxyhemefibrinogen (COHF) were used. Hemodialysis patients (n = 45) had abnormally increased COHb concentrations of 2.2 ± 1.9%, indicative of Hmox upregulation. Coagulation and fibrinolytic parameter normal values were determined with normal individual (n = 30) plasma. Thirty-seven patients of the hemodialysis cohort had COHF formation (82.2%, [67.9%-92.0%]; mean, [95% confidence interval]), and many of this group of patients had abnormally great velocity of clot growth (73.3%, [58.1%-85.4%]) and strength (75.6%, [60.5%-87.1%]). Furthermore, over half of COHF positive patients had a hypofibrinolytic state, evidenced by an abnormally prolonged time to maximum rate of lysis (53.3%, [37.9%-68.6%]) and clot lysis time (64.4%, [48.8%-78.1%]). Carbon monoxide enhanced coagulation and diminished fibrinolytic vulnerability in hemodialysis patients. Future investigation of hemodialysis, CO-related thrombophilia is warranted.

  16. [Palm oil derivatives with different concentration of palmitic acid and antioxidants. Effects upon plasmatic lipids and platelet aggregation].

    PubMed

    Scorza, T; Martucci, A; Torrealba de Ron, A T

    1999-03-01

    It was evaluated the effect of diet rich with cholesterol (0.1%) and different concentration of palmitic acid (16:0) and antioxidants (vitamin C, alpha tocopherol and retinol) upon plasmatic lipids and platelet aggregability in rabbits. The animals were distributed in three groups: I. Standard chow meal (Rp Conejarina) + cholesterol (chol) 0.1%; II. Standard chow meal + chol 0.1% + semipurified palm oil 10% (16:0 = 39.8%, oleic acid 48.7%, linoleic acid 11.4%, retinol 7.3 ug/dL, alpha tocopherol 157.6 ug/dL; III. Standard chow meal + chol 0.1% + crude palm oil 10% (16:0 = 45.3%, oleic acid 46.3%, linoleic acid 7.9%, retinol 96.4 ug/dL, alpha tocopherol 322.8 ug/dL). Monthly determination of plasmatic lipids were done (Enzymatic methods) and at ten months platelet aggregability with ADP, plasmatic vitamin C, retinol and, alpha tocopherol determination were done. Total plasmatic cholesterol (TC) and LDLc increased significantly in the three groups of animals. Significant differences between groups were not found. Platelet aggregability was lower in the animals fed with palmitic acid rich diet (groups II and III) (P = 0.002 and 0.001). Retinol, alpha tocopherol plasmatic concentrations revealed no significant differences. Vitamin C in the groups I was lower than groups II and III (P < 0.05 < 0.02). In this study hypercholesterolemic rabbits fed with rich diets (crude and semipurified) had lower platelet aggregability without changes in plasmatic lipids concentrations.

  17. Heme oxygenase derived carbon monoxide and iron mediated plasmatic hypercoagulability in a patient with calcific mitral valve disease.

    PubMed

    Thompson, Jess L; Nielsen, Vance G; Castro, Allison R; Chen, Andrew

    2015-05-01

    We present a case of a patient with calcific mitral valve stenosis and plasmatic hypercoagulability. Using thrombelastography, the patient was determined to have an abnormally large velocity of plasma thrombus growth and strength with reduced vulnerability to lysis. Critically, increased carboxyhemoglobin concentration (2.4 %) was present, likely secondary to hemolysis from mitral stenosis and engagement of systemic heme oxygenase. It was determined that the patient's plasmatic hypercoagulability was in part due to carboxyhemefibrinogen formation and iron-enhancement of coagulation via two thrombelastographic methods. In conclusion, future investigation of the involvement of both carbon monoxide and iron mediated hypercoagulability in the setting of stenotic valve disease is warranted.

  18. Variants and Haplotypes in Angiotensinogen Gene Are Associated With Plasmatic Angiotensinogen Level in Mexican Population

    PubMed Central

    Balam-Ortiz, Eros; Esquivel-Villarreal, Adolfo; Alfaro-Ruiz, Luis; Carrillo, Karol; Elizalde, Adela; Gil, Trinidad; Urushihara, Maki; Kobori, Hiroyuki; Jimenez-Sanchez, Gerardo

    2011-01-01

    Introduction The plasmatic angiotensinogen (AGT) level has been associated with essential hypertension. Linkage analysis has found a relationship between the AGT gene locus and hypertension in the Mexican-American population, but studies have failed to identify genetic variants associated with hypertension or plasma AGT levels. This study analyzes the relationship between polymorphisms in the AGT gene and plasmatic AGT levels in Mexican population. Methods Nine polymorphisms in AGT gene were genotyped, and plasma AGT level was determined by enzyme-linked immunosorbent assay. Results Differences in AGT plasma levels were associated with 2 polymorphisms: T-20G, TT = 25.3 ± 8.3 versus TG + GG = 21.6 ± 8.8 μg/mL; P = 0.008 and C3389T (T174M), CC = 25.8 ± 9.9 versus TC + TT = 20.5 ± 5.4 μg/mL; P = 0.0002. Haplotype 2 was associated with low plasma AGT (−5.1 μg/mL [95% confidence interval: −8.6 to −1.6], P = 0.004) and Haplotype 8 was associated with high plasma AGT (6.5 μg/mL [95% confidence interval: 2.5 to 10.6], P = 0.001). This association remained after adjustment for covariates. A Likelihood Ratio Test for haplotype-phenotype association adjusted for covariates resulted in χ2 = 38.9, P = 0.0005. The total effect of the haplotypes on plasma AGT level variance was 19.5%. No association was identified between haplotypes and quantitative traits of blood pressure. Conclusions Two polymorphisms (T-20G and C3389T) and 2 haplotypes (H2 and H8) showed an association with plasma AGT levels in Mexican population. PMID:21629041

  19. 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.

  20. The Papillomavirus E2 Proteins

    PubMed Central

    McBride, Alison A.

    2013-01-01

    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. PMID:23849793

  1. LPS-induced c-Fos activation in NTS neurons and plasmatic cortisol increases in septic rats are suppressed by bilateral carotid chemodenervation.

    PubMed

    Reyes, Edison-Pablo; Abarzúa, Sebastián; Martin, Aldo; Rodríguez, Jorge; Cortés, Paula P; Fernández, Ricardo

    2012-01-01

    Lipopolysaccharide (LPS) administered I.P. increases significantly the activation of c-Fos in neurons of the nucleus of the solitary tract (NTS), which in turn activates hypothalamus-pituitary-adrenal axis. The vagus nerve appears to play a role in conveying cytokines signals to the central nervous system (CNS), since -in rodent models of sepsis- bilateral vagotomy abolishes increases in plasmatic glucocorticoid levels, but does not suppress c-Fos NTS activation. Considering that NTS also receives sensory inputs from carotid body chemoreceptors, we evaluated c-Fos activation and plasmatic cortisol levels 90 min after I.P. administration of 15 mg/kg LPS. Experiments were performed in male Sprague-Dawley rats, in control conditions and after bilateral carotid neurotomy (BCN). LPS administration significantly increases the number of c-Fos positive NTS neurons and plasmatic cortisol levels in animals with intact carotid/sinus nerves. When LPS was injected after BCN, the number of c-Fos positive NTS neurons, and plasmatic cortisol levels were not significantly modified. Our data suggest that carotid body chemoreceptors might mediate CNS activation during sepsis.

  2. Effects of selected xenobiotics on hepatic and plasmatic biomarkers in juveniles of Solea senegalensis.

    PubMed

    Solé, Montserrat; Fortuny, Anna; Mañanós, Evaristo

    2014-11-01

    In recent years, Solea senegalensis has increasingly been used in pollution monitoring studies. In order to assess its response to some particular widespread pollutants, juveniles of S. senegalensis were administered an intraperitoneal injection of the model aryl hydrocarbon receptor agonist β-naphtoflavone (βNF) and chemicals of environmental concern, such as the fungicide ketoconazole (KETO), the lipid regulator gemfibrozil (GEM), the surfactant nonylphenol (NP) and the synthetic hormone ethinylestradiol (EE2). Two days after injection, the effect of these chemicals was followed up as alterations of hepatic microsomal activities of the cytochrome P450 (CYPs) and associated reductases, carboxylesterases (CbEs) and the conjugation enzyme uridine diphosphate glucuronyltransferase (UDPGT). In the cytosolic fraction of the liver, the effect on CbEs, glutathione S-transferase (GST) and antioxidant activities was also considered. Alterations on the endocrine reproductive system were evaluated by plasma levels of vitellogenin (VTG) and the sex steroids estradiol (E2), testosterone (T), 11-ketotestosterone (11KT) and the progestin 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P). Injection with the model compound βNF induced the hydrolysis rate of the seven CYP substrates assayed. The xenobiotic GEM induced three CYP-related activities (e.g. ECOD) and UDPGT, but depressed antioxidant defenses. EE2 induced four CYPs, more significantly ECOD and BFCOD activities. The xenoestrogens NP and EE2 altered the activities of CbE in microsomes and catalase, and were the only treatments that induced de novo VTG synthesis. In addition, the progestin 17,20β-P, was induced in NP-injected fish. None of the treatments caused statistically significant effects on steroid plasma levels. In conclusion, the CYP substrates assayed responded specifically to treatments and juveniles of S. senegalensis appear good candidates for assessing xenobiotics exposure. PMID:25462670

  3. Effects of selected xenobiotics on hepatic and plasmatic biomarkers in juveniles of Solea senegalensis.

    PubMed

    Solé, Montserrat; Fortuny, Anna; Mañanós, Evaristo

    2014-11-01

    In recent years, Solea senegalensis has increasingly been used in pollution monitoring studies. In order to assess its response to some particular widespread pollutants, juveniles of S. senegalensis were administered an intraperitoneal injection of the model aryl hydrocarbon receptor agonist β-naphtoflavone (βNF) and chemicals of environmental concern, such as the fungicide ketoconazole (KETO), the lipid regulator gemfibrozil (GEM), the surfactant nonylphenol (NP) and the synthetic hormone ethinylestradiol (EE2). Two days after injection, the effect of these chemicals was followed up as alterations of hepatic microsomal activities of the cytochrome P450 (CYPs) and associated reductases, carboxylesterases (CbEs) and the conjugation enzyme uridine diphosphate glucuronyltransferase (UDPGT). In the cytosolic fraction of the liver, the effect on CbEs, glutathione S-transferase (GST) and antioxidant activities was also considered. Alterations on the endocrine reproductive system were evaluated by plasma levels of vitellogenin (VTG) and the sex steroids estradiol (E2), testosterone (T), 11-ketotestosterone (11KT) and the progestin 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P). Injection with the model compound βNF induced the hydrolysis rate of the seven CYP substrates assayed. The xenobiotic GEM induced three CYP-related activities (e.g. ECOD) and UDPGT, but depressed antioxidant defenses. EE2 induced four CYPs, more significantly ECOD and BFCOD activities. The xenoestrogens NP and EE2 altered the activities of CbE in microsomes and catalase, and were the only treatments that induced de novo VTG synthesis. In addition, the progestin 17,20β-P, was induced in NP-injected fish. None of the treatments caused statistically significant effects on steroid plasma levels. In conclusion, the CYP substrates assayed responded specifically to treatments and juveniles of S. senegalensis appear good candidates for assessing xenobiotics exposure.

  4. Effects of the presence of indium on the mammary gland ultrastructure, body weight, food intake and plasmatic prolactin concentration.

    PubMed

    Ayadi, Ahlem; Maghraoui, Samira; Kammoun, Sayda; Tekaya, Leila

    2014-10-01

    Several studies have demonstrated the toxic effect of indium. This element induces impairments in many organs such as spleen, lungs and testicles after its systemic administration. Teratogenic and embryotoxic effects of this element have also been established. In the present study, we attempt to investigate the histological and the ultrastructural consequences of the presence of this element in mammary gland tissue using conventional transmission electron microscopy and to evaluate the incidences of its presence on the food intake, body weight and prolactin plasmatic concentration of lactating female rats. Our study showed that this element induced a significant decrease in food intake and body weight, and caused some cellular damage in the glandular epithelial cell such as cytoplasmic vacuolization and expansion of the ergastoplasm. The ultrastructural observations also showed many electron-dense surcharges in the lysosomes of the glandular epithelial cells. The electron probe microanalysis showed that these deposits are composed of indium and phosphorus. The lysosomes, known for their protective role of sequestrating foreign elements to avoid their diffusion in the blood, failed to stop the toxic effect of indium.

  5. Drospirenone intake alters plasmatic steroid levels and cyp17a1 expression in gonads of juvenile sea bass.

    PubMed

    Blanco, Maria; Fernandes, Denise; Medina, Paula; Blázquez, Mercedes; Porte, Cinta

    2016-06-01

    Drospirenone (DRO) is one of the most widely used progestins in contraceptive treatments and hormone replacement therapies. The pharmacokinetics and potential toxicological effects of DRO were investigated in juvenile sea bass (Dicentrarchus labrax) exposed through the diet (0.01-10 μg DRO/g) for up to 31 days. DRO was detected in the blood (4-27 ng/mL) of fish exposed to the highest concentration, with no significant bioaccumulation over time and no alteration of hepatic metabolizing enzymes, namely, CYP1A and CYP3A-catalysed activities and UDP-glucuronyltransferase (UGT). Pregnenolone (P5), progesterone (P4), 17α-hydroxyprogesterone (17P4), 17α-hydroxypregnenolone (17P5), androstenedione (AD) and testosterone (T) were determined in plasma and gene expression of cyp17a1, cyp19a1a and cyp11β analysed by qRT-PCR in gonads. The significant increase in plasmatic levels of 17P5, 17P4 and AD detected after 31 days exposure to 10 ng DRO/g together with the increased expression of cyp17a1 in females evidence the ability of DRO to alter steroid synthesis at low intake concentrations (7 ng DRO/day). However, the potential consequences of this steroid shift for female reproduction remain to be investigated. PMID:26995450

  6. [Radioimmunologic determination of plasmatic triiodothyronine. Verification of operative parameters; 1st clinical results].

    PubMed

    Dotti, C

    1975-06-01

    .01 e + 0.08, n = 10, r = 0.999 for ANS and f = 1.08 e -- 1.64, n = 9, r = 0.995 for Merth, implying a practically quantitative recovery in both cases. Thyroxine (T4) cross reaction study has been undertaken under a quite new optics re-calculating the regression lines -- equations of the T3 recovery in the presence of added T4; in that case, the following equations are valid: f = 1.05 e + 0,13, n = 10, r = 0.992 for ANS and f = 1.07 e + 2.26, n = 18, r = 0,985 for Merth. Reducing the maximum added T4 to 1 ng/tube, the cross reaction can be considered as negligible. T3 levels for normal subjects are finally reported: 1.50 +/- 0.80, n= 37 for ANS and 1.40 +/- 0.68, n = 40 for Merth (ng T3/ml, means +/- 2SD). PMID:1223940

  7. Mice Survival and Plasmatic Cytokine Secretion in a "Two Hit" Model of Sepsis Depend on Intratracheal Pseudomonas Aeruginosa Bacterial Load.

    PubMed

    Restagno, Damien; Venet, Fabienne; Paquet, Christian; Freyburger, Ludovic; Allaouchiche, Bernard; Monneret, Guillaume; Bonnet, Jeanne-Marie; Louzier, Vanessa

    2016-01-01

    Sepsis is characterized by pro- and anti-inflammatory responses following infection. While inflammation is responsible for widespread organ damage, anti-inflammatory mediators lead to immunoparalysis increasing susceptibility to secondary infections (nosocomial pneumonia). We aimed to investigate the impact of bacterial load on survival and cytokine release in a two-hit murine (C57BL/6J) model of CLP followed by P. aeruginosa pneumonia. Plasmatic TNFα, IL-6, IL-10, sTNFr I and II were quantified until 13 days. At D5, splenocytes were processed for immunological assays or mice were intratracheally instilled with Pseudomonas aeruginosa (5.106, 2.107 and 108 CFU) to evaluate survival and cytokines production. TNFα, sTNFrs, IL-6 and IL-10 increased 2h post CLP. TNFα and sTNFrs declined respectively one and two days later. In CLP mice, IL-6 and IL-10 remained high for the whole experiment, as compared to Sham. At D5, for CLP mice, whereas total T cells population (CD3+) decreased, Treg fraction (CD4+/CD25+) increased. In parallel, T cells proliferation and LPS-stimulated splenocytes ability to release TNFα decreased. At D13, survival was 100% after 5.106 CFU, 50% for CLP mice after 2.107 CFU and 0% for CLP and Sham after 108 CFU. After instillation, IL-10 and IL-6 increased and appeared to be dose and time dependent. Pseudomonas was detected in all CLP and Sham's lungs; in spleen and liver only in CLP at 2.107 CFU, and in CLP and Sham at 108 CFU. We demonstrated that post-CLP immunosuppression followed by Pseudomonas aeruginosa lung instillation increases mortality reactivates cytokines secretion and is associated with systemic dissemination in septic mice depending on bacterial load. PMID:27574993

  8. Effect of Iron and Carbon Monoxide on Fibrinogenase-like Degradation of Plasmatic Coagulation by Venoms of Six Agkistrodon Species.

    PubMed

    Nielsen, Vance G; Redford, Daniel T; Boyle, Patrick K

    2016-05-01

    Annually, thousands suffer poisonous snakebite, often from defibrinogenating species. It has been demonstrated that iron and carbon monoxide change the ultrastructure of plasma thrombi and improve coagulation kinetics. Thus, this investigation sought to determine whether pre-treatment of plasma with iron and carbon monoxide could attenuate venom-mediated catalysis of fibrinogen obtained from Agkistrodon species with fibrinogenase activity. Human plasma was pre-treated with ferric chloride (0-10 μM) and carbon monoxide-releasing molecule-2 (CORM-2, 0-100 μM) prior to exposure to 0.5-11 μg/ml of six different Agkistrodon species' venom. The amount of venom used for experimentation needed to decrease coagulation function of one or more kinetic parameters by at least 50% of normal values for (e.g. half the normal speed of clot formation). Coagulation kinetics were determined with thrombelastography. All six snake venoms degraded plasmatic coagulation kinetics to a significant extent, especially prolonging the onset to clot formation and diminishing the speed of clot growth. Pre-treatment of plasma with iron and carbon monoxide attenuated these venom-mediated coagulation kinetic changes in a species-specific manner, with some venom effects markedly abrogated while others were only mildly decreased. Further in vitro investigation of other pit viper venoms that possess fibrinogenolytic activity is indicated to identify species amenable to or resistant to iron and carbon monoxide-mediated attenuation of venom-mediated catalysis of fibrinogen. Lastly, future pre-clinical investigation with animal models (e.g. rabbit ear-bleed model) is planned to determine whether iron and carbon monoxide can be used therapeutically after envenomation.

  9. Effect of Iron and Carbon Monoxide on Fibrinogenase-like Degradation of Plasmatic Coagulation by Venoms of Six Agkistrodon Species.

    PubMed

    Nielsen, Vance G; Redford, Daniel T; Boyle, Patrick K

    2016-05-01

    Annually, thousands suffer poisonous snakebite, often from defibrinogenating species. It has been demonstrated that iron and carbon monoxide change the ultrastructure of plasma thrombi and improve coagulation kinetics. Thus, this investigation sought to determine whether pre-treatment of plasma with iron and carbon monoxide could attenuate venom-mediated catalysis of fibrinogen obtained from Agkistrodon species with fibrinogenase activity. Human plasma was pre-treated with ferric chloride (0-10 μM) and carbon monoxide-releasing molecule-2 (CORM-2, 0-100 μM) prior to exposure to 0.5-11 μg/ml of six different Agkistrodon species' venom. The amount of venom used for experimentation needed to decrease coagulation function of one or more kinetic parameters by at least 50% of normal values for (e.g. half the normal speed of clot formation). Coagulation kinetics were determined with thrombelastography. All six snake venoms degraded plasmatic coagulation kinetics to a significant extent, especially prolonging the onset to clot formation and diminishing the speed of clot growth. Pre-treatment of plasma with iron and carbon monoxide attenuated these venom-mediated coagulation kinetic changes in a species-specific manner, with some venom effects markedly abrogated while others were only mildly decreased. Further in vitro investigation of other pit viper venoms that possess fibrinogenolytic activity is indicated to identify species amenable to or resistant to iron and carbon monoxide-mediated attenuation of venom-mediated catalysis of fibrinogen. Lastly, future pre-clinical investigation with animal models (e.g. rabbit ear-bleed model) is planned to determine whether iron and carbon monoxide can be used therapeutically after envenomation. PMID:26467642

  10. Plasmatic concentration of organochlorine lindane acts as metabolic disruptors in HepG2 liver cell line by inducing mitochondrial disorder

    SciTech Connect

    Benarbia, Mohammed el Amine; Macherel, David; Faure, Sébastien; Jacques, Caroline; Andriantsitohaina, Ramaroson; Malthièry, Yves

    2013-10-15

    Lindane (LD) is a persistent environmental pollutant that has been the subject of several toxicological studies. However, concentrations used in most of the reported studies were relatively higher than those found in the blood of the contaminated area residents and effects of low concentrations remain poorly investigated. Moreover, effects on cell metabolism and mitochondrial function of exposure to LD have received little attention. This study was designed to explore the effects of low concentrations of LD on cellular metabolism and mitochondrial function, using the hepatocarcinoma cell line HepG2. Cells were exposed to LD for 24, 48 and 72 h and different parameters linked with mitochondrial regulation and energy metabolism were analyzed. Despite having any impact on cellular viability, exposure to LD at plasmatic concentrations led to an increase of maximal respiratory capacity, complex I activity, intracellular ATP and NO release but decreased uncoupled respiration to ATP synthesis and medium lactate levels. In addition, LD exposure resulted in the upregulation of mitochondrial biogenesis genes. We suggest that, at plasmatic concentrations, LD acts as a metabolic disruptor through impaired mitochondrial function and regulation with an impact on cellular energetic metabolism. In addition, we propose that a cellular assay based on the analysis of mitochondria function, such as described here for LD, may be applicable for larger studies on the effects of low concentrations of xenobiotics, because of the exquisite sensitivity of this organelle. - Highlights: Our data clearly demonstrated in HepG2 cells that exposure at plasmatic low concentrations of LD were able to: • Impair mitochondrial function • Caused alteration on nucleo-mitochondrial cross-talk • Increase nitric oxide release and protein nitration • Impair cellular energetic metabolism and lipid accumulation.

  11. Plasmatic retinol-binding protein 4 and glial fibrillary acidic protein as biomarkers to differentiate ischemic stroke and intracerebral hemorrhage.

    PubMed

    Llombart, Víctor; García-Berrocoso, Teresa; Bustamante, Alejandro; Giralt, Dolors; Rodriguez-Luna, David; Muchada, Marian; Penalba, Anna; Boada, Cristina; Hernández-Guillamon, Mar; Montaner, Joan

    2016-01-01

    A rapid differentiation of acute ischemic stroke and intracerebral hemorrhage (ICH) is essential for an adequate treatment and to promote a better outcome. Our aim was to identify new plasma biomarkers to differentiate stroke subtypes and to combine their diagnostic ability with other biomarkers already described for this clinical indication. Plasma samples of ischemic stroke patients (36) and ICH patients (10) were screened using a 177 antibodies library, and 11 showed different concentrations among stroke subtypes (p < 0.05), mainly chemokines, growth factors and angiogenic factors. Five proteins were selected for replication in 16 ischemic stroke patients and 16 ICH patients, and retinol-binding protein 4 (RPB4), apolipoprotein B100 and pigment epithelial-derived factor were replicated (p < 0.05). These proteins, together with glial fibrillary acidic protein (GFAP) and receptor for advanced glycation end product, were tested in 38 ischemic stroke and 28 ICH samples. Finally, RBP4 >61 μg/mL and GFAP <0.07 ng/mL showed a specificity of 100% for both subtypes. Moreover, after multivariate logistic regression analysis, RBP4 >48.75 μg/mL (ORadj : 6.09 (1.3-28.57), p = 0.02) and GFAP <0.07 ng/mL (ORadj : 0.03 (0.003-0.31), p = 0.003) resulted in independent predictors of stroke subtype, improving discrimination by 29% (p < 0.0001). Both biomarkers might be useful as diagnostic biomarkers to differentiate ischemic stroke and ICH. A rapid differentiation of ischemic stroke from intracerebral hemorrhage is essential to provide the appropriate treatment. We describe the discovery and subsequent replications of RBP4 and its combination with circulating GFAP as plasmatic biomarkers for hyperacute stroke subtype differentiation. The combination of these biomarkers and others might aid to speed up the discrimination of both stroke subtypes improving the outcome of patients.

  12. Evolutionary variation of papillomavirus E2 protein and E2 binding sites

    PubMed Central

    2011-01-01

    Background In an effort to identify the evolutionary changes relevant to E2 function, within and between papillomavirus genera, we evaluated the E2 binding sites (E2BS)s inside the long-control-region (LCR), and throughout the genomes. We identified E2BSs in the six largest genera of papillomaviruses: Alpha, Beta, Gamma, Delta, Lambda, and Xi-papillomaviruses (128 genomes), by comparing the sequences with a model consensus we created from known functional E2BSs (HPV16, HPV18, BPV1). We analyzed the sequence conservation and nucleotide content of the 4-nucleotide spacer within E2BSs. We determined that there is a statistically significant difference in GC content of the four-nucleotide E2BS spacer, between Alpha and Delta-papillomaviruses, as compared to each of the other groups. Additionally, we performed multiple alignments of E2 protein sequences using members of each genus in order to identify evolutionary changes within the E2 protein. Results When a phylogenetic tree was generated from E2 amino acid sequences, it was discovered that the alpha-papillomavirus genera segregates into two distinct subgroups (α1 and α2). When these subgroups were individually analyzed, it was determined that the subgroup α1 consensus E2BS favored a spacer of AAAA, whereas subgroup α2 favored the opposite orientation of the same spacer; TTTT. This observation suggests that these conserved inverted linkers could have functional importance. PMID:21806797

  13. Aspirin inhibits androgen response to chorionic gonadotropin in humans.

    PubMed

    Conte, D; Romanelli, F; Fillo, S; Guidetti, L; Isidori, A; Franceschi, F; Latini, M; di Luigi, L

    1999-12-01

    Eicosanoids play an important role in the regulation of the hypothalamic-pituitary axis; less clear is their role in testicular steroidogenesis. To evaluate the involvement of cyclooxygenase metabolites, such as prostaglandins, in the regulation of human testicular steroidogenesis, we examined the effects of a prostaglandin-blocker, aspirin, on plasma testosterone, pregnenolone, progesterone, 17OH-progesterone, androstenedione, dehydroepiandrosterone, and 17beta-estradiol response to human chorionic gonadotropin (hCG) in normal male volunteers in a placebo-controlled, single-blinded study. To test the efficacy of aspirin, seminal prostaglandin E(2) levels were also determined. hCG stimulation increased peripheral levels of testosterone, 17OH-progesterone, androstenedione, dehydroepiandrosterone, and 17beta-estradiol, without affecting circulating pregnenolone and progesterone values. Aspirin significantly lowered seminal prostaglandin E(2) levels, whereas it did not modify steroid concentrations not exposed to exogenous hCG. Moreover, the drug significantly reduced the response of testosterone, 17OH-progesterone, androstenedione, and dehydroepiandrosterone to hCG, as assessed by the mean integrated area under the curve, whereas it did not influence 17beta-estradiol response. In conclusion, aspirin treatment inhibits androgen response to chorionic gonadotropin stimulation in normal humans. The action of aspirin is probably mediated via an effective arachidonate cyclooxygenase block.

  14. Estrogen modulates the mRNA levels for cancellous bone protein of ovariectomized rats.

    PubMed

    Salih, M A; Liu, C C; Arjmandi, B H; Kalu, D N

    1993-12-01

    This study was undertaken to examine the effects of ovariectomy and 17 beta-estradiol (E2) on the gene expression of type 1 collagen, osteocalcin and the protooncogen, c-myc, in cancellous bone. Female Sprague-Dawley rats, aged 95 days, were divided into 4 groups. Group 1 was sham operated and Groups 2-4 were ovariectomized. Groups 3 and 4 received daily injections of 160 ng and 1600 ng E2/kg body weight, respectively. Groups 1 and 2 received the solvent vehicle. All animals were sacrificed after 14 days. The femurs were dissected out and cancellous bone scraped from the distal metaphysis. RNA was isolated from the cancellous bone, immobilized on filters or size-fractionated by agarose gel electrophoresis and adsorbed on filters which were then hybridized with specific cDNA probes. Ovariectomy resulted in a significant increase in the mRNAs of type 1 collagen, osteocalcin and c-myc. The increase was suppressed in animals that received 17 beta-estradiol injections. In addition, ovariectomy caused the expected decrease in cancellous bone in the proximal tibia and increased osteoclast and osteoblast numbers. The ovariectomy-induced changes were prevented by 17 beta-estradiol administration. These findings suggest that the lack of ovarian hormones shortly after ovariectomy up-regulates and estrogen administration down-regulates the expression of important cancellous bone matrix proteins as well as the protooncogen, c-myc.

  15. Compared binding properties between resveratrol and other polyphenols to plasmatic albumin: consequences for the health protecting effect of dietary plant microcomponents.

    PubMed

    Latruffe, Norbert; Menzel, Matthias; Delmas, Dominique; Buchet, René; Lançon, Allan

    2014-01-01

    Phytophenols are considered to have beneficial effects towards human physiology. They are food microcomponents with potent chemopreventive properties towards the most three frequent contemporary human diseases, e.g., cardiovascular alterations, cancer and neurodegenerative pathologies. Related to this, the plasmatic form and plasmatic level of plant polyphenols in the body circulation are crucial for their efficiency. Thus, determinations of the binding process of resveratrol and of common flavonoids produced by major edible plants, berries and fruits to plasma proteins are essential. The interactions between resveratrol and albumin, a major plasma protein, were compared with those already published, involving curcumin, genistein, quercetin and other well-known food-containing polyphenols. The approaches used are usually intrinsic fluorescence intensity changes, quenching of protein intrinsic fluorescence and infrared spectroscopy. It appears that: (1) all of the studied polyphenols interact with albumin; (2) while most of the studied polyphenols interact at one albumin binding site, there are two different types of resveratrol binding sites for bovine serum albumin, one with the highest affinity (apparent KD of 4 µM) with a stoichiometry of one per monomer and a second with a lower affinity (apparent KD of 20 µM) with also a stoichiometry of one per monomer; (3) at least one binding site is in the vicinity of one tryptophanyl residue of bovine serum albumin; and (4) resveratrol binding to bovine serum albumin produces a very small structural conformation change of the polypeptide chain. These results support a role played by polyphenols-albumin interactions in the plasma for the bio-activities of these food microcomponents in the body. PMID:25347454

  16. 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

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

    PubMed

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

    2016-01-01

    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.

  18. 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

  19. Mice Survival and Plasmatic Cytokine Secretion in a “Two Hit” Model of Sepsis Depend on Intratracheal Pseudomonas Aeruginosa Bacterial Load

    PubMed Central

    Restagno, Damien; Venet, Fabienne; Paquet, Christian; Freyburger, Ludovic; Allaouchiche, Bernard; Monneret, Guillaume; Bonnet, Jeanne-Marie; Louzier, Vanessa

    2016-01-01

    Sepsis is characterized by pro- and anti-inflammatory responses following infection. While inflammation is responsible for widespread organ damage, anti-inflammatory mediators lead to immunoparalysis increasing susceptibility to secondary infections (nosocomial pneumonia). We aimed to investigate the impact of bacterial load on survival and cytokine release in a two-hit murine (C57BL/6J) model of CLP followed by P. aeruginosa pneumonia. Plasmatic TNFα, IL-6, IL-10, sTNFr I and II were quantified until 13 days. At D5, splenocytes were processed for immunological assays or mice were intratracheally instilled with Pseudomonas aeruginosa (5.106, 2.107 and 108 CFU) to evaluate survival and cytokines production. TNFα, sTNFrs, IL-6 and IL-10 increased 2h post CLP. TNFα and sTNFrs declined respectively one and two days later. In CLP mice, IL-6 and IL-10 remained high for the whole experiment, as compared to Sham. At D5, for CLP mice, whereas total T cells population (CD3+) decreased, Treg fraction (CD4+/CD25+) increased. In parallel, T cells proliferation and LPS-stimulated splenocytes ability to release TNFα decreased. At D13, survival was 100% after 5.106 CFU, 50% for CLP mice after 2.107 CFU and 0% for CLP and Sham after 108 CFU. After instillation, IL-10 and IL-6 increased and appeared to be dose and time dependent. Pseudomonas was detected in all CLP and Sham’s lungs; in spleen and liver only in CLP at 2.107 CFU, and in CLP and Sham at 108 CFU. We demonstrated that post-CLP immunosuppression followed by Pseudomonas aeruginosa lung instillation increases mortality reactivates cytokines secretion and is associated with systemic dissemination in septic mice depending on bacterial load. PMID:27574993

  20. Interplay between Arabidopsis Activating Factors E2Fb and E2Fa in Cell Cycle Progression and Development1[W

    PubMed Central

    Sozzani, Rosangela; Maggio, Caterina; Varotto, Serena; Canova, Sabrina; Bergounioux, Catherine; Albani, Diego; Cella, Rino

    2006-01-01

    Eukaryotic E2Fs are conserved transcription factors playing crucial and antagonistic roles in several pathways related to cell division, DNA repair, and differentiation. In plants, these processes are strictly intermingled at the growing zone to produce postembryonic development in response to internal signals and environmental cues. Of the six AtE2F proteins found in Arabidopsis (Arabidopsis thaliana), only AtE2Fa and AtE2Fb have been clearly indicated as activators of E2F-responsive genes. AtE2Fa activity was shown to induce S phase and endoreduplication, whereas the function of AtE2Fb and the interrelationship between these two transcription factors was unclear. We have investigated the role played by the AtE2Fb gene during cell cycle and development performing in situ RNA hybridization, immunolocalization of the AtE2Fb protein in planta, and analysis of AtE2Fb promoter activity in transgenic plants. Overexpression of AtE2Fb in transgenic Arabidopsis plants led to striking modifications of the morphology of roots, cotyledons, and leaves that can be ascribed to stimulation of cell division. The accumulation of the AtE2Fb protein in these lines was paralleled by an increased expression of E2F-responsive G1/S and G2/M marker genes. These results suggest that AtE2Fa and AtE2Fb have specific expression patterns and play similar but distinct roles during cell cycle progression. PMID:16514015

  1. E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

    PubMed Central

    Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

    2015-01-01

    Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53−/− mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy. PMID:25656653

  2. Associations among Inflammatory Biomarkers in the Circulating, Plasmatic, Salivary and Intraluminal Anatomical Compartments in Apparently Healthy Preschool Children from the Western Highlands of Guatemala

    PubMed Central

    Soto-Méndez, María José; Romero-Abal, María Eugenia; Aguilera, Concepción María; Rico, María Cruz; Solomons, Noel W.; Schümann, Klaus; Gil, Angel

    2015-01-01

    Background Undernutrition and inflammation are related in many ways; for instance, non-hygienic environments are associated with both poor growth and immunostimulation in children. Objective To describe any existing interaction among different inflammation biomarkers measured in the distinct anatomical compartments of whole blood, feces, plasma and saliva. Methods In this descriptive, cross-sectional study, samples of whole blood, feces, plasma and saliva were collected on the 8th and last week of observation among 87 attendees (42 girls and 45 boys) of 3 daycare centers offering a common 40-day rotating menu in Guatemala’s Western Highlands. Analyses included white blood cell count (WBC), fecal calprotectin, and plasmatic and salivary cytokines including IL-1B, IL-6, IL-8, IL-10 and TNF-α. Associations were assessed using Spearman rank-order and goodness-of-fit correlations, as indicated, followed by backwards-elimination multiple regression analyses to determine predictor variables for IL-10 in both anatomical compartments. Results Of a total of 66 cross-tabulations in the Spearman hemi-matrix, 22 (33%) were significantly associated. All 10 paired associations among the salivary cytokines had a significant r value, whereas 7 of 10 possible associations among plasma cytokines were significant. Associations across anatomical compartments, however, were rarely significant. IL-10 in both biological fluids were higher than corresponding reference values. When a multiple regression model was run in order to determine independent predictors for IL-10 in each anatomical compartment separately, IL-6, IL-8 and TNF-α emerged as predictors in plasma (r2 = 0.514) and IL-1B, IL-8 and TNF-α remained as independent predictors in saliva (r2 = 0.762). Significant cross-interactions were seen with WBC, but not with fecal calprotectin. Conclusion Interactions ranged from robust within the same anatomical compartment to limited to nil across distinct anatomical compartments. The

  3. 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

  4. Nematicidal activity of (E,E)-2,4-decadienal and (E)-2-decenal from Ailanthus altissima against Meloidogyne javanica.

    PubMed

    Caboni, Pierluigi; Ntalli, Nikoletta G; Aissani, Nadhem; Cavoski, Ivana; Angioni, Alberto

    2012-02-01

    Methanol extracts of various plant parts of Ailanthus altissima were tested against the root knot nematode Meloidogyne javanica . Extracts of bark (ABE), wood (AWE), roots (ARE), and leaves (ALE) from A. altissima were investigated against freshly hatched second-stage juveniles (J(2)). AWE was the most active extract, with EC(50/3d) of 58.9 mg/L, while ALE, ARE, and ABE did not show nematicidal activity. The chemical composition of the extracts of A. altissima was determined by gas chromatography-mass spectrometry, and (E,E)-2,4-decadienal, (E)-2-undecenal, (E)-2-decenal, hexanal, nonanal, and furfural were the most prominent constituents. (E,E)-2,4-Decadienal, (E)-2-decenal, and furfural showed the highest nematicidal activity against M. javanica , with EC(50/1d) = 11.7, 20.43, and 21.79 mg/L, respectively, while the other compounds were inactive at the concentrations tested. The results obtained showed that AWE and its constituents (E,E)-2,4-decadienal and (E)-2-decenal could be considered as potent botanical nematicidal agents. PMID:22224661

  5. Radiation hardness of COTS EPROMs and E2PROMs

    NASA Astrophysics Data System (ADS)

    Vujisić, Miloš; Stanković, Koviljka; Dolićanin, Edin; Osmokrović, Predrag

    2010-05-01

    This paper examines and compares the effects of exposing commercial, off the shelf erasable programmable read-only memory (EPROM) and electrically erasable programmable read-only memory (E2PROM) components to gamma rays. Results obtained for CMOS-based EPROM (NM27C010) and E2PROM (NM93CS46) components provide evidence that EPROMs have a greater radiation hardness than E2PROMs. Moreover, the changes in EPROMs are reversible, and after erasure and reprogramming all EPROM components restore their functionality. On the other hand, changes in E2PROMs are irreversible. The obtained results are analyzed and interpreted on the basis of gamma ray interaction with the CMOS structure.

  6. A MUB E2 structure reveals E1 selectivity between cognate ubiquitin E2s in eukaryotes

    PubMed Central

    Lu, Xiaolong; Malley, Konstantin R.; Brenner, Caitlin C.; Koroleva, Olga; Korolev, Sergey; Downes, Brian P.

    2016-01-01

    Ubiquitin (Ub) is a protein modifier that controls processes ranging from protein degradation to endocytosis, but early-acting regulators of the three-enzyme ubiquitylation cascade are unknown. Here we report that the prenylated membrane-anchored ubiquitin-fold protein (MUB) is an early-acting regulator of subfamily-specific E2 activation. An AtMUB3:AtUBC8 co-crystal structure defines how MUBs inhibit E2∼Ub formation using a combination of E2 backside binding and a MUB-unique lap-bar loop to block E1 access. Since MUBs tether Arabidopsis group VI E2 enzymes (related to HsUbe2D and ScUbc4/5) to the plasma membrane, and inhibit E2 activation at physiological concentrations, they should function as potent plasma membrane localized regulators of Ub chain synthesis in eukaryotes. Our findings define a biochemical function for MUB, a family of highly conserved Ub-fold proteins, and provide an example of selective activation between cognate Ub E2s, previously thought to be constitutively activated by E1s. PMID:27550514

  7. A MUB E2 structure reveals E1 selectivity between cognate ubiquitin E2s in eukaryotes.

    PubMed

    Lu, Xiaolong; Malley, Konstantin R; Brenner, Caitlin C; Koroleva, Olga; Korolev, Sergey; Downes, Brian P

    2016-01-01

    Ubiquitin (Ub) is a protein modifier that controls processes ranging from protein degradation to endocytosis, but early-acting regulators of the three-enzyme ubiquitylation cascade are unknown. Here we report that the prenylated membrane-anchored ubiquitin-fold protein (MUB) is an early-acting regulator of subfamily-specific E2 activation. An AtMUB3:AtUBC8 co-crystal structure defines how MUBs inhibit E2∼Ub formation using a combination of E2 backside binding and a MUB-unique lap-bar loop to block E1 access. Since MUBs tether Arabidopsis group VI E2 enzymes (related to HsUbe2D and ScUbc4/5) to the plasma membrane, and inhibit E2 activation at physiological concentrations, they should function as potent plasma membrane localized regulators of Ub chain synthesis in eukaryotes. Our findings define a biochemical function for MUB, a family of highly conserved Ub-fold proteins, and provide an example of selective activation between cognate Ub E2s, previously thought to be constitutively activated by E1s. PMID:27550514

  8. A MUB E2 structure reveals E1 selectivity between cognate ubiquitin E2s in eukaryotes

    NASA Astrophysics Data System (ADS)

    Lu, Xiaolong; Malley, Konstantin R.; Brenner, Caitlin C.; Koroleva, Olga; Korolev, Sergey; Downes, Brian P.

    2016-08-01

    Ubiquitin (Ub) is a protein modifier that controls processes ranging from protein degradation to endocytosis, but early-acting regulators of the three-enzyme ubiquitylation cascade are unknown. Here we report that the prenylated membrane-anchored ubiquitin-fold protein (MUB) is an early-acting regulator of subfamily-specific E2 activation. An AtMUB3:AtUBC8 co-crystal structure defines how MUBs inhibit E2~Ub formation using a combination of E2 backside binding and a MUB-unique lap-bar loop to block E1 access. Since MUBs tether Arabidopsis group VI E2 enzymes (related to HsUbe2D and ScUbc4/5) to the plasma membrane, and inhibit E2 activation at physiological concentrations, they should function as potent plasma membrane localized regulators of Ub chain synthesis in eukaryotes. Our findings define a biochemical function for MUB, a family of highly conserved Ub-fold proteins, and provide an example of selective activation between cognate Ub E2s, previously thought to be constitutively activated by E1s.

  9. Interaction of E2F7 transcription factor with E2F1 and C-terminal-binding protein (CtBP) provides a mechanism for E2F7-dependent transcription repression.

    PubMed

    Liu, Beiyu; Shats, Igor; Angus, Steven P; Gatza, Michael L; Nevins, Joseph R

    2013-08-23

    Previous work has identified distinct functions for E2F proteins during a cellular proliferative response including a role for E2F1-3 in the activation of transcription at G1/S and a role for E2F4-8 in repressing the same group of E2F1-3 target genes as cells progress through S phase. We now find that E2F7 and E2F8, which are induced by E2F1-3 at G1/S, can form a heterodimer with E2F1 through interactions involving the DNA-binding domains of the two proteins. In vitro DNA interaction assays demonstrate the formation of an E2F1-E2F7 complex, as well as an E2F7-E2F7 complex on adjacent E2F-binding sites. We also show that E2F7 recruits the co-repressor C-terminal-binding protein (CtBP) and that CtBP2 is essential for E2F7 to repress E2F1 transcription. Taken together, these findings suggest a mechanism for the repression of transcription by E2F7.

  10. 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.

  11. Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2).

    PubMed Central

    Cha, S; Leung, P S; Van de Water, J; Tsuneyama, K; Joplin, R E; Ansari, A A; Nakanuma, Y; Schatz, P J; Cwirla, S; Fabris, L E; Neuberger, J M; Gershwin, M E; Coppel, R L

    1996-01-01

    Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8855289

  12. 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

  13. 26 CFR 31.3231(e)-2 - Contribution base.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 15 2010-04-01 2010-04-01 false Contribution base. 31.3231(e)-2 Section 31.3231... Contribution base. The term compensation does not include any remuneration paid during any calendar year by an employer to an employee for services rendered in excess of the applicable contribution base. For...

  14. 26 CFR 31.3231(e)-2 - Contribution base.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 15 2013-04-01 2013-04-01 false Contribution base. 31.3231(e)-2 Section 31.3231... Contribution base. The term compensation does not include any remuneration paid during any calendar year by an employer to an employee for services rendered in excess of the applicable contribution base. For...

  15. 26 CFR 31.3231(e)-2 - Contribution base.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 15 2012-04-01 2012-04-01 false Contribution base. 31.3231(e)-2 Section 31.3231... Contribution base. The term compensation does not include any remuneration paid during any calendar year by an employer to an employee for services rendered in excess of the applicable contribution base. For...

  16. 26 CFR 31.3231(e)-2 - Contribution base.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 15 2011-04-01 2011-04-01 false Contribution base. 31.3231(e)-2 Section 31.3231... Contribution base. The term compensation does not include any remuneration paid during any calendar year by an employer to an employee for services rendered in excess of the applicable contribution base. For...

  17. 26 CFR 31.3231(e)-2 - Contribution base.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 15 2014-04-01 2014-04-01 false Contribution base. 31.3231(e)-2 Section 31.3231... Contribution base. The term compensation does not include any remuneration paid during any calendar year by an employer to an employee for services rendered in excess of the applicable contribution base. For...

  18. 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.

  19. Reducing prostaglandin E2 production to raise cancer immunogenicity.

    PubMed

    Zelenay, Santiago; Reis E Sousa, Caetano

    2016-05-01

    Cyclooxygenases (COX), commonly upregulated in numerous cancers, generate prostaglandin E2 (PGE2), which has been implicated in key aspects of malignant growth including proliferation, invasion and angiogenesis. Recently, we showed that production of PGE2 by cancer cells dominantly enables progressive tumor growth via immune escape and that cyclooxygenase inhibitors synergize with immunotherapy to enhance tumor eradication. PMID:27467936

  20. Endocrine disrupter--estradiol--in Chesapeake Bay tributaries.

    PubMed

    Dorabawila, Nelum; Gupta, Gian

    2005-04-11

    Exogenous chemicals that interfere with natural hormonal functions are considered endocrine disrupting chemicals (EDCs). Estradiol (17beta-estradiol or E2) is the most potent of all xenoestrogens. Induction of vitellogenin (VTG) production in male fish occurs at E2 concentrations as low as 1 ng l-1. E2 reaches aquatic systems mainly through sewage and animal waste disposal. Surface water samples from ponds, rivers (Wicomico, Manokin and Pocomoke), sewage treatment plants (STPs), and coastal bays (Assawoman, Monie, Chincoteague, and Tangier Sound-Chesapeake Bay) on the Eastern Shore of Maryland were analyzed for E2 using enzyme linked immuno-sorbent assay (ELISA). E2 concentrations in river waters varied between 1.9 and 6.0 ng l-1. Highest E2 concentrations in river waters were observed immediately downstream of STPs. E2 concentrations in all the coastal bays tested were 2.3-3.2 ng l-1. PMID:15811666

  1. 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.

  2. 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.

  3. 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.

  4. Multicilin drives centriole biogenesis via E2f proteins.

    PubMed

    Ma, Lina; Quigley, Ian; Omran, Heymut; Kintner, Chris

    2014-07-01

    Multiciliate cells employ hundreds of motile cilia to produce fluid flow, which they nucleate and extend by first assembling hundreds of centrioles. In most cells, entry into the cell cycle allows centrioles to undergo a single round of duplication, but in differentiating multiciliate cells, massive centriole assembly occurs in G0 by a process initiated by a small coiled-coil protein, Multicilin. Here we show that Multicilin acts by forming a ternary complex with E2f4 or E2f5 and Dp1 that binds and activates most of the genes required for centriole biogenesis, while other cell cycle genes remain off. This complex also promotes the deuterosome pathway of centriole biogenesis by activating the expression of deup1 but not its paralog, cep63. Finally, we show that this complex is disabled by mutations in human Multicilin that cause a severe congenital mucociliary clearance disorder due to reduced generation of multiple cilia. By coopting the E2f regulation of cell cycle genes, Multicilin drives massive centriole assembly in epithelial progenitors in a manner required for multiciliate cell differentiation.

  5. 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.

  6. Poster 14: Explorer of Enceladus and Titan (E2T)

    NASA Astrophysics Data System (ADS)

    Mitri, Giuseppe; Tobie, Gabriel; Postberg, Frank; Soderblom, Jason M.; Wurz, Peter; Barnes, Jason W.; Berga, Marco; Coustenis, Athena; D'Ottavio, Andrea Hayes, Alexander G.; Hayne, Paul O.; Lebreton, Jean-Pierre; Lorenz, Ralph D.; Martelli, Andrea; Petropoulos, Anastassios E.; Yen, Chen-wan L.; Reh, Kim R.; Sotin, Christophe; Srama, Ralf; Tortora, Paolo

    2016-06-01

    The NASA-ESA Cassini-Huygens mission has revealed Titan and Enceladus to be two of the most enigmatic worlds in the Solar System. Titan, with its organically rich and dynamic atmosphere and geology, and Enceladus, with its active plume, both harboring subsurface oceans, are prime environments in which to investigate the conditions for the emergence of life and the habitability of Ocean Worlds. Explorer of Enceladus and Titan (E2T) is dedicated to investigating the evolution and habitability of these Saturnian satellites and will be proposed as a medium-class mission led by ESA in collaboration with NASA in response to ESA's M5 Call. E2T has a focused payload that will provide in-situ sampling and high-resolution imaging during multiple flybys of Enceladus and Titan using a solar-electric powered spacecraft in orbit around Saturn. The E2T mission will provide high-resolution mass spectroscopy of the plume emanating from Enceladus' south polar terrain (SPT) and of Titan's upper atmosphere as well as high-resolution IR imaging of the plume and the source fractures on Enceladus' SPT, and it will detail Titan's geomorphology at 50-100 m resolution. The E2T mission has three scientific goals: 1) Investigate the origin and evolution of volatile-rich icy worlds by examining both Enceladus and Titan, 2) Investigate the habitability and potential for life in ocean worlds on both Enceladus and Titan and 3) Investigate Titan as an Earth-like world with an evolving climate and landscape. These investigations will be accomplished by measuring the nature, abundance and isotopic properties of solid- and vapor-phase species in Enceladus' plume and Titan's upper atmosphere. E2T's high-resolution time-of-flight mass spectrometers will enable us to untangle the ambiguities left by Cassini regarding the identification of low-mass organic species, identify high-mass organic species for the first time, further constrain trace species such as the noble gases, and clarify the evolution of

  7. 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.

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

    PubMed

    Sahin, Fikret; Sladek, Todd L

    2010-07-04

    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, G(0) accumulation, and target gene experiments.

  9. (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.

  10. 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.

  11. 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.

  12. B-lymphocyte development is regulated by the combined dosage of three basic helix-loop-helix genes, E2A, E2-2, and HEB.

    PubMed Central

    Zhuang, Y; Cheng, P; Weintraub, H

    1996-01-01

    B-lymphocyte development requires the basic helix-loop-helix proteins encoded by the E2A gene. In this study, the control mechanism of E2A was further explored by disruption of the E2A-related genes, E2-2 and HEB. In contrast to E2A, E2-2 and HEB are not essential for the establishment of the B-cell lineage. However, both E2-2 and HEB are required for the generation of the normal numbers of pro-B cells in mouse embryos. Breeding tests among mice carrying different mutations revealed that E2-2 and HEB interact with E2A in many developmental processes including generation of B cells. Specifically, mice transheterozygous for any two mutations of these three genes produced fewer pro-B cells than the singly heterozygous littermates. This study indicates that B-cell development is dependent not only on an essential function provided by the E2A gene but also on a combined dosage set by E2A, E2-2, and HEB. PMID:8649400

  13. 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.

  14. E2 transition probabilities in {sup 114}Te: A conundrum

    SciTech Connect

    Moeller, O.; Warr, N.; Jolie, J.; Dewald, A.; Fitzler, A.; Linnemann, A.; Zell, K.O.; Garrett, P.E.; Yates, S.W.

    2005-06-01

    Lifetimes in {sup 114}Te were determined using the recoil distance Doppler-shift technique with a plunger device coupled to five HP Ge detectors enhanced by one Euroball cluster detector. The experiment was carried out at the Cologne FN Tandem facility using the {sup 93}Nb({sup 24}Mg,p2n) reaction at 90 MeV. The differential decay curve method in coincidence mode was employed to derive lifetimes for seven excited states, whereas the lifetime of an isomeric state was obtained in singles mode. The resulting E2 transition probabilities are shown to be very anomalous in comparison with the vibrational energy spacings of the ground-state band.

  15. E2 Transition Probabilities in 114Te: a Conundrum

    SciTech Connect

    Moller, O; Warr, N; Jolie, J; Dewald, A; Fitzler, A; Linnemann, A; Zell, K O; Garrett, P E; Yates, S W

    2005-05-13

    Lifetimes in {sup 114}Te were determined using the recoil distance Doppler-shift technique with a plunger device coupled to five HP Ge detectors enhanced by one Euroball Cluster detector. The experiment was carried out at the Cologne FN Tandem facility using the {sup 93}Nb({sup 24}Mg,p2n) reaction at 90 MeV. The differential decay curve method in coincidence mode was employed to derive lifetimes for seven excited states, while the lifetime of an isomeric state was obtained in singles mode. The resulting E2 transition probabilities are shown to be very anomalous in comparison with the vibrational energy spacings of the ground state band.

  16. Spectrin’s chimeric E2/E3 enzymatic activity

    PubMed Central

    Goodman, Steven R; Petrofes Chapa, Rachel

    2015-01-01

    In this minireview, we cover the discovery of the human erythrocyte α spectrin E2/E3 ubiquitin conjugating/ligating enzymatic activity and the specific cysteines involved. We then discuss the consequences when this activity is partially inhibited in sickle cell disease and the possibility that the same attenuation is occurring in multiple organ dysfunction syndrome. We finish by discussing the reasons for believing that nonerythroid α spectrin isoforms (I and II) also have this activity and the importance of testing this hypothesis. If correct, this would suggest that the nonerythroid spectrin isoforms play a major role in protein ubiquitination in all cell types. This would open new fields in experimental biology focused on uncovering the impact that this enzymatic activity has upon protein–protein interactions, protein turnover, cellular signaling, and many other functions impacted by spectrin, including DNA repair. PMID:26283706

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

    NASA Astrophysics Data System (ADS)

    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.

    2012-06-01

    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.

  18. 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.

  19. Arsenic and 17-β-estradiol bind to each other and neutralize each other's signaling effects.

    PubMed

    Kumar, Sukhdeep; Mukherjee, Tapan K; Guptasarma, Purnananda

    2016-09-01

    We report that arsenic trioxide (ATO) and 17-beta-estradiol (E2) abolish each other's independent cell signaling effects in respect of cell survival and proliferation/migration of breast cancer (MCF-7) cells. The possibility that this is due to binding of ATO to E2 was confirmed through difference absorption spectroscopy, chromatography-coupled voltammometry and 1-D (1)H and (13)C NMR spectroscopy. Binding leads to attenuation of E2's hydroxyl (1)H peaks at its C17 and C3 carbon positions. The results suggest that ATO and E2 can titrate each other's levels, potentially explaining why sustained arsenic exposure tends to be associated with delays in age of menarche, advanced age of menopause, poorer sperm quality, higher overall morbidity in men, and lower incidences of breast cancer in women in some arsenic-contaminated areas. PMID:27346132

  20. Chronic estrogen exposure maintains elevated levels of progesterone receptor mRNA in guinea pig hypothalamus.

    PubMed

    Bayliss, D A; Millhorn, D E

    1991-05-01

    We performed in situ hybridization on hypothalamic sections from ovariectomized guinea pig using a cocktail of three 35S-labeled oligonucleotides complementary to mammalian progesterone receptor (PR) cDNA. PR mRNA was readily detected in hypothalamic neurons from guinea pigs pretreated with 17 beta-estradiol benzoate (E2B), but not from animals which did not receive supplemental E2B. The distribution of PR mRNA-containing cells corresponded well with previous localizations of PR in guinea pig. In contrast to earlier reports of E2B regulation of PR mRNA in rat hypothalamus, however, we found that PR mRNA remained elevated during chronic exposure to E2B (up to 10 days) in guinea pig. PMID:2072827

  1. ERK activation is regulated by E2F1 and is essential for E2F1-induced S phase entry.

    PubMed

    Korotayev, Katya; Chaussepied, Marie; Ginsberg, Doron

    2008-06-01

    The E2F family of transcription factors regulates a diverse array of cellular functions including cell cycle progression, cell differentiation and apoptosis. Recent studies indicate that E2F1 influences the activity of signal transduction pathways. We identify here a novel link between E2F1 and the Ras/Raf/MEK/ERK signaling pathway, namely that E2F1 levels affect growth factor-induced ERK phosphorylation. Specifically, downregulating E2F1 inhibits PDGF-induced ERK phosphorylation and ectopic expression of E2F1 sensitizes cells to PDGF. We demonstrate that E2F1 induces ERK activation via a transcriptional mechanism and upregulates the expression of two guanine nucleotide exchange factors, RASGRP1 and RASGEF1B, which promote Ras activation. Furthermore, we show that E2F1-induced ERK activity is essential for E2F1-induced S phase entry. Current literature dictates that the cyclin D/pRB/E2F pathway lies downstream of the mitogenically activated Ras/Raf/MEK/ERK cascade. Our results indicate that the relationship between these signaling modules is not a simple unidirectional linear one and suggests there exists a positive feedback loop that may enhance both ERK signaling and E2F1 activity. PMID:18396012

  2. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation.

    PubMed

    Pei, Yonggang; Banerjee, Shuvomoy; Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S

    2016-08-01

    Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies. PMID:27548379

  3. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation.

    PubMed

    Pei, Yonggang; Banerjee, Shuvomoy; Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S

    2016-08-01

    Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies.

  4. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation

    PubMed Central

    Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S.

    2016-01-01

    Epstein–Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies. PMID:27548379

  5. Observation of an E2 (Ubc9)-homodimer by crystallography.

    PubMed

    Alontaga, Aileen Y; Ambaye, Nigus D; Li, Yi-Jia; Vega, Ramir; Chen, Chih-Hong; Bzymek, Krzysztof P; Williams, John C; Hu, Weidong; Chen, Yuan

    2016-06-01

    Post-translational modifications by the small ubiquitin-like modifiers (SUMO), in particular the formation of poly-SUMO-2 and -3 chains, regulates essential cellular functions and its aberration leads to life-threatening diseases (Geoffroy and Hay, 2009) [1]. It was shown previously that the non-covalent interaction between SUMO and the conjugating enzyme (E2) for SUMO, known as Ubc9, is required for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. However, the structure of SUMO-Ubc9 non-covalent complex, by itself, could not explain how the poly-SUMO-2/3 chain forms and consequently a Ubc9 homodimer, although never been observed, was proposed for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. Here, we solved the crystal structure of a heterotrimer containing a homodimer of Ubc9 and the RWD domain from RWDD3. The asymmetric Ubc9 homodimer is mediated by the N-terminal region of one Ubc9 molecule and a surface near the catalytic Cys of the second Ubc9 molecule (Fig. 1A). This N-terminal surface of Ubc9 that is involved in the homodimer formation also interacts with the RWD domain, the ubiquitin-fold domain of the SUMO activating enzyme (E1), SUMO, and the E3 ligase, RanBP2 (Knipscheer et al., 2007; Tong et al.. 1997; Tatham et al., 2005; Reverter and Lima, 2005; Capili and Lima, 2007; Wang et al., 2009, 2010; Wang and Chen, 2010; Alontaga et al., 2015) [2], [3], [4], [5], [6], [7], [8], [9], [10]. The existence of the Ubc9 homodimer in solution is supported by previously published solution NMR studies of rotational correlation time and chemical shift perturbation (Alontaga et al., 2015; Yuan et al., 1999) [10], [11]. Site-directed mutagenesis and biochemical analysis suggests that this dimeric arrangement of Ubc9 is likely important for poly-SUMO chain formation (Fig. 1B and C). The asymmetric Ubc9 homodimer described for the first time in this work could provide the critical missing link in the poly-SUMO chain formation mechanism. The

  6. Observation of an E2 (Ubc9)-homodimer by crystallography.

    PubMed

    Alontaga, Aileen Y; Ambaye, Nigus D; Li, Yi-Jia; Vega, Ramir; Chen, Chih-Hong; Bzymek, Krzysztof P; Williams, John C; Hu, Weidong; Chen, Yuan

    2016-06-01

    Post-translational modifications by the small ubiquitin-like modifiers (SUMO), in particular the formation of poly-SUMO-2 and -3 chains, regulates essential cellular functions and its aberration leads to life-threatening diseases (Geoffroy and Hay, 2009) [1]. It was shown previously that the non-covalent interaction between SUMO and the conjugating enzyme (E2) for SUMO, known as Ubc9, is required for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. However, the structure of SUMO-Ubc9 non-covalent complex, by itself, could not explain how the poly-SUMO-2/3 chain forms and consequently a Ubc9 homodimer, although never been observed, was proposed for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. Here, we solved the crystal structure of a heterotrimer containing a homodimer of Ubc9 and the RWD domain from RWDD3. The asymmetric Ubc9 homodimer is mediated by the N-terminal region of one Ubc9 molecule and a surface near the catalytic Cys of the second Ubc9 molecule (Fig. 1A). This N-terminal surface of Ubc9 that is involved in the homodimer formation also interacts with the RWD domain, the ubiquitin-fold domain of the SUMO activating enzyme (E1), SUMO, and the E3 ligase, RanBP2 (Knipscheer et al., 2007; Tong et al.. 1997; Tatham et al., 2005; Reverter and Lima, 2005; Capili and Lima, 2007; Wang et al., 2009, 2010; Wang and Chen, 2010; Alontaga et al., 2015) [2], [3], [4], [5], [6], [7], [8], [9], [10]. The existence of the Ubc9 homodimer in solution is supported by previously published solution NMR studies of rotational correlation time and chemical shift perturbation (Alontaga et al., 2015; Yuan et al., 1999) [10], [11]. Site-directed mutagenesis and biochemical analysis suggests that this dimeric arrangement of Ubc9 is likely important for poly-SUMO chain formation (Fig. 1B and C). The asymmetric Ubc9 homodimer described for the first time in this work could provide the critical missing link in the poly-SUMO chain formation mechanism. The

  7. Characterization of Prostaglandin E2 Production by Candida albicans▿

    PubMed Central

    Erb-Downward, John R.; Noverr, Mairi C.

    2007-01-01

    Candida albicans produces lipid metabolites that are functionally similar to host prostaglandins. These studies, using mass spectrometry, demonstrate that C. albicans produces authentic prostaglandin E2 (PGE2) from arachidonic acid. Maximal PGE2 production was achieved at 37°C in stationary-phase culture supernatants and in cell-free lysates generated from stationary-phase cells. Interestingly, PGE2 production is inhibited by both nonspecific cyclooxygenase and lipoxygenase inhibitors but not by inhibitors specific for the cyclooxygenase 2 isoenzyme. The C. albicans genome does not possess a cyclooxygenase homolog; however, several genes that may play a role in prostaglandin production from C. albicans were investigated. It was found that a C. albicans fatty acid desaturase homolog (Ole2) and a multicopper oxidase homolog (Fet3) play roles in prostaglandin production, with ole2/ole2 and fet3/fet3 mutant strains exhibiting reduced PGE2 levels compared with parent strains. This work demonstrates that the synthesis of PGE2 in C. albicans proceeds via novel pathways. PMID:17470538

  8. 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

  9. 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.

  10. Prostaglandin E2 protects lower airways against bronchoconstriction.

    PubMed

    Hartney, John M; Coggins, Kenneth G; Tilley, Stephen L; Jania, Leigh A; Lovgren, Alysia Kern; Audoly, Laurent P; Koller, Beverly H

    2006-01-01

    Prostaglandin E2 (PGE2), similar to beta-adrenergic receptor agonists, can protect airways from bronchoconstriction and resulting increase in airway resistance induced by a number of agents, including cholinergic receptor agonists and antigen. We examined the impact of sustained alterations in PGE2 pathways on changes in airway resistance. Genetic methods were utilized to alter PGE2 metabolism and signal transduction in the murine lung. PGE2 levels were elevated by generating mice lacking 15-hydroxyprostaglandin (Hpgd-/-), the major catabolic enzyme of PGE2, and by generating a transgenic line in which mouse PGE2 synthase (Ptges) expression is driven by a human lung-specific promoter, hSP-C. Conversely, to determine the impact of loss of PGE2 on airway reactivity, we examined mice lacking this synthase (Ptges-/-) and receptors that mediate the actions of PGE2, particularly the PGE2 EP2 receptor (Ptger2). Diminished capacity to produce and respond to PGE2 did not alter the response of mice to cholinergic stimuli. In contrast, the responsiveness to cholinergic stimulation was dramatically altered in animals with elevated PGE2 levels. The Hpgd-/- and hSP-C-Ptges transgenic lines both showed attenuated airway responsiveness to methacholine as measured by lung resistance. Thus, whereas compromise of the Ptges/PGE2/Ptger2 pathway does not alter airway responsiveness, genetic modulation that elevates PGE2 levels in the lung attenuates airway responsiveness. PMID:16113047

  11. (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.

  12. 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.

  13. Structure of the Rb C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F release.

    PubMed

    Rubin, Seth M; Gall, Anne-Laure; Zheng, Ning; Pavletich, Nikola P

    2005-12-16

    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. PMID:16360038

  14. Laser-assisted (e,2e) collisions in helium

    SciTech Connect

    Khalil, D.; Maquet, A.; Taieeb, R.; Joachain, C.J.; Makhoute, A.

    1997-12-01

    We have studied the influence of a strong laser on the dynamics of fast (e,2e) collisions in helium, in asymmetric, coplanar geometry. The interaction of the laser field with the unbound electrons is treated in a nonperturbative way. The wave functions of the incident and scattered electrons in the laser field are treated as Volkov waves, while that of the ejected electron moving in the combined field of the residual He{sup +} ion and of the laser is obtained by generalizing the ansatz proposed by Joachain {ital et al.} [Phys. Rev. Lett. {bold 61}, 165 (1988)] for the case of atomic hydrogen. On the other hand, the interaction of bound electrons with the laser field is treated by using first-order perturbation theory, assuming that the electric-field strength is much less than the atomic unit e/a{sub 0}{sup 2}{approx_equal}5{times}10{sup 9} Vcm{sup {minus}1}. The required scattering amplitudes are evaluated by using two different implementations of the Dalgarno and Lewis method. The first approach uses a technique proposed by Zernik and Klopfenstein, based on Laplace transforms and analytic continuation procedures. The second approach is based on a Sturmian basis expansion. The influence of the laser parameters (photon energy and intensity and direction of polarization) on the angular distribution of the ejected electron is analyzed, and several illustrative examples are discussed. We find that in general the triple-differential cross sections are strongly dependent on the dressing of the projectile and the target by the laser field. {copyright} {ital 1997} {ital The American Physical Society}

  15. Tropical Cyclones in the GISS ModelE2

    NASA Technical Reports Server (NTRS)

    Camargo, Suzana J.; Sobel, Adam H.; Del Genio, Anthony; Jonas, Jeffrey A.; Kelley, Maxwell; Lu, Yun; Shaevitz, Daniel; Henderson, Naomi

    2016-01-01

    The authors describe the characteristics of tropical cyclone (TC) activity in the GISS general circulation ModelE2 with a horizontal resolution 1deg x 1deg. Four model simulations are analyzed. In the first, the model is forced with sea surface temperature (SST) from the recent historical climatology. The other three have different idealized climate change simulations, namely (1) a uniform increase of SST by 2 deg., (2) doubling of the CO2 concentration and (3) a combination of the two. These simulations were performed as part of the US Climate Variability and Predictability Program Hurricane Working Group. Diagnostics of standard measures of TC activity are computed from the recent historical climatological SST simulation and compared with the same measures computed from observations. The changes in TC activity in the three idealized climate change simulations, by comparison with that in the historical climatological SST simulation, are also described. Similar to previous results in the literature, the changes in TC frequency in the simulation with a doubling CO2 and an increase in SST are approximately the linear sum of the TC frequency in the other two simulations. However, in contrast with previous results, in these simulations the effects of CO2 and SST on TC frequency oppose each other. Large-scale environmental variables associated with TC activity are then analyzed for the present and future simulations. Model biases in the large-scale fields are identified through a comparison with ERA-Interim reanalysis. Changes in the environmental fields in the future climate simulations are shown and their association with changes in TC activity discussed.

  16. 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 M F; 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.

  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

  18. Characterization of competing distortions in YF e2O4

    NASA Astrophysics Data System (ADS)

    Blasco, J.; Lafuerza, S.; García, J.; Subías, G.; Cuartero, V.; García-Muñoz, J. L.; Popescu, C.; Peral, I.

    2016-05-01

    We report the structural changes of three YF e2O4 -δ (δ <0.1 ) specimens using high resolution synchrotron x-ray powder diffraction between 80 and 300 K. All samples adopt a rhombohedral cell at room temperature (space group R 3 ¯m ). This cell becomes unstable for the three samples on cooling, and the oxygen-poor specimen (δ ˜0.1 ) shows a single transition at 240 K. The nearly stoichiometric (δ ≤0.03 ) compounds exhibit two structural transitions with decreasing temperature at about 240 and 200 K. Each transition is revealed by an anomaly in the heat capacity measurements and a jump in the electric resistivity. Below 240 K, a strong splitting of some diffraction peaks is accompanied by the occurrence of superstructure peaks that follow the propagation vector k =(1 /7 ,-2 /7 ,9 /7 ) . The cell symmetry is then triclinic, and the structural transition is characterized by an expansion of the c axis coupled to a contraction of the other two lattice parameters. There are 49 nonequivalent sites for Fe atoms with a maximum charge disproportionation of ˜0.5 e- . Upon cooling at 200 K, the previous superstructure peaks begin to vanish, and finally they are replaced by a new set of superstructure peaks following the propagation vector k =(1 /4 ,1 /2 ,1 /4 ) with respect to the rhombohedral cell. The transition is also reflected in sudden changes in the lattice parameters that seem to smooth the changes observed in the previous transition. The new cell is also triclinic, and there are 48 nonequivalent Fe sites with a maximum charge disproportionation of ˜0.7 e- . Both phases coexist in a wide temperature range because this second transition is not completed at 80 K. A symmetry mode analysis indicates a complicated pattern for the charge distribution in the Fe sublattice of both distorted structures but clearly discard any bimodal distribution of only two types of Fe cations. Therefore, the sharp jumps in the electric resistivity at the phase transitions are

  19. 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.

  20. [Site-directed mutagensis of the major antigen E2 gene of CSFV, its high level expression in Escherichia coli and the immunonicity of recombinant E2 protein].

    PubMed

    Yu, Xing-Long; Tu, Chang-Chun; Xu, Xing-Ran; Zhang, Mao-Lin; Chen, Yi-Xiang; Liu, Bo-Hua

    2003-07-01

    Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The

  1. The human papillomavirus E7-E2 interaction mechanism in vitro reveals a finely tuned system for modulating available E7 and E2 proteins.

    PubMed

    Smal, Clara; Wetzler, Diana E; Dantur, Karina I; Chemes, Lucia B; Garcia-Alai, María M; Dellarole, Mariano; Alonso, Leonardo G; Gaston, Kevin; de Prat-Gay, Gonzalo

    2009-12-22

    Transcription of the human papillomavirus E7 oncoprotein is negatively controlled by the viral E2 protein, and loss of this repression leads to irreversible transformation and carcinogenesis. Here we show that interaction of the HPV16 E7 protein with the DNA binding domain of the E2 protein (E2C) leads to ionic strength-dependent hetero-oligomerization even at the lowest concentrations measurable. Titration experiments followed by light scattering and native gel electrophoresis show insoluble oligomeric complexes with a >or=2000 nm diameter and intermediate soluble complexes 40 and 115 nm in diameter, respectively, formed in excess of E2C. A discrete oligomeric soluble complex formed in excess of E7 displays a diameter of 12 nm. The N-terminal domain of E7 interacts with E2C with a K(D) of 0.1 muM, where the stretch of residues 25-40 of E7, encompassing both a PEST motif and phosphorylation sites, is sufficient for the interaction. Displacement of the soluble E7-E2C complex by an E2 site DNA duplex and site-directed mutagenesis indicate that the protein-protein interface involves the DNA binding helix of E2. The formation of complexes of different sizes and properties in excess of either of the viral proteins reveals a finely tuned mechanism that could regulate the intracellular levels of both proteins as infection and transformation progress. Sequestering E2 into E7-E2 oligomers provides a possible additional route to uncontrolled E7 expression, in addition and prior to the disruption of the E2 gene during viral integration into the host genome.

  2. Transformation properties of the E2a-Pbx1 chimeric oncoprotein: fusion with E2a is essential, but the Pbx1 homeodomain is dispensable.

    PubMed Central

    Monica, K; LeBrun, D P; Dedera, D A; Brown, R; Cleary, M L

    1994-01-01

    The t(1;19) chromosomal translocation in acute lymphoblastic leukemias creates chimeric E2a-Pbx1 oncoproteins that can act as DNA-binding activators of transcription. A structural analysis of the functional domains of E2a-Pbx1 showed that portions of both E2a and Pbx1 were essential for transformation of NIH 3T3 cells and transcriptional activation of synthetic reporter genes containing PBX1 consensus binding sites. Hyperexpression of wild-type or experimentally truncated Pbx1 proteins was insufficient for transformation, consistent with their inability to activate transcription. When fused with E2a, the Pbx-related proteins Pbx2 and Pbx3 were also transformation competent, demonstrating that all known members of this highly similar subfamily of homeodomain proteins have latent oncogenic potential. The oncogenic contributions of E2a to the chimeras were localized to transactivation motifs AD1 and AD2, as their mutation significantly impaired transformation. Either the homeodomain or Pbx1 amino acids flanking this region could mediate transformation when fused to E2a. However, the homeodomain was not essential for transformation, since a mutant E2a-Pbx1 protein (E2a-Pbx delta HD) lacking the homeodomain efficiently transformed fibroblasts and induced malignant lymphomas in transgenic mice. Thus, transformation mediated by the chimeric oncoprotein E2a-Pbx1 is absolutely dependent on motifs acquired from E2a but the Pbx1 homeodomain is optional. The latter finding suggests that E2a-Pbx1 may interact with cellular proteins that assist or mediate alterations in gene expression responsible for oncogenesis even in the absence of homeodomain-DNA interactions. Images PMID:7969166

  3. 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.

  4. ELL inhibits E2F1 transcriptional activity by enhancing E2F1 deacetylation via recruitment of histone deacetylase 1.

    PubMed

    Zhang, Wei; Ji, Wei; Liu, Xing; Ouyang, Gang; Xiao, Wuhan

    2014-02-01

    ELL (eleven-nineteen lysine-rich leukemia protein) was first identified as a translocation partner of MLL in acute myeloid leukemia; however, the exact mechanism of its action has remained elusive. In this study, we identified ELL as a direct downstream target gene of E2F1. Coimmunoprecipitation assays showed that ELL interacted with E2F1 in vitro and in vivo, leading to inhibition of E2F1 transcriptional activity. In addition, ELL enhanced E2F1 deacetylation via recruitment of histone deacetylase 1 (HDAC1). Notably, the MLL-ELL fusion protein lost the inhibitory role of ELL in E2F1 transcriptional activity. Furthermore, DNA damage induced ELL in an E2F1-dependent manner and ELL protected cells against E2F1-dependent apoptosis. Our findings not only connect ELL to E2F1 function and uncover a novel role of ELL in response to DNA damage but also provide an insight into the mechanism for MLL-ELL-associated leukemogenesis.

  5. 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 ...

  6. Disruption of RB/E2F-1 interaction by single point mutations in E2F-1 enhances S-phase entry and apoptosis.

    PubMed Central

    Shan, B; Durfee, T; Lee, W H

    1996-01-01

    The retinoblastoma protein (RB) has been proposed to function as a negative regulator of cell proliferation by complexing with cellular proteins such as the transcription factor E2F. To study the biological consequences of the RB/E2F-1 interaction, point mutants of E2F-1 which fail to bind to RB were isolated by using the yeast two-hybrid system. Sequence analysis revealed that within the minimal 18-amino acid peptide of E2F-1 required for RB binding, five residues, Tyr (position 411), Glu (419), and Asp-Leu-Phe (423-425), are critical. These amino acids are conserved among the known E2F family members. While mutation of any of these five amino acids abolished binding to RB, all mutants retained their full transactivation potential. Expression of mutated E2F-1, when compared with that of wild-type, significantly accelerated entry into S phase and subsequent apoptosis. These results provide direct genetic evidence for the biological significance of the RB/E2F interaction and strongly suggest that the interplay between RB and E2F is critical for proper cell cycle progression. Images Fig. 3 Fig. 4 PMID:8570615

  7. 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...

  8. E2F1 Regulates Cellular Growth by mTORC1 Signaling

    PubMed Central

    Real, Sebastian; Meo-Evoli, Nathalie; Espada, Lilia; Tauler, Albert

    2011-01-01

    During cell proliferation, growth must occur to maintain homeostatic cell size. Here we show that E2F1 is capable of inducing growth by regulating mTORC1 activity. The activation of cell growth and mTORC1 by E2F1 is dependent on both E2F1's ability to bind DNA and to regulate gene transcription, demonstrating that a gene induction expression program is required in this process. Unlike E2F1, E2F3 is unable to activate mTORC1, suggesting that growth activity could be restricted to individual E2F members. The effect of E2F1 on the activation of mTORC1 does not depend on Akt. Furthermore, over-expression of TSC2 does not interfere with the effect of E2F1, indicating that the E2F1-induced signal pathway can compensate for the inhibitory effect of TSC2 on Rheb. Immunolocalization studies demonstrate that E2F1 induces the translocation of mTORC1 to the late endosome vesicles, in a mechanism dependent of leucine. E2F1 and leucine, or insulin, together affect the activation of S6K stronger than alone suggesting that they are complementary in activating the signal pathway. From these studies, E2F1 emerges as a key protein that integrates cell division and growth, both of which are essential for cell proliferation. PMID:21283628

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. Novel retinoblastoma mutation abrogating the interaction to E2F2/3, but not E2F1, led to selective suppression of thyroid tumors

    PubMed Central

    Toki, Hideaki; Inoue, Maki; Minowa, Osamu; Motegi, Hiromi; Saiki, Yuriko; Wakana, Shigeharu; Masuya, Hiroshi; Gondo, Yoichi; Shiroishi, Toshihiko; Yao, Ryoji; Noda, Tetsuo

    2014-01-01

    Mutant mouse models are indispensable tools for clarifying gene functions and elucidating the pathogenic mechanisms of human diseases. Here, we describe novel cancer models bearing point mutations in the retinoblastoma gene (Rb1) generated by N-ethyl-N-nitrosourea mutagenesis. Two mutations in splice sites reduced Rb1 expression and led to a tumor spectrum and incidence similar to those observed in the conventional Rb1 knockout mice. The missense mutant, Rb1D326V/+, developed pituitary tumors, but thyroid tumors were completely suppressed. Immunohistochemical analyses of thyroid tissue revealed that E2F1, but not E2F2/3, was selectively inactivated, indicating that the mutant Rb protein (pRb) suppressed thyroid tumors by inactivating E2F1. Interestingly, Rb1D326V/+ mice developed pituitary tumors that originated from the intermediate lobe of the pituitary, despite selective inactivation of E2F1. Furthermore, in the anterior lobe of the pituitary, other E2F were also inactivated. These observations show that pRb mediates the inactivation of E2F function and its contribution to tumorigenesis is highly dependent on the cell type. Last, by using a reconstitution assay of synthesized proteins, we showed that the D326V missense pRb bound to E2F1 but failed to interact with E2F2/3. These results reveal the effect of the pRb N-terminal domain on E2F function and the impact of the protein on tumorigenesis. Thus, this mutant mouse model can be used to investigate human Rb family-bearing mutations at the N-terminal region. PMID:25088905

  15. Regulation of human genome expression and RNA splicing by human papillomavirus 16 E2 protein.

    PubMed

    Gauson, Elaine J; Windle, Brad; Donaldson, Mary M; Caffarel, Maria M; Dornan, Edward S; Coleman, Nicholas; Herzyk, Pawel; Henderson, Scott C; Wang, Xu; Morgan, Iain M

    2014-11-01

    Human papillomavirus 16 (HPV16) is causative in human cancer. The E2 protein regulates transcription from and replication of the viral genome; the role of E2 in regulating the host genome has been less well studied. We have expressed HPV16 E2 (E2) stably in U2OS cells; these cells tolerate E2 expression well and gene expression analysis identified 74 genes showing differential expression specific to E2. Analysis of published gene expression data sets during cervical cancer progression identified 20 of the genes as being altered in a similar direction as the E2 specific genes. In addition, E2 altered the splicing of many genes implicated in cancer and cell motility. The E2 expressing cells showed no alteration in cell growth but were altered in cell motility, consistent with the E2 induced altered splicing predicted to affect this cellular function. The results present a model system for investigating E2 regulation of the host genome.

  16. 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

  17. 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.

  18. 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.

  19. Loop 7 of E2 enzymes: an ancestral conserved functional motif involved in the E2-mediated steps of the ubiquitination cascade.

    PubMed

    Papaleo, Elena; Casiraghi, Nicola; Arrigoni, Alberto; Vanoni, Marco; Coccetti, Paola; De Gioia, Luca

    2012-01-01

    The ubiquitin (Ub) system controls almost every aspect of eukaryotic cell biology. Protein ubiquitination depends on the sequential action of three classes of enzymes (E1, E2 and E3). E2 Ub-conjugating enzymes have a central role in the ubiquitination pathway, interacting with both E1 and E3, and influencing the ultimate fate of the substrates. Several E2s are characterized by an extended acidic insertion in loop 7 (L7), which if mutated is known to impair the proper E2-related functions. In the present contribution, we show that acidic loop is a conserved ancestral motif in E2s, relying on the presence of alternate hydrophobic and acidic residues. Moreover, the dynamic properties of a subset of family 3 E2s, as well as their binary and ternary complexes with Ub and the cognate E3, have been investigated. Here we provide a model of L7 role in the different steps of the ubiquitination cascade of family 3 E2s. The L7 hydrophobic residues turned out to be the main determinant for the stabilization of the E2 inactive conformations by a tight network of interactions in the catalytic cleft. Moreover, phosphorylation is known from previous studies to promote E2 competent conformations for Ub charging, inducing electrostatic repulsion and acting on the L7 acidic residues. Here we show that these active conformations are stabilized by a network of hydrophobic interactions between L7 and L4, the latter being a conserved interface for E3-recruitment in several E2s. In the successive steps, L7 conserved acidic residues also provide an interaction interface for both Ub and the Rbx1 RING subdomain of the cognate E3. Our data therefore suggest a crucial role for L7 of family 3 E2s in all the E2-mediated steps of the ubiquitination cascade. Its different functions are exploited thank to its conserved hydrophobic and acidic residues in a finely orchestrate mechanism.

  20. 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

  1. 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

  2. The contribution of E2F-regulated transcription to Drosophila PCNA gene function.

    PubMed

    Thacker, Stephen A; Bonnette, Peter C; Duronio, Robert J

    2003-01-01

    E2F proteins control cell cycle progression by predominantly acting as either activators or repressors of transcription. How the antagonizing activities of different E2Fs are integrated by cis-acting control regions into a final transcriptional output in an intact animal is not well understood. E2F function is required for normal development in many species, but it is not completely clear for which genes E2F-regulated transcription provides an essential biological function. To address these questions, we have characterized the control region of the Drosophila PCNA gene. A single E2F binding site within a 100-bp enhancer is necessary and sufficient to direct the correct spatiotemporal program of G1-S-regulated PCNA expression during development. This dynamic program requires both E2F-mediated transcriptional activation and repression, which, in Drosophila, are thought to be carried out by two distinct E2F proteins. Our data suggest that functional antagonism between these different E2F proteins can occur in vivo by competition for the same binding site. An engineered PCNA gene with mutated E2F binding sites supports a low level of expression that can partially rescue the lethality of PCNA null mutants. Thus, E2F regulation of PCNA is dispensable for viability, but is nonetheless important for normal Drosophila development. PMID:12526745

  3. 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

  4. Constitutive E2F1 Overexpression Delays Endochondral Bone Formation by Inhibiting Chondrocyte Differentiation

    PubMed Central

    Scheijen, Blanca; Bronk, Marieke; van der Meer, Tiffany; Bernards, René

    2003-01-01

    Longitudinal bone growth results from endochondral ossification, a process that requires proliferation and differentiation of chondrocytes. It has been shown that proper endochondral bone formation is critically dependent on the retinoblastoma family members p107 and p130. However, the precise functional roles played by individual E2F proteins remain poorly understood. Using both constitutive and conditional E2F1 transgenic mice, we show that ubiquitous transgene-driven expression of E2F1 during embryonic development results in a dwarf phenotype and significantly reduced postnatal viability. Overexpression of E2F1 disturbs chondrocyte maturation, resulting in delayed endochondral ossification, which is characterized by reduced hypertrophic zones and disorganized growth plates. Employing the chondrogenic cell line ATDC5, we investigated the effects of enforced E2F expression on the different phases of chondrocyte maturation that are normally required for endochondral ossification. Ectopic E2F1 expression strongly inhibits early- and late-phase differentiation of ATDC5 cells, accompanied by diminished cartilage nodule formation as well as decreased type II collagen, type X collagen, and aggrecan gene expression. In contrast, overexpression of E2F2 or E2F3a results in only a marginal delay of chondrocyte maturation, and increased E2F4 levels have no effect. These data are consistent with the notion that E2F1 is a regulator of chondrocyte differentiation. PMID:12724423

  5. 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.

  6. 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.

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

    PubMed

    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

  8. The Mitotic Checkpoint Gene, SIL is Regulated by E2F1

    PubMed Central

    Erez, Ayelet; Chaussepied, Marie; Tina, Colaizzo-Anas; Aplan, Peter; Ginsberg, Doron; Izraeli, Shai

    2009-01-01

    The SIL gene expression is increased in multiple cancers and correlates with the expression of mitotic spindle checkpoint genes and with increased metastatic potential. SIL regulates mitotic entry, organization of the mitotic spindle and cell survival. The E2F transcription factors regulate cell cycle progression by controlling the expression of genes mediating the G1/S transition. More recently E2F has been shown to regulate mitotic spindle checkpoint genes as well. As SIL expression correlates with mitotic checkpoint genes we hypothesized that SIL is regulated by E2F. We mined raw data of published experiments and performed new experiments by modification of E2F expression in cell lines, reporter assays and chromatin immunoprecipitation. Ectopic expression or endogenous activation of E2F induced the expression of SIL, while knockdown of E2F by shRNA, downregulated SIL expression. E2F activated SIL promoter by reporter assay and bound to SIL promoter in-vivo. Taken together these data demonstrate that SIL is regulated by E2F. As SIL is essential for mitotic entry, E2F may regulate G2/M transition through the induction of SIL. Furthermore, as silencing of SIL cause apoptosis in cancer cells, these finding may have therapeutic relevance in tumors with constitutive activation of E2F. PMID:18649360

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

    PubMed

    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.

  10. Unusual properties of adenovirus E2E transcription by RNA polymerase III.

    PubMed

    Huang, Wenlin; Flint, S J

    2003-04-01

    In adenovirus type 5-infected cells, RNA polymerase III transcription of a gene superimposed on the 5' end of the E2E RNA polymerase II transcription unit produces two small (<100-nucleotide) RNAs that accumulate to low steady-state concentrations (W. Huang, R. Pruzan, and S. J. Flint, Proc. Natl. Acad. Sci. USA 91:1265-1269, 1984). To gain a better understanding of the function of this RNA polymerase III transcription, we have examined the properties of the small E2E RNAs and E2E RNA polymerase III transcription in more detail. The accumulation of cytoplasmic E2E RNAs and the rates of E2E transcription by the two RNA polymerases during the infectious cycle were analyzed by using RNase T(1) protection and run-on transcription assays, respectively. Although the RNA polymerase III transcripts were present at significantly lower concentrations than E2E mRNA throughout the period examined, E2E transcription by RNA polymerase III was found to be at least as efficient as that by RNA polymerase II. The short half-lifes of the small E2E RNAs estimated by using the actinomycin D chase method appear to account for their limited accumulation. The transcription of E2E sequences by RNA polymerase II and III in cells infected by recombinant adenoviruses carrying ectopic E2E-CAT (chloramphenicol transferase) reporter genes with mutations in E2E promoter sequences was also examined. The results of these experiments indicate that recognition of the E2E promoter by the RNA polymerase II transcriptional machinery in infected cells limits transcription by RNA polymerase III, and vice versa. Such transcriptional competition and the properties of E2E RNAs made by RNA polymerase III suggest that the function of this viral RNA polymerase III transcription unit is unusual. PMID:12634361

  11. E2f8 mediates tumor suppression in postnatal liver development

    PubMed Central

    Kent, Lindsey N.; Rakijas, Jessica B.; Pandit, Shusil K.; Westendorp, Bart; Chen, Hui-Zi; Huntington, Justin T.; Tang, Xing; Bae, Sooin; Srivastava, Arunima; Senapati, Shantibhusan; Martin, Chelsea K.; Cuitino, Maria C.; Perez, Miguel; Clouse, Julian M.; Chokshi, Veda; Shinde, Neelam; Kladney, Raleigh; Sun, Daokun; Perez-Castro, Antonio; Matondo, Ramadhan B.; Nantasanti, Sathidpak; Mokry, Michal; Machiraju, Raghu; Fernandez, Soledad; Rosol, Thomas J.; Pohar, Kamal S.; Pipas, James M.; Schmidt, Carl R.; de Bruin, Alain

    2016-01-01

    E2F-mediated transcriptional repression of cell cycle–dependent gene expression is critical for the control of cellular proliferation, survival, and development. E2F signaling also interacts with transcriptional programs that are downstream of genetic predictors for cancer development, including hepatocellular carcinoma (HCC). Here, we evaluated the function of the atypical repressor genes E2f7 and E2f8 in adult liver physiology. Using several loss-of-function alleles in mice, we determined that combined deletion of E2f7 and E2f8 in hepatocytes leads to HCC. Temporal-specific ablation strategies revealed that E2f8’s tumor suppressor role is critical during the first 2 weeks of life, which correspond to a highly proliferative stage of postnatal liver development. Disruption of E2F8’s DNA binding activity phenocopied the effects of an E2f8 null allele and led to HCC. Finally, a profile of chromatin occupancy and gene expression in young and tumor-bearing mice identified a set of shared targets for E2F7 and E2F8 whose increased expression during early postnatal liver development is associated with HCC progression in mice. Increased expression of E2F8-specific target genes was also observed in human liver biopsies from HCC patients compared to healthy patients. In summary, these studies suggest that E2F8-mediated transcriptional repression is a critical tumor suppressor mechanism during postnatal liver development. PMID:27454291

  12. Inhibition of E2F-mediated transcription by p202.

    PubMed Central

    Choubey, D; Li, S J; Datta, B; Gutterman, J U; Lengyel, P

    1996-01-01

    Many of the antimicrobial, immunomodulatory and cell growth inhibitory activities of the interferons are mediated by interferon-inducible proteins. Earlier we characterized an interferon-inducible murine protein, p202, whose expression in transfected cells inhibits cell proliferation and which can form a complex with retinoblastoma protein (pRb). Here we report that in transfected cells expression of p202 inhibits E2F-stimulated transcription of a reporter gene and of endogenous genes. Inhibition of the transcriptional activity of E2F by p202 does not depend on fully functional pRb and is correlated with inhibition of the sequence-specific DNA binding of E2F. p202 interacts with the transcription factor E2F (E2F-1/DP-1) in vitro and in vivo. Inhibition of E2F activity by p202 may contribute to growth inhibition by the interferons. Images PMID:8896460

  13. Cooperative activation of tissue-specific genes by pRB and E2F1.

    PubMed

    Flowers, Stephen; Xu, Fuhua; Moran, Elizabeth

    2013-04-01

    The retinoblastoma tumor suppressor protein pRB is conventionally regarded as an inhibitor of the E2F family of transcription factors. Conversely, pRB is also recognized as an activator of tissue-specific gene expression along various lineages including osteoblastogenesis. During osteoblast differentiation, pRB directly targets Alpl and Bglap, which encode the major markers of osteogenesis alkaline phosphatase and osteocalcin. Surprisingly, p130 and repressor E2Fs were recently found to cooccupy and repress Alpl and Bglap in proliferating osteoblast precursors before differentiation. This raises the further question of whether these genes convert to E2F activation targets when differentiation begins, which would constitute a remarkable situation wherein pRB and E2F would be cotargeting genes for activation. Chromatin immunoprecipitation analysis in an osteoblast differentiation model shows that Alpl and Bglap are indeed targeted by an activator E2F, i.e., is E2F1. Promoter occupation of Alpl and Bglap by E2F1 occurs specifically during activation, and depletion of E2F1 severely impairs their induction. Mechanistically, promoter occupation by E2F1 and pRB is mutually dependent, and without this cooperative effect, activation steps previously shown to be dependent on pRB, including recruitment of RNA polymerase II, are impaired. Myocyte- and adipocyte-specific genes are also cotargeted by E2F1 and pRB during differentiation along their respective lineages. The finding that pRB and E2F1 cooperate to activate expression of tissue-specific genes is a paradigm distinct from the classical concept of pRB as an inhibitor of E2F1, but is consistent with the observed roles of these proteins in physiological models.

  14. S Phase–Coupled E2f1 Destruction Ensures Homeostasis in Proliferating Tissues

    PubMed Central

    Davidson, Jean M.; Duronio, Robert J.

    2012-01-01

    Precise control of cell cycle regulators is critical for normal development and tissue homeostasis. E2F transcription factors are activated during G1 to drive the G1-S transition and are then inhibited during S phase by a variety of mechanisms. Here, we genetically manipulate the single Drosophila activator E2F (E2f1) to explore the developmental requirement for S phase–coupled E2F down-regulation. Expression of an E2f1 mutant that is not destroyed during S phase drives cell cycle progression and causes apoptosis. Interestingly, this apoptosis is not exclusively the result of inappropriate cell cycle progression, because a stable E2f1 mutant that cannot function as a transcription factor or drive cell cycle progression also triggers apoptosis. This observation suggests that the inappropriate presence of E2f1 protein during S phase can trigger apoptosis by mechanisms that are independent of E2F acting directly at target genes. The ability of S phase-stabilized E2f1 to trigger apoptosis requires an interaction between E2f1 and the Drosophila pRb homolog, Rbf1, and involves induction of the pro-apoptotic gene, hid. Simultaneously blocking E2f1 destruction during S phase and inhibiting the induction of apoptosis results in tissue overgrowth and lethality. We propose that inappropriate accumulation of E2f1 protein during S phase triggers the elimination of potentially hyperplastic cells via apoptosis in order to ensure normal development of rapidly proliferating tissues. PMID:22916021

  15. 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.

  16. Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA.

    PubMed

    Sen, P; Pazour, G J; Anderson, D; Das, A

    1989-05-01

    The VirE2 protein of Agrobacterium tumefaciens Ti plasmid pTiA6 is a single-stranded-DNA-binding protein. Density gradient centrifugation studies showed that it exists as a tetramer in solution. Monomeric VirE2 active in DNA binding could also be obtained by using a different protein isolation procedure. VirE2 was found to be thermolabile; brief incubation at 37 degrees C abolished its DNA-binding activity. It was insensitive to the sulfhydryl-specific reagent N-ethylmaleimide. Removal of the carboxy-terminal 37 residues of the 533-residue VirE2 polypeptide led to complete loss of DNA-binding activity; however, chimeric fusion proteins containing up to 125 residues of the VirE2 C terminus were inactive in DNA binding. In nuclease protection studies, VirE2 protected single-stranded DNA against degradation by DNase I. Analysis of the DNA-VirE2 complex by electron microscopy demonstrated that VirE2 coats a single-stranded DNA molecule and that the binding of VirE2 to its substrate is cooperative. PMID:2708313

  17. Polymorphic genetic characterization of E2 gene of bovine viral diarrhea virus in China.

    PubMed

    Lang, Yifei; Gao, Shandian; Du, Junzheng; Shao, Junjun; Cong, Guozheng; Lin, Tong; Zhao, Furong; Liu, Lihong; Chang, Huiyun

    2014-12-01

    Bovine viral diarrhea virus (BVDV) is one of the wide distributed pathogenic viruses of livestock and wild animals worldwide. E2 glycoprotein is a major structural component of the BVDV virion and plays a key role in viral attachment to host cells and inducing immune responses against viral infection. In order to gain detailed information of the E2 coding region of BVDV circulating in China, 46 positive samples were tested by RT-PCR for the E2 coding region. The 1122 nt nucleotide sequences of full-length E2 were harvested and analyzed. The results suggested that full-length E2 was an ideal target for BVDV genotyping and divided the domestic BVDV isolates into 9 subgenotypes, namely BVDV-1a, -1b1, -1c, -1d, -1o, -1m, -1p, -1q and BVDV-2a, showing great diversity. The difference of nonsynonymous and synonymous substitution rates (dN-dS) inferred both positive and purifying selection of the E2. However, combination of positive and purifying selection at different points indicated purifying selection within the complete E2. Protein properties analysis based on glycosylation sites and epitope prediction demonstrated that the biological character of E2 among individual BVDV subgenotype was similar, but may alter due to amino acid changes. For the first time, the comprehensive collection of E2 sequences of Chinese BVDV isolates was elucidated, which would provide information for future vaccine design and BVD control in China.

  18. 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.

  19. 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

  20. Fate of endocrine disrupting compounds in membrane bioreactor systems.

    PubMed

    Hu, J Y; Chen, X; Tao, G; Kekred, K

    2007-06-01

    Yeast estrogen screen (YES) bioassay and liquid chromatography-mass spectrum-mass spectrum (LC-MS-MS) analysis were performed to investigate the fate of active and potential endocrine disrupting compounds in 3 pilot-scale and 2 lab-scale membrane bioreactor (MBR) systems. Compared with the overall estrogenicities of sewage treatment plant (STP) effluents from references, the MBR systems studied have relatively good performance in the removal of estrogenicity. Estrone (E1) was removed with relatively high efficiency (80.2-91.4%), but 17beta-estradiol (E2) was removed with moderate efficiency (49.3-66.5%) by the MBRs. However, the experimental results indicated that after the treatment by MBR, substantial amounts of E1, estrone-3-sulfate (E1-3S), estrone-3-glucuronide (E1-3G), and 17beta-estradiol-glucuronides (E2-G) passed through treatment systems and entered into the aquatic environment. The reduction in the levels of overall equivalent E1 (68.4%) and that of overall equivalent E2 (80.8%) was demonstrated for the pilot-scale MBR-B. For alkylphenol compounds, bisphenol A (BPA) was removed well with a removal efficiency of 68.9 -90.1%, but 4-nonylphenol (4-NP) concentration was amplified (removal efficiency of -439.5 to -161.1%) after MBR treatment which could be caused by the transformation of its parent compounds, nonylphenol polyethoxylates (NPnEOs). The amounts of adsorbed estrogens per kg dry mass was relatively low, due to short hydraulic retention time and high mixed liquor suspended solids in MBRs, compared to that in STPs. PMID:17612196

  1. 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

  2. Variations in the association of papillomavirus E2 proteins with mitotic chromosomes

    PubMed Central

    Oliveira, Jaquelline G.; Colf, Leremy A.; McBride, Alison A.

    2006-01-01

    The E2 protein segregates episomal bovine papillomavirus (BPV) genomes to daughter cells by tethering them to mitotic chromosomes, thus ensuring equal distribution and retention of viral DNA. To date, only the BPV1 E2 protein has been shown to bind to mitotic chromosomes. We assessed the localization of 13 different animal and human E2 proteins from seven papillomavirus genera, and we show that most of them are stably bound to chromosomes throughout mitosis. Furthermore, in contrast to the random association of BPV1 E2 with mitotic chromosomes, several of these proteins appear to bind to more specific regions of mitotic chromosomes. Using human papillomavirus (HPV) type 8 E2, we show that this region is adjacent to centromeres/kinetochores. Therefore, E2 proteins from both HPV and animal papillomavirus bind to mitotic chromosomes, and there are variations in the specificity of this binding. Only the α-papillomavirus E2 proteins do not stably associate with mitotic chromatin throughout mitosis. These proteins closely associate with prophase chromosomes and bind to chromosomes in telophase but not in metaphase. However, extraction of mitotic cells before fixation results in α-E2 proteins binding to the pericentromeric region of metaphase chromosomes, as observed for HPV8 E2. We postulate that this is the authentic target of these E2 proteins but that additional factors or a specialized cellular environment is required to stabilize this association. Thus, E2-mediated tethering of viral genomes to mitotic chromosomes is a common strategy of papillomaviruses, but different viruses have evolved different variations of this theme. PMID:16415162

  3. 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.

  4. Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens.

    PubMed

    Volokhina, Irina; Sazonova, Inna; Velikov, Vladimir; Chumakov, Mikhail

    2005-01-01

    Bacteria of the genus Agrobacterium can transfer a portion of their Ti plasmid (T-DNA) in complex with the VirE2 and VirD2 proteins into the plant-cell nucleus and cause it to be integrated in the host-cell chromosomes. The mechanism of T-DNA transfer across the plant-cell membrane and cytoplasm is unknown. The aim of this study was to isolate the virulence protein VirE2 in order to explore its role in T-DNA transfer across the eukaryotic-cell membrane and cytoplasm. To obtain VirE2, we cloned the virE2 gene into plasmid pQE31 in Escherichia coli cells. VirE2 protein was isolated from E. coli XL-1 blue cells containing a recombinant plasmid, pQE31-virE2. The cells were ultrasonically disrupted, and the protein containing six histidine residues at the N-terminal end was isolated by affinity chromatography on Ni-NTA agarose. The purified preparation was tested by immunodot, by using polyclonal rabbit antibodies and miniantibodies produced toward VirE2. The capacity of the recombinant protein VirE2 for interacting with single-stranded DNA was tested by the formation of complexes, recorded by agarose-gel electrophoresis. In summary, A. tumefaciens virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into the plant cell, was isolated, purified, and partially characterized. Anti-VirE2 miniantibodies were obtained, and direct labeling of VirE2 with colloidal gold was done for the first time. PMID:15782940

  5. E2F1 enhances glycolysis through suppressing Sirt6 transcription in cancer cells.

    PubMed

    Wu, Minghui; Seto, Edward; Zhang, Jingsong

    2015-05-10

    The fast proliferation of cancer cells requires reprogramming of its energy metabolism with aerobic glycolysis as a major energy source. Sirt6, a class III histone deacetylase, has been shown to down regulate glycolysis by inhibiting the expression of several key glycolytic genes. Based on the published study on the metabolic phenotype of E2F1 -/- mice and SIRT6 -/- mice, we hypothesize that E2F1 enhances glycolysis and inhibits the expression of Sirt6. Indeed, over-expressing of E2F1, but not its DNA binding deficient mutant, significantly enhanced glucose uptake and lactate production in bladder and prostate cancer cell lines. E2F1 over-expression also suppressed Sirt6 expression and function. Moreover, E2F1 directly bound to Sirt6 promoter and suppressed Sirt6 promoter activity under both normoxic and hypoxic culture conditions. E2F1 siRNA blocked the up-regulation of E2F1 under hypoxia, increased Sirt6 expression and decreased glycolysis compared to those of scrambled siRNA transected cells. Furthermore, HDAC1 deacetylated E2F1 and diminished its transcription suppression of Sirt6 promoter. Treatment with the HDAC inhibitor, trichostatin A (TSA), suppressed Sirt6 promoter activity with increased binding of acetylated E2F1 to Sirt6 promoter. Mutating the E2F1 binding site on the proximal Sirt6 promoter abolished the suppression of Sirt6 transcription by TSA. These data indicate a novel oncogenic role of E2F1, i.e. enhancing glycolysis by suppressing Sirt6 transcription.

  6. Active nuclear import and export pathways regulate E2F-5 subcellular localization.

    PubMed

    Apostolova, Margarita D; Ivanova, Iordanka A; Dagnino, Carla; D'Souza, Sudhir J A; Dagnino, Lina

    2002-09-13

    Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130-154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein.

  7. E2F transcription factors and digestive system malignancies: how much do we know?

    PubMed

    Xanthoulis, Athanasios; Tiniakos, Dina G

    2013-06-01

    E2F family of transcription factors regulates various cellular functions related to cell cycle and apoptosis. Its individual members have traditionally been classified into activators and repressors, based on in vitro studies. However their contribution in human cancer is more complicated and difficult to predict. We review current knowledge on the expression of E2Fs in digestive system malignancies and its clinical implications for patient prognosis and treatment. E2F1, the most extensively studied member and the only one with prognostic value, exhibits a tumor-suppressing activity in esophageal, gastric and colorectal adenocarcinoma, and in hepatocellular carcinoma (HCC), whereas in pancreatic ductal adenocarcinoma and esophageal squamous cell carcinoma may function as a tumor-promoter. In the latter malignancies, E2F1 immunohistochemical expression has been correlated with higher tumor grade and worse patient survival, whereas in esophageal, gastric and colorectal adenocarcinomas is a marker of increased patient survival. E2F2 has only been studied in colorectal cancer, where its role is not considered significant. E2F4's role in colorectal, gastric and hepatic carcinogenesis is tumor-promoting. E2F8 is strongly upregulated in human HCC, thus possibly contributing to hepatocarcinogenesis. Adenoviral transfer of E2F as gene therapy to sensitize pancreatic cancer cells for chemotherapeutic agents has been used in experimental studies. Other therapeutic strategies are yet to be developed, but it appears that targeted approaches using E2F-agonists or antagonists should take into account the tissue-dependent function of each E2F member. Further understanding of E2Fs' contribution in cellular functions in vivo would help clarify their role in carcinogenesis.

  8. Leukotriene D4-induced Caco-2 cell proliferation is mediated by prostaglandin E2 synthesis

    PubMed Central

    Cabral, Marisol; Martín-Venegas, Raquel; Moreno, Juan J

    2015-01-01

    Leukotriene D4 (LTD4) is a pro-inflammatory mediator formed from arachidonic acid through the action of 5-lipoxygenase (5-LOX). Its biological effects are mediated by at least two G-coupled plasmatic cysteinyl LT receptors (CysLT1-2R). It has been reported an upregulation of the 5-LOX pathway in tumor tissue unlike in normal colon mucosa. Colon tumors generally have an increased expression of CysLT1R and colon cancer patients with high expression levels of CysLT1R have poor prognosis. We previously observed that the cyclooxygenase pathway is involved in the control of intestinal epithelial cancer cell growth through PGE2 production. The aim of this study was therefore to assess the effect of LTD4 binding with CysLT1R on Caco-2 cell growth. We note a number of key findings from this research. We observed that at a concentration similar to that found under inflammatory conditions, LTD4 was able to induce Caco-2 cell proliferation and DNA synthesis. Moreover, with the use of a specific receptor antagonist this study has demonstrated that the effect of LTD4 is a result of its interaction with CystLT1R. We also note the possible participation of the PLC-IP3-Ca2+/DAG-PKC signaling pathways in cytosolic PLA2 and [3H]AA release induced by LTD4-CystLT1R interaction. Finally, we found that the resulting activation of the AA cascade and the production of PGE2 eicosanoid could be related to the activation of cell signaling pathways such as ERK and CREB. These findings will help facilitate our understanding of how inflammatory mediators can affect the survival and dissemination of intestinal carcinoma cells. PMID:26216432

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

    PubMed Central

    Wang, Jimin; Li, Yue; Modis, Yorgo

    2014-01-01

    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. PMID:24725935

  10. Destructive interference of E2 matrix elements in a triaxial rotor model

    NASA Astrophysics Data System (ADS)

    Allmond, J. M.; Wood, J. L.; Kulp, W. D.

    2010-05-01

    A triaxial rotor model with independent inertia and electric quadrupole tensors is applied to nuclei that have certain E2 matrix elements equal to zero. It is shown that such vanishing E2 matrix elements are explained by the model as a destructive interference effect. The example of Pt196 is considered.

  11. Destructive interference of E2 matrix elements in a triaxial rotor model

    SciTech Connect

    Allmond, James M; Wood, J. L.; Kulp, W. D.

    2010-01-01

    A triaxial rotor model with independent inertia and electric quadrupole tensors is applied to nuclei that have certain E2 matrix elements equal to zero. It is shown that such vanishing E2 matrix elements are explained by the model as a destructive interference effect. The example of 196Pt is considered.

  12. 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

  13. Supramolecular complexes of the Agrobacterium tumefaciens virulence protein VirE2.

    PubMed

    Volokhina, I V; Gusev, Yu S; Mazilov, S I; Chumakov, M I

    2011-11-01

    Virulence protein VirE2 from Agrobacterium tumefaciens is involved in plant infection by transferring a fragment of agrobacterial Ti plasmid ssT-DNA in complex with VirE2-VirD2 proteins into the plant cell nucleus. The VirE2 protein interactions with ssDNA and formation of VirE2 protein complexes in vitro and in silico have been studied. Using dynamic light scattering we found that purified recombinant protein VirE2 exists in buffer solution in the form of complexes of 2-4 protein molecules of 12-18 nm size. We used computer methods to design models of complexes consisting of two and four individual VirE2 proteins, and their dimensions were estimated. Dimensions of VirE2 complexes with ssDNA (550 and 700 nucleotide residues) were determined using transmission electron microscopy and dynamic light scattering. We found that in vitro, upon interaction with ssDNA recombinant protein, VirE2 is able to alter conformation of the latter by shortening the initial length of the ssDNA. PMID:22117554

  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. 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

  16. 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.

  17. 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.

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

    PubMed

    Wang, Jimin; Li, Yue; Modis, Yorgo

    2014-04-01

    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.

  19. 17 CFR 270.6e-2 - Exemptions for certain variable life insurance separate accounts.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... extent necessary with Rule 7d-1 (17 CFR 270.7d-1) under the Act; (2) The assets of the separate account... variable life insurance separate accounts. 270.6e-2 Section 270.6e-2 Commodity and Securities Exchanges...-2 Exemptions for certain variable life insurance separate accounts. (a) A separate account, and...

  20. 17 CFR 270.6e-2 - Exemptions for certain variable life insurance separate accounts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... extent necessary with Rule 7d-1 (17 CFR 270.7d-1) under the Act; (2) The assets of the separate account... variable life insurance separate accounts. 270.6e-2 Section 270.6e-2 Commodity and Securities Exchanges...-2 Exemptions for certain variable life insurance separate accounts. (a) A separate account, and...

  1. 17 CFR 270.6e-2 - Exemptions for certain variable life insurance separate accounts.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... extent necessary with Rule 7d-1 (17 CFR 270.7d-1) under the Act; (2) The assets of the separate account... variable life insurance separate accounts. 270.6e-2 Section 270.6e-2 Commodity and Securities Exchanges...-2 Exemptions for certain variable life insurance separate accounts. (a) A separate account, and...

  2. 17 CFR 270.6e-2 - Exemptions for certain variable life insurance separate accounts.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... extent necessary with Rule 7d-1 (17 CFR 270.7d-1) under the Act; (2) The assets of the separate account... variable life insurance separate accounts. 270.6e-2 Section 270.6e-2 Commodity and Securities Exchanges...-2 Exemptions for certain variable life insurance separate accounts. (a) A separate account, and...

  3. Dual level inhibition of E2F-1 activity by adeno-associated virus Rep78.

    PubMed

    Batchu, R B; Shammas, M A; Wang, J Y; Munshi, N C

    2001-06-29

    E2F-1, a major cellular transcription factor, plays a pivotal role in regulating the cell cycle. The activity of E2F-1 is negatively regulated by its interaction with retinoblastoma protein (pRB), and disruption of the pRB-E2F-1 complex, a hallmark of cellular transformation by DNA tumor viruses, leads to cell proliferation. Adeno-associated virus-2 (AAV) is known to have onco-suppressive properties against DNA tumor viruses. Here we provide, for the first time, the molecular basis for antioncogenic activity of AAV. Rep78, a major regulatory protein of AAV, interacts at the protein level with E2F-1 and stabilizes the pRB-E2F-1 complex. At the DNA level, Rep78 binds to a putative site on the E2F-1 promoter and down-regulates the adenovirus-induced E2F-1 transcription. This dual level of Rep78 activity leads to decreased cellular levels of free E2F-1, leading to its onco-suppressive properties.

  4. 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.

  5. E2F1 mediates sustained lipogenesis and contributes to hepatic steatosis

    PubMed Central

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

    2015-01-01

    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

  6. Emerging roles of E2Fs in cancer: an exit from cell cycle control

    PubMed Central

    Chen, Hui-Zi; Tsai, Shih-Yin; Leone, Gustavo

    2012-01-01

    Mutations of the retinoblastoma tumour suppressor gene (RB1) or components regulating the RB pathway have been identified in almost every human malignancy. The E2F transcription factors function in cell cycle control and are intimately regulated by RB. Studies of model organisms have revealed conserved functions for E2Fs during development, suggesting that the cancer-related proliferative roles of E2F family members represent a recent evolutionary adaptation. However, given that some human tumours have concurrent RB1 inactivation and E2F amplification and overexpression, we propose that there are alternative tumour-promoting activities for the E2F family, which are independent of cell cycle regulation. PMID:19851314

  7. DEK Expression is controlled by E2F and deregulated in diverse tumor types.

    PubMed

    Carro, Maria Stella; Spiga, Fabio Mario; Quarto, Micaela; Di Ninni, Valentina; Volorio, Sara; Alcalay, Myriam; Müller, Heiko

    2006-06-01

    Deregulation of the retinoblastoma (pRB) tumor suppressor pathway associated with aberrant activity of E2F transcription factors is frequently observed in human cancer. Microarray based analyses have revealed a large number of potential downstream mediators of the tumor suppressing activity of pRB, including DEK, a fusion partner of CAN found in a subset of acute myeloid leukaemia (AML) patients carrying a (6; 9) translocation. Here we report that the expression of DEK is under direct control of E2F transcription factors. Chromatin immunoprecipitation assays show that the DEK promoter is bound by endogenous E2F in vivo. The DEK promoter is transactivated by E2F and mutation of E2F binding sites eliminates this effect. Expression levels of DEK in human tumors have been investigated by tissue micro array analysis. We find that DEK is overexpressed in many solid tumors such as colon cancer, larynx cancer, bladder cancer, and melanoma.

  8. Nano-optical imaging of WS e2 waveguide modes revealing light-exciton interactions

    NASA Astrophysics Data System (ADS)

    Fei, Z.; Scott, M. E.; Gosztola, D. J.; Foley, J. J.; Yan, J.; Mandrus, D. G.; Wen, H.; Zhou, P.; Zhang, D. W.; Sun, Y.; Guest, J. R.; Gray, S. K.; Bao, W.; Wiederrecht, G. P.; Xu, X.

    2016-08-01

    We report on a nano-optical imaging study of WS e2 thin flakes with scanning near-field optical microscopy (NSOM). The NSOM technique allows us to visualize in real space various waveguide photon modes inside WS e2 . By tuning the excitation laser energy, we are able to map the entire dispersion of these waveguide modes both above and below the A exciton energy of WS e2 . We found that all the modes interact strongly with WS e2 excitons. The outcome of the interaction is that the observed waveguide modes shift to higher momenta right below the A exciton energy. At higher energies, on the other hand, these modes are strongly damped due to adjacent B excitons or band-edge absorptions. The mode-shifting phenomena are consistent with polariton formation in WS e2 .

  9. 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.

  10. 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

  11. E2F Transcription Factors Control the Roller Coaster Ride of Cell Cycle Gene Expression.

    PubMed

    Thurlings, Ingrid; de Bruin, Alain

    2016-01-01

    Initially, the E2F transcription factor was discovered as a factor able to bind the adenovirus E2 promoter and activate viral genes. Afterwards it was shown that E2F also binds to promoters of nonviral genes such as C-MYC and DHFR, which were already known at that time to be important for cell growth and DNA metabolism, respectively. These findings provided the first clues that the E2F transcription factor might be an important regulator of the cell cycle. Since this initial discovery in 1987, several additional E2F family members have been identified, and more than 100 targets genes have been shown to be directly regulated by E2Fs, the majority of these are important for controlling the cell cycle. The progression of a cell through the cell cycle is accompanied with the increased expression of a specific set of genes during one phase of the cell cycle and the decrease of the same set of genes during a later phase of the cell cycle. This roller coaster ride, or oscillation, of gene expression is essential for the proper progression through the cell cycle to allow accurate DNA replication and cell division. The E2F transcription factors have been shown to be critical for the temporal expression of the oscillating cell cycle genes. This review will focus on how the oscillation of E2Fs and their targets is regulated by transcriptional, post-transcriptional and post-translational mechanism in mammals, yeast, flies, and worms. Furthermore, we will discuss the functional impact of E2Fs on the cell cycle progression and outline the consequences when E2F expression is disturbed. PMID:26254918

  12. Functional interaction of the bovine papillomavirus E2 transactivation domain with TFIIB.

    PubMed

    Yao, J M; Breiding, D E; Androphy, E J

    1998-02-01

    Induction of gene expression by the papillomavirus E2 protein requires its approximately 220-amino-acid amino-terminal transactivation domain (TAD) to interact with cellular factors that lead to formation of an activated RNA polymerase complex. These interaction partners have yet to be identified and characterized. The E2 protein localizes the transcription complex to the target promoter through its carboxy-terminal sequence-specific DNA binding domain. This domain has been reported to bind the basal transcription factors TATA-binding protein and TFIIB. We present evidence establishing a direct interaction between amino acids 74 to 134 of the E2 TAD and TFIIB. Within this region, the E2 point mutant N127Y was partially defective and W99C was completely defective for TFIIB binding in vitro, and these mutants displayed reduced or no transcriptional activity, respectively, upon transfection into C33A cells. Overexpression of TFIIB specifically restored transactivation by N127Y to close to wild-type levels, while W99C remained inactive. To further demonstrate the functional interaction of TFIIB with the wild-type E2 TAD, this region was fused to a bacterial DNA binding domain (LexA:E2:1-216). Upon transfection with increasing amounts of LexA:E2:1-216, there was reduction of its transcriptional activity, a phenomenon thought to result from titration of limiting factors, or squelching. Squelching of LexA:E2:1-216, or the wild-type E2 activator, was partially relieved by overexpression of TFIIB. We conclude that a specific region of the E2 TAD functionally interacts with TFIIB.

  13. Absence of pRb facilitates E2F1-induced apoptosis in breast cancer cells.

    PubMed

    Sun, Baohua; Wingate, Hannah; Swisher, Stephen G; Keyomarsi, Khandan; Hunt, Kelly K

    2010-03-15

    The transcription factor E2F1 is known for its interaction with pRb, controlling cell proliferation; however, E2F1 also has a pivotal role in regulating apoptosis.  The relationship between pRb and E2F1 balances cell proliferation and apoptosis giving pRb tumor suppressive properties. The intricacies of the pRb/E2F1 relationship and thus the regulation of cell fate is cell context dependent. To explore the role of pRb in the E2F1-induced apoptosis of human breast cancer cells, we examined cell growth and apoptosis induction in isogenic cell systems of immortalized breast epithelial cells lacking either pRb (76NE7) or p53 (76NE6). We found that E2F1 caused accumulation of cells in G2 and S phases of the cell cycle along with apoptosis in 76NE7 but not 76NE6 cells.  Variants of 76NE6 cells with functional p53 did not rescue the apoptotic response in these cells, whereas knocking down pRb resulted in significant E2F1-induced apoptosis. We also determined that the effect of E2F1 overexpression in two breast cancer cell lines, MDA-MB-436 and MDA-MB-468, which lack pRb and functional p53, was accumulation of cells in G2/S phase and apoptosis. However, E2F did not cause apotosis  in MCF-7 cells which harbor a functional pRb. Therefore, we conclude that in the absence of Rb, E2F1 overexpression results in apoptosis, not proliferation, and that this effect is independent of p53.

  14. Direct visualization of Agrobacterium-delivered VirE2 in recipient cells

    PubMed Central

    Li, Xiaoyang; Yang, Qinghua; Tu, Haitao; Lim, Zijie; Pan, Shen Q

    2014-01-01

    Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single-stranded DNA molecules (T–DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split-GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2-GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1–10) was expressed in recipient cells. Upon delivery of VirE2-GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2-GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium-delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf-infiltration assay; most of VirE2 moved at a speed of 1.3–3.1 μm sec−1 in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2-GFP formed filamentous structures of different lengths, even in the absence of T-DNA. As a non-natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium-delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T-DNA transformation for a non-natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non-natural host recipient. The split-GFP approach could enable the real-time visualization of VirE2 trafficking inside recipient cells. PMID:24299048

  15. Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2.

    PubMed

    Dombek, P; Ream, W

    1997-02-01

    The transferred DNA (T-DNA) portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid enters infected plant cells and integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events liberate the lower strand of the T-DNA from the Ti plasmid, producing single-stranded DNA molecules (T strands) that are covalently linked to VirD2 at their 5' ends. A. tumefaciens appears to transfer T-DNA into plant cells as a T-strand-VirD2 complex. The bacterium also transports VirE2, a cooperative single-stranded DNA-binding protein, into plant cells during infection. Both VirD2 and VirE2 contain nuclear localization signals that may direct these proteins, and bound T strands, into plant nuclei. Here we report the locations of functional regions of VirE2 identified by eight insertions of XhoI linker oligonucleotides, and one deletion mutation, throughout virE2. We examined the effects of these mutations on virulence, single-stranded DNA (ssDNA) binding, and accumulation of VirE2 in A. tumefaciens. Two of the mutations in the C-terminal half of VirE2 eliminated ssDNA binding, whereas two insertions in the N-terminal half altered cooperativity. Four of the mutations, distributed throughout virE2, decreased the stability of VirE2 in A. tumefaciens. In addition, we isolated a mutation in the central region of VirE2 that decreased tumorigenicity but did not affect ssDNA binding or VirE2 accumulation. This mutation may affect export of VirE2 into plant cells or nuclear localization of VirE2, or it may affect an uncharacterized activity of VirE2. PMID:9023198

  16. Direct visualization of Agrobacterium-delivered VirE2 in recipient cells.

    PubMed

    Li, Xiaoyang; Yang, Qinghua; Tu, Haitao; Lim, Zijie; Pan, Shen Q

    2014-02-01

    Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single-stranded DNA molecules (T-DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split-GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2-GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1-10) was expressed in recipient cells. Upon delivery of VirE2-GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2-GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium-delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf-infiltration assay; most of VirE2 moved at a speed of 1.3-3.1 μm sec⁻¹ in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2-GFP formed filamentous structures of different lengths, even in the absence of T-DNA. As a non-natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium-delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T-DNA transformation for a non-natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non-natural host recipient. The split-GFP approach could enable the real-time visualization of VirE2 trafficking inside recipient cells. PMID:24299048

  17. 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.

  18. The Retinoblastoma Tumor Suppressor Protein (pRb)/E2 Promoter Binding Factor 1 (E2F1) Pathway as a Novel Mediator of TGFβ-induced Autophagy.

    PubMed

    Korah, Juliana; Canaff, Lucie; Lebrun, Jean-Jacques

    2016-01-29

    TGFβ is a multifunctional cytokine that regulates cell proliferation, cell immortalization, and cell death, acting as a key homeostatic mediator in various cell types and tissues. Autophagy is a programmed mechanism that plays a pivotal role in controlling cell fate and, consequently, many physiological and pathological processes, including carcinogenesis. Although autophagy is often considered a pro-survival mechanism that renders cells viable in stressful conditions and thus might promote tumor growth, emerging evidence suggests that autophagy is also a tumor suppressor pathway. The relationship between TGFβ signaling and autophagy is context-dependent and remains unclear. TGFβ-mediated activation of autophagy has recently been suggested to contribute to the growth inhibitory effect of TGFβ in hepatocarcinoma cells. In the present study, we define a novel process of TGFβ-mediated autophagy in cancer cell lines of various origins. We found that autophagosome initiation and maturation by TGFβ is dependent on the retinoblastoma tumor suppressor protein/E2 promoter binding factor (pRb/E2F1) pathway, which we have previously established as a critical signaling axis leading to various TGFβ tumor suppressive effects. We further determined that TGFβ induces pRb/E2F1-dependent transcriptional activation of several autophagy-related genes. Together, our findings reveal that TGFβ induces autophagy through the pRb/E2F1 pathway and transcriptional activation of autophagy-related genes and further highlight the central relevance of the pRb/E2F1 pathway downstream of TGFβ signaling in tumor suppression.

  19. Insights into Nuclear Triaxiality from Interference Effects in E2 Matrix Elements

    NASA Astrophysics Data System (ADS)

    Allmond, J. M.; Wood, J. L.; Kulp, W. D.

    2007-10-01

    Recently, we have introduced [1] a triaxial rotor model with independent inertia and E2 tensors. The E2 matrix elements [2] of the osmium isotopes (186, 188, 190, and 192) are studied in the framework of this model (59 of 84 E2 matrix elements deviate by 30% or less). It is shown that interference effects in the inertia tensor (K-mixing) and the E2 tensor can lead to significant reductions in the diagonal E2 matrix elements. In some instances, the diagonal E2 matrix elements may decrease with increasing spin. Additionally, a sum rule for diagonal E2 matrix elements is shown and used to explore missing strength from K-admixtures. [1] J.L. Wood, A-M. Oros-Peusquens, R. Zaballa, J.M. Allmond, and W.D. Kulp, Phys. Rev. C 70, 024308 (2004). [2] C.Y. Wu, D. Cline, T. Czosnyka, A. Backlin, C. Baktash, R.M. Diamond, G.D. Dracoulis, L. Hasselgren, H. Kluge, et al., Nucl. Phys. A607, 178 (1996).

  20. 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.

  1. Control of e2f1 and PCNA by Drosophila transcription factor DREF.

    PubMed

    Kawamori, Akihito; Shimaji, Kouhei; Yamaguchi, Masamitsu

    2013-11-01

    DREF (DNA replication-related element-binding factor), a zinc finger type transcription factor required for proper cell cycle progression in both mitotic and endocycling cells, is a positive regulator of E2F1, an important transcription factor which regulates genes related to the S-phase of the cell cycle. DREF and E2F1 regulate similar sets of replication-related genes, including proliferating cell nuclear antigen (PCNA), and play roles in the G1 to S phase transition. However, the relationships between dref and e2f1 or PCNA during development are poorly understood. Here, we provided evidence for novel control of e2f1 and PCNA involving DREF in endocycling cells. Somatic clone analysis demonstrated that dref knockdown stabilized E2F1 expression at posttranscriptional levels in endocycling salivary gland cells. Similarly, PCNA expression was up-regulated in the endocycling salivary gland cells. Genetic interaction analysis indicated that the endoreplication defects are partly caused via possible enhancement of E2F1 activity. From these results and previous reports, we conclude that regulation of e2f1 and PCNA by DREF in vivo is complex and the regulation mechanism may differ with the tissue and/or positions in the tissue.

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

    PubMed

    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

    2016-02-01

    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

  3. 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

  4. E2 ubiquitin-conjugating enzymes regulate the deubiquitinating activity of OTUB1.

    PubMed

    Wiener, Reuven; DiBello, Anthony T; Lombardi, Patrick M; Guzzo, Catherine M; Zhang, Xiangbin; Matunis, Michael J; Wolberger, Cynthia

    2013-09-01

    OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin (Ub)-conjugating enzymes including UBC13 and UBCH5. OTUB1 binds E2~Ub thioester intermediates and prevents ubiquitin transfer, thereby noncatalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin. This stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 enzyme stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.

  5. Transcriptional regulation of BMCC1 mediated by E2F1 in neuroblastoma cells.

    PubMed

    Islam, Mohammad Sazzadul; Tatsumi, Yasutoshi; Takano, Ryo; Yokochi, Tomoki; Akter, Jesmin; Ozaki, Toshinori; Nakamura, Yohko; Ohira, Miki; Nakagawara, Akira

    2016-09-01

    BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1)/PRUNE2 is highly expressed in patients with favorable neuroblastoma (NB), encoding a multifunctional scaffold protein that modulates several signaling networks including RhoA and AKT pathways. Accumulating evidence suggests that BMCC1 acts as a tumor-suppressor. In this study, we addressed molecular mechanism underlying transcriptional regulation of BMCC1 in NBs. We found that transcription factor E2F1 was recruited to E2F-binding site in the promoter region of BMCC1 gene. Indeed, overexpression of E2F1 resulted in an increase in the expression level of BMCC1 in NB cell lines. On the other hand, knockdown of E2F1 in NB cells yielded down-regulation of BMCC1. Also, we showed that BMCC1 and E2F1 were simultaneously induced at G1 to S phase transition. Therefore, we conclude that E2F1 directly facilitated BMCC1 transcription. Taking together, these results suggest that BMCC1 induced by E2F1 acts as a tumor suppressor through its pro-apoptotic function, resulted in favorable prognosis of NB.

  6. Transcriptional regulation of BMCC1 mediated by E2F1 in neuroblastoma cells.

    PubMed

    Islam, Mohammad Sazzadul; Tatsumi, Yasutoshi; Takano, Ryo; Yokochi, Tomoki; Akter, Jesmin; Ozaki, Toshinori; Nakamura, Yohko; Ohira, Miki; Nakagawara, Akira

    2016-09-01

    BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1)/PRUNE2 is highly expressed in patients with favorable neuroblastoma (NB), encoding a multifunctional scaffold protein that modulates several signaling networks including RhoA and AKT pathways. Accumulating evidence suggests that BMCC1 acts as a tumor-suppressor. In this study, we addressed molecular mechanism underlying transcriptional regulation of BMCC1 in NBs. We found that transcription factor E2F1 was recruited to E2F-binding site in the promoter region of BMCC1 gene. Indeed, overexpression of E2F1 resulted in an increase in the expression level of BMCC1 in NB cell lines. On the other hand, knockdown of E2F1 in NB cells yielded down-regulation of BMCC1. Also, we showed that BMCC1 and E2F1 were simultaneously induced at G1 to S phase transition. Therefore, we conclude that E2F1 directly facilitated BMCC1 transcription. Taking together, these results suggest that BMCC1 induced by E2F1 acts as a tumor suppressor through its pro-apoptotic function, resulted in favorable prognosis of NB. PMID:27453342

  7. 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

  8. Genomic and Genetic Variation in E2F1 in Men with Non-Obstructive Azoospermia

    PubMed Central

    Jorgez, Carolina J.; Wilken, Nathan; Addai, Josephine B.; Newberg, Justin; Vangapandu, Hima V.; Pastuszak, Alexander W.; Mukherjee, Sarmistha; Rosenfeld, Jill A.; Lipshultz, Larry I.; Lamb, Dolores J.

    2014-01-01

    Objective To identify gene dosage changes associated with non-obstructive azoospermia (NOA) using array comparative genomic hybridization (aCGH). Design Prospective study. Patients 110 men with NOA and 78 fertile controls. Settings Medical School Interventions None Main Outcome Measure The study has four distinct analytic components: aCGH, a molecular karyotype that detects copy-number-variations (CNV), Taqman-CNV assays to validate CNVs, mutation identification by Sanger sequencing and histologic analyses of testicular tissues. Results A microduplication at 20q11.22 encompassing E2F1 was identified in one of eight men with NOA analyzed using aCGH. CNVs were confirmed and an additional 102 men with NOA screened using Taqman CNV assays for a total of 110 NOA men analyzed for CNVs in E2F1. Eight of 110 (7.3%) NOA men had microduplications or microdeletions of E2F1 that were absent in fertile controls. Conclusions E2F1 microduplications or microdeletions are present in NOA men (7.3%). Duplications or deletions of E2F1 occur very rarely in the general population (0.011%), but E2F1 gene dosage changes, previously reported only in cancers, are present in a subset of NOA men. These results recapitulate the infertility phenotype seen in mice lacking or over-expressing E2f1. PMID:25439843

  9. Structural Insights into the Conformation and Oligomerization of E2~Ubiquitin Conjugates

    PubMed Central

    Page, Richard C.; Pruneda, Jonathan N.; Amick, Joseph; Klevit, Rachel E.; Misra, Saurav

    2012-01-01

    Post-translational modification of proteins by ubiquitin (Ub) regulates a host of cellular processes including protein quality control, DNA repair, endocytosis and cellular signaling. In the ubiquitination cascade, a thioester-linked conjugate between the Ub C-terminus and the active site cysteine of a ubiquitin-conjugating enzyme (E2) is formed. The E2~Ub conjugate interacts with a ubiquitin ligase (E3) to transfer Ub to a lysine residue on a target protein. The flexibly-linked E2~Ub conjugates have been shown to form a range of structures in solution. In addition, select E2~Ub conjugates oligomerize through a noncovalent “backside” interaction between Ub and E2 components of different conjugates. Additional studies are needed to bridge the gap between the dynamic monomeric conjugates, E2~Ub oligomers and the mechanisms of ubiquitination. We present a new 2.35 Å crystal structure of an oligomeric UbcH5c~Ub conjugate. The conjugate forms a staggered linear oligomer that differs substantially from the “infinite spiral” helical arrangement of the sole previously reported structure of an oligomeric conjugate. Our structure also differs in intra-conjugate conformation from other structurally characterized conjugates. Despite these differences, we find that the backside interaction mode is conserved in different conjugate oligomers and is independent of intra-conjugate relative E2/Ub orientations. We delineate a common intra-conjugate E2-binding surface on Ub. In addition, we demonstrate that an E3 ligase CHIP (carboxyl terminus of Hsp70 interacting protein) interacts directly with UbcH5c~Ub oligomers, not only with conjugate monomers. These results provide insights into the conformational diversity of E2~Ub conjugates and conjugate oligomers, and into their compatibility and interactions with E3 ligases, which have important consequences for the ubiquitination process. PMID:22551455

  10. The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2

    PubMed Central

    DeSmet, Marsha; Kanginakudru, Sriramana; Rietz, Anne; Wu, Wai-Hong; Roden, Richard

    2016-01-01

    The origin recognition complex (ORC) coordinates a series of events that lead to initiation of DNA strand duplication. As a nuclear double stranded DNA plasmid, the papillomavirus (PV) genome resembles a mini-chromosome in infected cells. To initiate its replication, the viral E2 protein binds to and recruits the E1 DNA helicase at the viral origin. PV genome replication program exhibits three stages: initial amplification from a single genome upon infection to a few copies per cell, a cell cycle linked maintenance phase, and a differentiation dependent late stage where the genome is amplified to thousands of copies. Involvement of ORC or other pre-replication complex (pre-RC) factors has not been described. We report that human PV (HPV) and bovine PV (BPV-1) E2 proteins bind to ORC2, however, ORC2 was not detected at the viral origin. Depletion of ORC2 enhanced PV replication in a transient replication model and in keratinocytes stably maintaining viral episomes, while there was no effect on copy number in a cell line with integrated HPV genomes. Consistent with this, occupancy of E1 and E2 at the viral origin increased following ORC2 silencing. These data imply that ORC2 is not necessary for activation of the PV origin by E1 and E2 but instead suppresses E2 replicative function. Furthermore, we observed that over-expression of HPV E2 decreased ORC2 occupation at two known mammalian origins of replication, suggesting that E2 restricts pre-ORC assembly that could otherwise compete for host replication complexes necessary for viral genome amplification. We infer that the ORC2 complex with E2 restricts viral replication in the maintenance phase of the viral replication program and that elevated levels of E2 that occur during the differentiation dependent amplification stage subvert ORC loading and hence DNA synthesis at cellular origins. PMID:27701460

  11. [Isolation, purification, and identification of virulence protein VirE2 from Agrobacterium tumefaciens].

    PubMed

    Volokhina, I V; Sazonova, I A; Velikov, V A; Chumakov, M I

    2005-01-01

    Bacteria of the genus Agrobacterium are capable of transferring a fragment of their Ti-plasmid, T-DNA, in a complex with the proteins VirE2 and VirD2, into the nuclei of plant cells and incorporating it into the chromosome of the host. The mechanisms of T-DNA transportation through membrane and cytoplasm of the plant cell are unknown. The aim of this work was isolation of virulence protein VirE2 for studying its role in T-DNA transportation through the membrane and cytoplasm of eukaryotic cells. For VirE2 accumulation, virE2 gene was cloned into plasmid pQE31. VirE2 was isolated from the cells of E. coli strain XL1-blue, containing the recombinant plasmid pQE31-virE2. The cells were disrupted ultrasonically, and the protein with six histidine residues at the N-end was isolated by means of affinity chromatography on a Ni-NTA-superose column. The purified protein was tested by the immunodot method using polyclonal rabbit antibodies and anti-VirE2 miniantibodies. The ability of the recombinant protein VirE2 to bind to single-stranded DNA was judged from the formation of complexes detected by electrophoresis in agarose gel. Thus, we isolated, purified, and partially characterized the Agrobacterium tumefaciens virulence protein VirE2 which is capable of binding to single-stranded T-DNA upon transfer to the plant cell. PMID:15835784

  12. 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

  13. E2F transcription factors and digestive system malignancies: how much do we know?

    PubMed

    Evangelou, Konstantinos; Havaki, Sophia; Kotsinas, Athanassios

    2014-08-01

    The E2F proteins comprise a family of 8 members that function as transcription factors. They are key targets of the retinoblastoma protein (RB) and were initially divided into groups of activators and repressors. Accumulating data suggest that there is no specific role for each individual E2F member. Instead, each E2F can exert a variety of cellular effects, some of which represent opposing ones. For instance, specific E2Fs can activate transcription and repression, promote or hamper cell proliferation, augment or inhibit apoptosis, all being dependent on the cellular context. This complexity reflects the importance that these transcription factors have on a cell's fate. Thus, delineating the specific role for each E2F member in specific malignancies, although not easy, is a challenging and continuously pursued task, especially in view of potential E2F targeted therapies. Therefore, several reviews are continuously trying to evaluate available data on E2F status in various malignancies. Such reviews have attempted to reach a consensus, often in the simplistic form of oncogenes or tumor suppressor genes for the E2Fs. However they frequently miss spatial and temporal alterations of these factors during tumor development, which should also be considered in conjunction with the status of the regulatory networks that these factors participate in. In the current ''Letter to the Editor'', we comment on the flaws, misinterpretations and omissions in one such review article published recently in the World Journal of Gastroenterology regarding the role of E2Fs in digestive system malignancies.

  14. Effect of a dimer of nanoparticles on the linewidth of forbidden E2 transitions

    NASA Astrophysics Data System (ADS)

    Guzatov, D. V.; Klimov, V. V.

    2016-07-01

    In the framework of classical electrodynamics we have obtained and investigated analytical expressions for the radiation linewidth of forbidden E2 transitions in an atom located near a dimer of spherical particles. It is shown that the material of particles, their location and size have a significant effect on the linewidth of the E2 transition in the atom. It is found that in the gap between metal spherical nanoparticles, the linewidth of E2 transitions in the atom can take on substantially larger values than in the case of an atom near a single metal nanoparticle.

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

    PubMed

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

    2015-11-10

    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.

  16. Triaxial rotor model description of E2 properties in Os186,188,190,192

    NASA Astrophysics Data System (ADS)

    Allmond, J. M.; Zaballa, R.; Oros-Peusquens, A. M.; Kulp, W. D.; Wood, J. L.

    2008-07-01

    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 Os186,188,190,192. Most large and medium transition E2 matrix elements can be reproduced to within ~10%, and most diagonal elements to within ~30%. Most small transition matrix elements can be reproduced to within ~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 Os190,192 indicate significant admixtures of K=4 components. Attention is given to the Kπ=4+ bands in these nuclei and the controversial issue of whether they are of quadrupole or hexadecapole nature.

  17. 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.

  18. Structure of the CIAP2 Ring Domain Reveal Conformational Changes Associated With E2 Recruitment

    SciTech Connect

    Mace, P.D.; Linke, K.; Feltham, R.; Schumacher, F.-R.; Smith, C.A.; Vaux, D.L.; Silke, J.; Day, C.L.

    2009-05-19

    Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death that are highly expressed in many cancers. Cell death caused by antagonists that bind to IAP proteins is associated with their ubiquitylation and degradation. The RING domain at the C terminus of IAP proteins is pivotal. Here we report the crystal structures of the cIAP2 RING domain homodimer alone, and bound to the ubiquitin-conjugating (E2) enzyme UbcH5b. These structures show that small changes in the RING domain accompany E2 binding. By mutating residues at the E2-binding surface, we show that autoubiquitylation is required for regulation of IAP abundance. Dimer formation is also critical, and mutation of a single C-terminal residue abrogated dimer formation and E3 ligase activity was diminished. We further demonstrate that disruption of E2 binding, or dimerization, stabilizes IAP proteins against IAP antagonists in vivo.

  19. 260. Dennis Hill, Photographer April 1998 VIEW OF PIER E2 ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    260. Dennis Hill, Photographer April 1998 VIEW OF PIER E-2 FROM TORPEDO ASSEMBLY BUILDING, FACING SOUTHEAST. - San Francisco Oakland Bay Bridge, Spanning San Francisco Bay, San Francisco, San Francisco County, CA

  20. Human Papillomavirus E2 Protein: Linking Replication, Transcription, and RNA Processing.

    PubMed

    Graham, Sheila V

    2016-10-01

    The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the infected epithelium. This means that viral proteins must exert control over epithelial gene expression in order to optimize viral production. The HPV E2 protein controls replication, transcription, and viral genome partitioning during the viral infectious life cycle. It consists of a nucleic acid-binding domain and a protein-protein interaction domain separated by a flexible serine and arginine-rich hinge region. Over the last few years, mounting evidence has uncovered an important new role for E2 in viral and cellular RNA processing. This Gem discusses the role of E2 in controlling the epithelial cellular environment and how E2 might act to coordinate late events in the viral replication cycle.

  1. JAZ mediates G1 cell cycle arrest by interacting with and inhibiting E2F1

    PubMed Central

    Yang, Mingli; Wu, Song; Jia, Jinghua

    2011-01-01

    We discovered and reported JAZ as a unique dsRNA binding zinc finger protein that functions as a direct, positive regulator of p53 transcriptional activity to mediate G1 cell cycle arrest in a mechanism involving upregulation of the p53 target gene, p21. We now find that JAZ can also negatively regulate the cell cycle in a novel, p53-independent mechanism resulting from the direct interaction with E2F1, a key intermediate in regulating cell proliferation and tumor suppression. JAZ associates with E2F1's central DNA binding/dimerization region and its C-terminal transactivation domain. Functionally, JAZ represses E2F1 transcriptional activity in association with repression of cyclin A expression and inhibition of G1/S transition. This mechanism involves JAZ-mediated inhibition of E2F1's specific DNA binding activity. JAZ directly binds E2F1 in vitro in a dsRNA-independent manner, and JAZ's dsRNA binding ZF domains, which are necessary for localizing JAZ to the nucleus, are required for repression of transcriptional activity in vivo. Importantly for specificity, siRNA-mediated “knockdown” of endogenous JAZ increases E2F transcriptional activity and releases cells from G1 arrest, indicating a necessary role for JAZ in this transition. Although JAZ can directly inhibit E2F1 activity independently of p53, if functional p53 is expressed, JAZ may exert a more potent inhibition of cell cycle following growth factor withdrawal. Therefore, JAZ plays a dual role in cell cycle regulation by both repressing E2F1 transcriptional activity and activating p53 to facilitate efficient growth arrest in response to cellular stress, which may potentially be exploited therapeutically for tumor growth inhibition. PMID:21715977

  2. 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

  3. 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

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

    PubMed

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

    2015-11-10

    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.

  5. The E1-E2 center in gallium arsenide is the divacancy.

    PubMed

    Schultz, Peter A

    2015-02-25

    Based on defect energy levels computed from first-principles calculations, it is shown the E1-E2 center in irradiated GaAs cannot be due to an isolated arsenic vacancy. The only simple intrinsic defect with levels compatible with E1 and E2 is the divacancy. The arsenic monovacancy is reassigned to the E3 center in irradiated GaAs. These new assignments are shown to reconcile a number of seemingly contradictory experimental observations.

  6. Disrupted Differentiation and Oncogenic Transformation of Lymphoid Progenitors in E2A-HLF Transgenic Mice

    PubMed Central

    Smith, Kevin S.; Rhee, Joon Whan; Naumovski, Louie; Cleary, Michael L.

    1999-01-01

    The hepatic leukemia factor (HLF) gene codes for a basic region-leucine zipper (bZIP) protein that is disrupted by chromosomal translocations in a subset of pediatric acute lymphoblastic leukemias. HLF undergoes fusions with the E2A gene, resulting in chimeric E2a-Hlf proteins containing the E2a transactivation domains and the Hlf bZIP DNA binding and dimerization motifs. To investigate the in vivo role of this chimeric bZIP protein in oncogenic transformation, its expression was directed to the lymphoid compartments of transgenic mice. Within the thymus, E2a-Hlf induced profound hypoplasia, premature involution, and progressive accumulation of a T-lineage precursor population arrested at an early stage of maturation. In the spleen, mature T cells were present but in reduced numbers, and they lacked expression of the transgene, suggesting further that E2a-Hlf expression was incompatible with T-cell differentiation. In contrast, mature splenic B cells expressed E2a-Hlf but at lower levels and without apparent adverse or beneficial effects on their survival. Approximately 60% of E2A-HLF mice developed lymphoid malignancies with a mean latency of 10 months. Tumors were monoclonal, consistent with a requirement for secondary genetic events, and displayed phenotypes of either mid-thymocytes or, rarely, B-cell progenitors. We conclude that E2a-Hlf disrupts the differentiation of T-lymphoid progenitors in vivo, leading to profound postnatal thymic depletion and rendering B- and T-cell progenitors susceptible to malignant transformation. PMID:10330184

  7. Activation of BPV-1 replication in vitro by the transcription factor E2

    NASA Astrophysics Data System (ADS)

    Yang, Liu; Li, Rong; Mohr, Ian J.; Clark, Robin; Botchan, Michael R.

    1991-10-01

    Soluble extracts from uninfected murine cells supplemented with purified viral E1 and E2 proteins support the replication of exogenously added papilloma virus DNA. The E2 transactivator stimulates the binding of the E1 replication protein to the minimal origin of replication and activates DNA replication. These results support the concept that transcription factors have a direct role in the initiation of DNA replication in eukaryotes by participating in the assembly of a complex at the origin of replication.

  8. 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

  9. Target genes regulated by transcription factor E2F1 in small cell lung cancer.

    PubMed

    Li, Zun-Ling; Jiao, Fei; Ma, Ying; Yue, Zhen; Kong, Li-Jun

    2016-06-25

    Previously, we have reported that transcription factor E2F1 expression is up-regulated in approximately 95% of small cell lung cancer tissue samples and closely associated with invasion and metastasis, but few studies have investigated specific target genes regulated by E2F1 in this disease. The aim of this study was to clarify the target genes controlled by E2F1 in the small cell lung cancer cell line H1688. The results of chromatin immunoprecipitation sequencing (ChIP-seq) showed that total 5 326 potential target genes were identified, in which 4 700 were structural genes and 626 long non-coding RNAs (lncRNAs). Gene Ontology (GO) and enrichment map analysis results indicated that these target genes were associated with three main functions: (1) cell cycle regulation, (2) chromatin and histone modification, and (3) protein transport. MEME4.7.0 software was used to identify the E2F1 binding DNA motif, and six motifs were discovered for coding genes and lncRNAs. These results clarify the target genes of E2F1, and provide the experimental basis for further exploring the roles of E2F1 in tumorigenesis, development, invasion and metastasis, recurrence, and drug resistance in small cell lung cancer.

  10. 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.

  11. 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.

  12. 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.

  13. NATURE OF W51e2: MASSIVE CORES AT DIFFERENT PHASES OF STAR FORMATION

    SciTech Connect

    Shi Hui; Han, J. L.; Zhao Junhui E-mail: hjl@nao.cas.c

    2010-02-10

    We present high-resolution continuum images of the W51e2 complex processed from archival data of the Submillimeter Array (SMA) at 0.85 and 1.3 mm and the Very Large Array at 7 and 13 mm. We also made line images and profiles of W51e2 for three hydrogen radio recombination lines (RRLs; H26alpha, H53alpha, and H66alpha) and absorption of two molecular lines of HCN(4-3) and CO(2-1). At least four distinct continuum components have been detected in the 3'' region of W51e2 from the SMA continuum images at 0.85 and 1.3 mm with resolutions of 0.''3 x 0.''2 and 1.''4 x 0.''7, respectively. The west component, W51e2-W, coincides with the ultracompact H II region reported from previous radio observations. The H26alpha line observation reveals an unresolved hyper-compact ionized core (<0.''06 or <310 AU) with a high electron temperature of 1.2 x 10{sup 4} K, with the corresponding emission measure EM>7 x 10{sup 10} pc cm{sup -6} and the electron density N{sub e} >7 x 10{sup 6} cm{sup -3}. The inferred Lyman continuum flux implies that the H II region W51e2-W requires a newly formed massive star, an O8 star or a cluster of B-type stars, to maintain the ionization. W51e2-E, the brightest component at 0.85 mm, is located 0.''9 east from the hyper-compact ionized core. It has a total mass of {approx}140 M{sub sun} according to our spectral energy distribution analysis and a large infall rate of >1.3 x 10{sup -3} M{sub sun} yr{sup -1} inferred from the absorption of HCN. W51e2-E appears to be the accretion center in W51e2. Given the fact that no free-free emission and no RRLs have been detected, the massive core of W51e2-E appears to host one or more growing massive proto-stars. Located 2'' northwest from W51e2-E, W51e2-NW is detected in the continuum emission at 0.85 and 1.3 mm. No continuum emission has been detected at lambda>= 7 mm. Along with the maser activities previously observed, our analysis suggests that W51e2-NW is at an earlier phase of star formation. W51e2-N is

  14. 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

  15. Dissecting the Role of E2 Protein Domains in Alphavirus Pathogenicity

    PubMed Central

    Weger-Lucarelli, James; Aliota, Matthew T.; Wlodarchak, Nathan; Kamlangdee, Attapon; Swanson, Ryan

    2015-01-01

    ABSTRACT Alphaviruses represent a diverse set of arboviruses, many of which are important pathogens. Chikungunya virus (CHIKV), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the Caribbean and South America. In contrast to CHIKV, other related alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV), can cause encephalitic disease. E2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pathogenicity, and immunogenicity. Previous reports also have demonstrated that E2 contains residues important for host range expansions and monoclonal antibody binding; however, little is known about what role each protein domain (e.g., A, B, and C) of E2 plays on these factors. Therefore, we constructed chimeric cDNA clones between CHIKV and VEEV or SFV to probe the effect of each domain on pathogenicity in vitro and in vivo. CHIKV chimeras containing each of the domains of the E2 (ΔDomA, ΔDomB, and ΔDomC) from SFV, but not VEEV, were successfully rescued. Interestingly, while all chimeric viruses were attenuated compared to CHIKV in mice, ΔDomB virus showed similar rates of infection and dissemination in Aedes aegypti mosquitoes, suggesting differing roles for the E2 protein in different hosts. In contrast to CHIKV; ΔDomB, and to a lesser extent ΔDomA, caused neuron degeneration and demyelination in mice infected intracranially, suggesting a shift toward a phenotype similar to SFV. Thus, chimeric CHIKV/SFV provide insights on the role the alphavirus E2 protein plays on pathogenesis. IMPORTANCE Chikungunya virus (CHIKV) has caused large outbreaks of acute and chronic arthritis throughout Africa and Southeast Asia and has now become a massive public health threat in the Americas, causing an estimated 1.2 million human cases in just over a year. No approved vaccines or antivirals exist for human use against CHIKV or any other alphavirus. Despite the threat

  16. Antibodies to P450IID6, SLA, PDH-E2 and BCKD-E2 in Japanese patients with chronic hepatitis.

    PubMed

    Nishioka, M; Morshed, S A; Parveen, S; Kono, K; Matsuoka, H; Manns, M P

    1997-12-01

    Auto-antibodies specific to various antigens in chronic hepatitis (CH) have been detected but their specificities and implications were uncertain. The aims of the present study were to investigate the frequency and the significance of seropositivity of antibodies to P450IID6 or liver/kidney microsome 1 (LKM1), soluble liver antigen (SLA), pyruvate dehydrogenase (PDH) and branched-chain keto acid dehydrogenase (BCKD) in 188 Japanese patients with different forms of CH by western blot or enzyme immunoassay (EIA). Anti-LKM1 was also measured by indirect immunofluorescent test. Anti-P450IID6 was found in 6/188 (3.2%) CH patients including 5/104 (4.8%) with hepatitis C virus (C) infection and 1/12 (8.3%) CH-C patients with antibodies to nuclear and smooth muscle antigens and hypergammaglobulinaemia (> 2.5 g/dL). This patient was the only one diagnosed with autoimmune hepatitis (AIH). All CH patients with hepatitis B (B), hepatitis non-B non-C (NBNC) and AIH were seronegative for anti-LKM1. Antibodies to soluble liver antigen were found in two of 188 (1%) patients, one with AIH and one with CH-B. Anti-BCKD-E2 but not anti-PDH-E2 was found in four patients (2.5%), one with AIH, two with CH-C, and one with NBNC. There was no obvious difference in age, sex ratio and laboratory findings in patients with or without anti-SLA and anti-BCKD-E2. Antibodies to P450IID6, SLA, PDH-E2 and BCKD-E2 are uncommon in adult CH-C, CH-B, CH-NBNC and AIH patients in Japan. Some of these patients positive for auto-antibodies appear to have autoimmune features and might require a careful follow up. The heterogeneity of these antibodies in CH preclude further justification for subtyping of AIH by the presence of the distinct auto-antibodies.

  17. The Viral E8^E2C Repressor Limits Productive Replication of Human Papillomavirus 16

    PubMed Central

    Straub, Elke; Dreer, Marcel; Fertey, Jasmin; Iftner, Thomas

    2014-01-01

    Productive replication of human papillomavirus type 16 (HPV16) occurs only in differentiated keratinocyte cells. In addition to the viral E2 activator protein, HPV16 and related HPV types express transcripts coding for an E8^E2C fusion protein, which limits genome replication in undifferentiated keratinocytes. To address E8^E2C's role in productive replication of HPV16, stable keratinocyte cell lines containing wild-type (wt), E8^E2C knockout (E8−), or E8 KWK mutant (mt) genomes, in which conserved E8 residues were inactivated, were established. Copy numbers of E8− and E8 KWK mt genomes and amounts of early and late viral transcripts were greatly increased compared to those for the wt in undifferentiated keratinocytes, suggesting that HPV16 E8^E2C activities are highly dependent upon the E8 part. Upon differentiation in organotypic cultures, E8 mt genomes displayed higher early viral transcript levels, but no changes in cellular differentiation or virus-induced cellular DNA replication in suprabasal cells were observed. E8 mt genomes were amplified to higher copy numbers and showed increased L1 transcripts compared to wt genomes. Furthermore, the number of cells expressing the viral late protein E4 or L1 or amplifying viral genomes was greatly increased in E8 mt cell lines. In wild-type cells, E8^E2C transcript levels did not decrease by differentiation. Our data indicate that the E8^E2C repressor limits viral transcription and replication throughout the complete life cycle of HPV16. PMID:24198405

  18. 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

  19. Conservation of the E8 CDS of the E8^E2 protein among mammalian papillomaviruses.

    PubMed

    Puustusmaa, Mikk; Abroi, Aare

    2016-09-01

    Papillomaviridae are small dsDNA viruses with a limited coding capacity. To fulfill all of the functional requirements for propagation and spreading, papillomaviruses use double coding and alternative protein isoforms. E8 ^ E2 is an alternative E2 protein isoform that is generated by fusing the short E8 CDS that completely overlaps E1 to the 'hinge' and the DNA-binding region of the E2 protein via alternative transcription/splicing. The papillomaviruses in which E8 ^ E2 mRNA sequences have been described exhibit a sparse phylogenomic distribution. Thus, it is not clear whether E8 ^ E2 is an ancestral protein that has not been described for other papillomavirus types or whether it randomly appears because of the conservation of the E1 protein and occurs only coincidentally. We searched for potential E8 coding sequences in a non-redundant set of papillomaviruses and applied SynPlot2 and an in-house-developed algorithm (cRegions) to determine the most plausible of the above-mentioned scenarios. Beginning with nine experimentally described E8 ^ E2 mRNAs, we predicted the potential E8 CDSs for more than 300 mammalian papillomavirus genomes. According to our analysis, E8 ^ E2 is not a result of E1 coding and represents a protein in its own right, and it most likely has an ancestral origin that precedes the divergence of major mammalian papillomavirus genera.

  20. Conservation of the E8 CDS of the E8^E2 protein among mammalian papillomaviruses.

    PubMed

    Puustusmaa, Mikk; Abroi, Aare

    2016-09-01

    Papillomaviridae are small dsDNA viruses with a limited coding capacity. To fulfill all of the functional requirements for propagation and spreading, papillomaviruses use double coding and alternative protein isoforms. E8 ^ E2 is an alternative E2 protein isoform that is generated by fusing the short E8 CDS that completely overlaps E1 to the 'hinge' and the DNA-binding region of the E2 protein via alternative transcription/splicing. The papillomaviruses in which E8 ^ E2 mRNA sequences have been described exhibit a sparse phylogenomic distribution. Thus, it is not clear whether E8 ^ E2 is an ancestral protein that has not been described for other papillomavirus types or whether it randomly appears because of the conservation of the E1 protein and occurs only coincidentally. We searched for potential E8 coding sequences in a non-redundant set of papillomaviruses and applied SynPlot2 and an in-house-developed algorithm (cRegions) to determine the most plausible of the above-mentioned scenarios. Beginning with nine experimentally described E8 ^ E2 mRNAs, we predicted the potential E8 CDSs for more than 300 mammalian papillomavirus genomes. According to our analysis, E8 ^ E2 is not a result of E1 coding and represents a protein in its own right, and it most likely has an ancestral origin that precedes the divergence of major mammalian papillomavirus genera. PMID:27325292

  1. 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

  2. Effect of KCA-098 on the function of osteoblast-like cells and the formation of TRAP-positive multinucleated cells in a mouse bone marrow cell population.

    PubMed

    Kawashima, K; Inoue, T; Tsutsumi, N; Endo, H

    1996-01-26

    KCA-098 (3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinol ine-6-one), an analogue of coumestrol (a naturally occurring weak phytoestrogen), dose-dependently increased alkaline phosphatase activity of osteoblastic ROS 17/2.8 cells and freshly-isolated osteoblasts from neonatal mouse calvaria, and reduced cell proliferation. In addition, KCA-098 increased the synthesis of collagenese-digestible protein (CDP) of ROS 17/2.8 cells. On the other hand, KCA-098 had no effect on the basal synthesis of osteocalcin but reduced the 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)(2)D3)-induced increase in osteocalcin synthesized by ROS 17/2.8 cells. Therefore, KCA-098 had a bidirectional effect on the differentiation of osteoblasts (i.e., stimulating both alkaline phosphatase activity and synthesis of CDP and inhibiting osteocalcin synthesis). However, as KCA-098 stimulated the mineralization of chick embryonic bone in organ culture and recovered the bone density reduced by ovariectomy of rats, it would serve overall to stimulate the differentiation of osteoblasts. On the other hand, KCA-098 inhibited the formation of tartrate-resistant, acid phosphate-positive multinucleated cells (TRAP(+)MNC) induced by 1 alpha,25(OH)(2)D(3), parathyroid hormone (PTH), and prostaglandin E2 (PGE2) in cultures of mouse bone marrow cells, showing that it inhibits the formation of osteoclast-like cells. Coumestrol and 17beta-estradiol had no effect on the proliferation and alkaline phosphatase activity of ROS 17/2.8 cells. They did, however, dose-dependently inhibit osteoclast-like cell formation as well as KCA-098 did, indicating that the main action of coumestrol and 17beta-estradiol on bone tissue is the inhibition of bone resorption.

  3. An E2F1-HOXB9 Transcriptional Circuit Is Associated with Breast Cancer Progression

    PubMed Central

    Zhussupova, Aisulu; Hayashida, Tetsu; Takahashi, Maiko; Miyao, Kazuhiro; Okazaki, Hiroshi; Jinno, Hiromitsu; Kitagawa, Yuko

    2014-01-01

    Homeobox B9 (HOXB9), a member of the homeobox gene family, is overexpressed in breast cancer and promotes tumor progression and metastasis by stimulating epithelial-to-mesenchymal transition and angiogenesis within the tumor microenvironment. HOXB9 activates the TGFβ-ATM axis, leading to checkpoint activation and DNA repair, which engenders radioresistance in breast cancer cells. Despite detailed reports of the role of HOXB9 in breast cancer, the factors that regulate HOXB9 transcription have not been extensively examined. Here we uncover an underlying mechanism that may suggest novel targeting strategies for breast cancer treatment. To identify a transcription factor binding site (TFBS) in the HOXB9 promoter region, a dual luciferase reporter assay was conducted. Protein candidates that may directly attach to a TFBS of HOXB9 were examined by Q-PCR, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and mutation analysis. A HOXB9 promoter region from −404 to −392 was identified as TFBS, and E2F1 was a potential binding candidate in this region. The induction of HOXB9 expression by E2F1 was observed by Q-PCR in several breast cancer cell lines overexpressing E2F1. The stimulatory effect of E2F1 on HOXB9 transcription and its ability to bind the TFBS were confirmed by luciferase, EMSA and ChIP assay. Immunohistochemical analysis of 139 breast cancer tissue samples revealed a significant correlation between E2F1 and HOXB9 expression (p<0.001). Furthermore, a CDK4/6 inhibitor suppressed E2F1 expression and also reduced expression of HOXB9 and its downstream target genes. Our in vitro analysis identified the TFBS of the HOXB9 promoter region and suggested that E2F1 is a direct regulator of HOXB9 expression; these data support the strong correlation we found between E2F1 and HOXB9 in clinical breast cancer samples. These results suggest that targeting the E2F1/HOXB9 axis may be a novel strategy for the control or prevention of cancer

  4. 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.

  5. Redeployment of Myc and E2f1-3 drives Rb-deficient cell cycles.

    PubMed

    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

    2015-08-01

    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

  6. 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

  7. Three-dimensional reconstruction of Agrobacterium VirE2 protein with single-stranded DNA.

    PubMed

    Abu-Arish, Asmahan; Frenkiel-Krispin, Daphna; Fricke, Tobin; Tzfira, Tzvi; Citovsky, Vitaly; Wolf, Sharon Grayer; Elbaum, Michael

    2004-06-11

    Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. A single VirD2 molecule covalently binds to the 5'-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity. An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal ("telephone coil") organization of the VirE2-DNA complex. Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods. We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn. The inner face of the protein units contains a continuous wall and an inward protruding shelf. These structures appear to accommodate the DNA binding. Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors. Coexisting with the helices, we also found VirE2 tetrameric ring structures. A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction. PMID:15054095

  8. An (e,2e) Measurement of the Xe Photoelectron β Parameter

    NASA Astrophysics Data System (ADS)

    Childers, J. G.; Martin, N. L. S.; Thompson, D. B.

    2000-06-01

    We have carried out (e,2e) experiments on Xe in the autoionizing region between the ^2P_3/2 and ^2P_1/2 ionic limits. (e,2e) spectra were taken at 150 eV incident electron energy and 0^circ scattering angle corresponding to a momentum transfer of 0.14 au. The spectral range covered the (^2P_1/2)nd,ms (n>=6,m>=8) autoionizing resonances which have ejected electron energies between 0 and 1.3 eV. The (e,2e) spectrometer has two ejected electron detectors configured to allow the simultaneous collection of (e,2e) ejected-electron spectra 180^circ apart. The summation of these spectra eliminates the non-dipole effects due to dipole-monopole and dipole-quadrupole interference, leaving a spectrum that mimics a pure dipole photoelectron experiment. Two separate (e,2e) experiments, at ejected electron directions 60^circ (the minimum possible) and 90^circ away from the momentum transfer axis, enable the determination of the Xe β parameter. Our results are in quite good agreement with the true photoelectron experiments.(J.Z. Wu, S.B. Whitfield, C.D. Caldwell, M.O. Krause, P. van der Meulen and A. Fahlman, Phys.Rev.A 42), 1350 (1990).

  9. PCOS women show significantly higher homocysteine level, independent to glucose and E2 level

    PubMed Central

    Eskandari, Zahra; Sadrkhanlou, Rajab-Ali; Nejati, Vahid; Tizro, Gholamreza

    2016-01-01

    Background: It is reasonable to think that some biochemical characteristics of follicular fluid (FF) surrounding the oocyte may play a critical role in determining the quality of oocyte and the subsequent potential needed to achieve fertilization and embryo development. Objective: This study was carried out to evaluate the levels of FF homocysteine (Hcy) in IVF candidate polycystic ovary syndrome (PCOS) women and any relationships with FF glucose and estradiol (E2) levels. Materials and Methods: In this case control study which was performed in Dr. Tizro Day Care and IVF Center 70 infertile patients were enrolled in two groups: comprising 35 PCOS and 35 non PCOS women. Long protocol was performed for all patients. FF Hcy, glucose and E2 levels were analyzed at the time of oocyte retrieval. Results: It was observed that FF Hcy level was significantly higher in PCOS patients compared with non PCOSs (p<0.01). Observations demonstrated that in PCOS group, the Hcy level increased independent to E2, glucose levels, BMI and age, while the PCOS group showed significantly higher BMI compared with non-PCOS group (p=0.03). However, no significant differences were revealed between groups for FF glucose and E2 levels. Conclusion: Present data showed that although FF glucose and E2 levels were constant in PCOS and non PCOS patients, but the FF Hcy levels in PCOS were significantly increased (p=0.01). PMID:27679823

  10. Targeting the RB-E2F pathway in breast cancer

    PubMed Central

    Johnson, Jackie; Thijssen, Bram; McDermott, Ultan; Garnett, Mathew; Wessels, Lodewyk F.A.; Bernards, René

    2016-01-01

    Mutations of the retinoblastoma tumor suppressor gene (RB1) or components regulating the CDK-RB-E2F pathway have been identified in nearly every human malignancy. Re-establishing cell cycle control through CDK inhibition has therefore emerged as an attractive option in the development of targeted cancer therapy. The most successful example of this today is the use of the CDK4/6 inhibitor palbociclib combined with aromatase inhibitors for the treatment of estrogen receptor-positive breast cancers. Multiple studies have demonstrated that the CDK-RB-E2F pathway is critical for the control of cell proliferation. More recently, studies have highlighted additional roles of this pathway, especially E2F transcription factors themselves, in tumor progression, angiogenesis and metastasis. Specific E2Fs also have prognostic value in breast cancer, independent of clinical parameters. We discuss here recent advances in understanding of the RB-E2F pathway in breast cancer. We also discuss the application of genome-wide genetic screening efforts to gain insight into synthetic lethal interactions of CDK4/6 inhibitors in breast cancer for the development of more effective combination therapies. PMID:26923330

  11. Early thymocyte development is regulated by modulation of E2A protein activity.

    PubMed

    Engel, I; Johns, C; Bain, G; Rivera, R R; Murre, C

    2001-09-17

    The E2A gene encodes the E47 and E12 basic helix-loop-helix (bHLH) transcription factors. T cell development in E2A-deficient mice is partially arrested before lineage commitment. Here we demonstrate that E47 expression becomes uniformly high at the point at which thymocytes begin to commit towards the T cell lineage. E47 protein levels remain high until the double positive developmental stage, at which point they drop to relatively moderate levels, and are further downregulated upon transition to the single positive stage. However, stimuli that mimic pre-T cell receptor (TCR) signaling in committed T cell precursors inhibit E47 DNA-binding activity and induce the bHLH inhibitor Id3 through a mitogen-activated protein kinase kinase-dependent pathway. Consistent with these observations, a deficiency in E2A proteins completely abrogates the developmental block observed in mice with defects in TCR rearrangement. Thus E2A proteins are necessary for both initiating T cell differentiation and inhibiting development in the absence of pre-TCR expression. Mechanistically, these data link pre-TCR mediated signaling and E2A downstream target genes into a common pathway.

  12. 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.

  13. Relationship between vitamin D, IFN-γ, 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-γ), 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-γ 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-γ 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-γ and 25(OH)D3 (rs = -0.330; p = 0.03) and a positive correlation between IFN-γ and E2 (rs = 0.404; p = 0.007). This study demonstrates an interesting interplay between vitamin D, INF-γ, and E2 in SLE patients with inactive disease.

  14. Acetylation of Conserved Lysines in Bovine Papillomavirus E2 by p300

    PubMed Central

    Quinlan, Edward J.; Culleton, Sara P.; Wu, Shwu-Yuan; Chiang, Cheng-Ming

    2013-01-01

    The p300, CBP, and pCAF lysine acetyltransferase (KAT) proteins have been reported to physically interact with bovine (BPV) and human (HPV) papillomavirus E2 proteins. While overexpression of these KAT proteins enhances E2-dependent transcription, the mechanism has not been determined. Using RNA interference (RNAi) to deplete these factors, we demonstrated that E2 transcriptional activity requires physiological levels of p300, CBP, and pCAF. Each protein appears to have a unique function in E2-dependent transcription, since overexpression of one KAT failed to compensate for RNAi knockdown of another KAT. Using an in vitro acetylation assay, we identified highly conserved lysines that are targeted by p300 for acetylation. The conservative changes of lysines at positions 111 and 112 to arginine were of particular interest. The K111R and the K111R/K112R mutants showed reduced transcriptional activity that was not responsive to p300 overexpression, while the K112R mutant retained activity. p300 and CBP were detected at the viral promoter; however, pCAF was not. We propose a model by which E2 transcriptional activity is controlled by p300-mediated acetylation of lysine 111. This model represents a novel mechanism regulating papillomavirus gene expression. PMID:23152516

  15. PCOS women show significantly higher homocysteine level, independent to glucose and E2 level

    PubMed Central

    Eskandari, Zahra; Sadrkhanlou, Rajab-Ali; Nejati, Vahid; Tizro, Gholamreza

    2016-01-01

    Background: It is reasonable to think that some biochemical characteristics of follicular fluid (FF) surrounding the oocyte may play a critical role in determining the quality of oocyte and the subsequent potential needed to achieve fertilization and embryo development. Objective: This study was carried out to evaluate the levels of FF homocysteine (Hcy) in IVF candidate polycystic ovary syndrome (PCOS) women and any relationships with FF glucose and estradiol (E2) levels. Materials and Methods: In this case control study which was performed in Dr. Tizro Day Care and IVF Center 70 infertile patients were enrolled in two groups: comprising 35 PCOS and 35 non PCOS women. Long protocol was performed for all patients. FF Hcy, glucose and E2 levels were analyzed at the time of oocyte retrieval. Results: It was observed that FF Hcy level was significantly higher in PCOS patients compared with non PCOSs (p<0.01). Observations demonstrated that in PCOS group, the Hcy level increased independent to E2, glucose levels, BMI and age, while the PCOS group showed significantly higher BMI compared with non-PCOS group (p=0.03). However, no significant differences were revealed between groups for FF glucose and E2 levels. Conclusion: Present data showed that although FF glucose and E2 levels were constant in PCOS and non PCOS patients, but the FF Hcy levels in PCOS were significantly increased (p=0.01).

  16. E2F1 in renal cancer: Mr Hyde disguised as Dr Jekyll?

    PubMed

    Tian, Weihua; Cui, Fenggong; Esteban, Miguel A

    2013-10-01

    The transcription factor E2F1 has both oncogenic and tumour suppressor properties, depending on the context. Clarifying the function of E2F1 in different types of cancer is relevant because in those situations in which it acts as an oncogene there may be a route for therapeutic interference. Renal cell carcinoma is the most frequent form of kidney cancer in adults and inactivation of the von Hippel-Lindau (VHL) gene underlies most cases. This malignancy represents a challenge for standard therapies due to drug- and radio-resistance, effects that fit well within the scope of functions of E2F1. A new report by Mans et al postulates that up-regulation of E2F1 in VHL-defective renal cell carcinoma induces cell senescence and can thus be considered a good prognostic factor. Here we discuss these findings in a wider context and propose that E2F1 may actually not play a uniform role in renal cell carcinoma but rather an ambiguous one whose deeper understanding could have practical implications. PMID:23868058

  17. E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification

    SciTech Connect

    Huang, D.T.; Ayrault, O.; Hunt, H.W.; Taherbhoy, A.M.; Duda, D.M.; Scott, D.C.; Borg, L.A.; Neale, G.; Murray, P.J.; Roussel, M.F.; Schulman, B.A.

    2009-03-27

    Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation.

  18. Seasonal reproductive biology of two species of freshwater catfish from the Venezuelan floodplains.

    PubMed

    Marcano, Dayssi; Cardillo, Elizabeth; Rodriguez, Christian; Poleo, Germán; Gago, Nathalie; Guerrero, Hilda Y

    2007-01-01

    Oxydoras sifontesi and Pimelodus blochii are seasonal breeder fish. Spawn occurs once a year over a short interval of time, at the beginning of the rainy season. The gonadosomatic index (GSI), and plasma levels of steroid hormones of P. blochii and O. sifontesi were studied from fish samples, collected from the Portuguesa River (Portuguesa State, Venezuela) in 1998 and 2004-2005, respectively. Gonadal tissue samples were obtained and processed for histology. A macroscopic classification of the degree of gonadal maturation was performed using a six-stage maturity scale. Data was analyzed and compared as a function of the gonadal maturation stage. The GSI of both O. sifontesi and P. blochii increases from stages II to V (preparatory and prespawning periods) and decreases in the stage VI (postspawning). In males, the GSI was usually lower than in females. In both species, the higher plasmatic concentration of 17beta-estradiol (17beta-E) and testosterone (T) were obtained from specimens in stages IV or V. A significant decrease in both hormones was observed in stage VI, except for the males of P. blochii where T concentration did not change between gonadal stages IV-VI. The relevance of these results is discussed in relation to the seasonality and the particular hydrological conditions of the region.

  19. E2F1 modulates p38 MAPK phosphorylation via transcriptional regulation of ASK1 and Wip1.

    PubMed

    Hershko, Tzippi; Korotayev, Katya; Polager, Shirley; Ginsberg, Doron

    2006-10-20

    The E2F family of transcription factors regulates a diverse array of cellular functions, including cell proliferation, cell differentiation, and apoptosis. Recent studies indicate that E2F can also regulate transcription of upstream components of signal transduction pathways. We show here that E2F1 modulates the activity of the p38 MAPK pathway via E2F1-induced transient up-regulation of p38 MAPK phosphorylation. The mechanism by which E2F1 modulates p38 MAPK phosphorylation involves transcriptional induction of the kinase ASK1, a member of the MAPKKK family that phosphorylates p38 MKKs. Subsequent E2F-dependent down-regulation of the p38 signaling pathway is achieved through E2F-induced up-regulation of Wip1, a phosphatase that dephosphorylates and inactivates p38. Both ASK1 and Wip1 are essential mediators of the E2F-p38 connection: knock down of ASK1 inhibits E2F1-induced phosphorylation of p38, whereas knock down of Wip1 prolongs E2F1-induced p38 phosphorylation. Furthermore, Wip1 knock down enhances E2F1-induced apoptosis. Therefore, our data reveal a novel link between a central signaling pathway and the transcription factor E2F and identify Wip1 as a modulator of E2F1-induced apoptosis. PMID:16912047

  20. On the equivalence of experimental B(E2) values determined by various techniques

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

    We establish the equivalence of the various techniques for measuring B(E2) values using a statistical analysis. Data used in this work come from the recent compilation by B. Pritychenko et al. (2016) [5]. We consider only those nuclei for which the B(E2) values were measured by at least two different methods, with each method being independently performed at least twice. Our results indicate that most prevalent methods of measuring B(E2) values are equivalent, with some weak evidence that Doppler-shift attenuation method (DSAM) measurements may differ from Coulomb excitation (CE) and nuclear resonance fluorescence (NRF) measurements. However, such an evidence appears to arise from discrepant DSAM measurements of the lifetimes for 60Ni and some Sn nuclei rather than a systematic deviation in the method itself.

  1. 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

  2. Linked genes for calmodulin and E2 ubiquitin-conjugating enzyme in Trichomonas vaginalis.

    PubMed

    Keeling, P J; Doherty-Kirby, A L; Teh, E M; Doolittle, W F

    1996-01-01

    In searching the genomes of early-diverging protists to study whether the possession of calmodulin is ancestral to all eukaryotes, the gene for calmodulin was identified in Trichomonas vaginalis. This flagellate is a member of the Parabasalia, one of the earliest lineages of recognized eukaryotes to have diverged. This sequence was used to isolate a homologous 1.250-kb fragment from the T. vaginalis genome by inverse polymerase chain reaction. This fragment was also completely sequenced and shown to contain the 3' end of the single-copy calmodulin gene and the 3' end of a gene encoding a protein with high similarity to E2 ubiquitin-conjugating enzymes, a family which has previously only been identified in animals, plants, and fungi. Phylogenetic analysis of 50 members of the E2 family distinguishes at least nine separate subfamilies one of which includes the T. vaginalis E2-homologue and an uncharacterized gene from yeast chromosome XII.

  3. 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.

  4. [Research progress in roles of high-risk human papillomavirus E2 protein].

    PubMed

    Wu, En-Qi; Tang, Yuan-Yu

    2014-03-01

    High-risk human papillomavirus (HPV) is the principal cause of various cancers including cervical cancer, anal cancer, vulvar cancer, and some head and neck cancers. In the viral life cycle, by interacting with both viral and host DNA and proteins, the HPV E2 protein plays a pivotal role in viral transcriptional regulation and DNA replication, and it is also associated with modification of various cellular processes, including host gene transcription, RNA processing, apoptosis, ubiquitination, and intracellular trafficking, to create a convenient environment for a replicative cycle of the virus and contribute to the HPV pathogenesis. Elucidating the roles of E2 protein throughout the viral life cycle will improve our understanding of the viral life cycle and pathogenesis and help us identify novel antiviral agents with therapeutic potential. This article reviews the research progress in the structure, roles, and activity of high-risk HPV E2 protein, particularly that of HPV-16.

  5. Development of single dilution immunoassay to detect E2 protein specific classical swine fever virus antibody.

    PubMed

    Kumar, Rakesh; Barman, Nagendra N; Khatoon, Elina; Kumar, Sachin

    2016-04-01

    Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease in swine. The disease is endemic in different parts of the world and vaccination is the only way to protect pigs from CSFV infection. The virus surface protein E2 is the major immunogenic protein eliciting protective immunity against CSFV infection in swine. The whole virus antigen cannot differentiate CSFV from other pestiviruses as it cross reacts with border disease and bovine viral diarrhoea viruses. Commercial available ELISA is based on the whole CSFV particle and can lead to false positive results. Moreover, the available commercial ELISA is not cost effective. In the present study, a recombinant E2 protein based single serum dilution ELISA was developed which showed enhanced sensitivity, specificity and accuracy as compared to commercial CSFV detection ELISA. The recombinant E2 protein based ELISA could be an alternate to existing diagnostics against CSFV infection in pigs. PMID:27032503

  6. 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.

  7. 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

  8. Production and Actions of the Anandamide Metabolite Prostamide E2 in the Renal Medulla

    PubMed Central

    Li, Cao; Xia, Min; Poklis, Justin L.; Lichtman, Aron H.; Abdullah, Rehab A.; Dewey, William L.; Li, Pin-Lan

    2012-01-01

    Medullipin has been proposed to be an antihypertensive lipid hormone released from the renal medulla in response to increased arterial pressure and renal medullary blood flow. Because anandamide (AEA) possesses characteristics of this purported hormone, the present study tested the hypothesis that AEA or one of its metabolites represents medullipin. AEA was demonstrated to be enriched in the kidney medulla compared with cortex. Western blotting and enzymatic analyses of renal cortical and medullary microsomes revealed opposite patterns of enrichment of two AEA-metabolizing enzymes, with fatty acid amide hydrolase higher in the renal cortex and cyclooxygenase-2 (COX-2) higher in the renal medulla. In COX-2 reactions with renal medullary microsomes, prostamide E2, the ethanolamide of prostaglandin E2, was the major product detected. Intramedullarily infused AEA dose-dependently increased urine volume and sodium and potassium excretion (15–60 nmol/kg/min) but had little effect on mean arterial pressure (MAP). The renal excretory effects of AEA were blocked by intravenous infusion of celecoxib (0.1 μg/kg/min), a selective COX-2 inhibitor, suggesting the involvement of a prostamide intermediate. Plasma kinetic analysis revealed longer elimination half-lives for AEA and prostamide E2 compared with prostaglandin E2. Intravenous prostamide E2 reduced MAP and increased renal blood flow (RBF), actions opposite to those of angiotensin II. Coinfusion of prostamide E2 inhibited angiotensin II effects on MAP and RBF. These results suggest that AEA and/or its prostamide metabolites in the renal medulla may represent medullipin and function as a regulator of body fluid and MAP. PMID:22685343

  9. E2C(R2) Periodic Benefit-Risk Evaluation Report and E2C(R2) Periodic Benefit-Risk Evaluation Report--Questions and Answers; International Council for Harmonisation; Guidances for Industry; Availability. Notice.

    PubMed

    2016-07-19

    The Food and Drug Administration (FDA or Agency) is announcing the availability of guidances for industry entitled ``E2C(R2) Periodic Benefit-Risk Evaluation'' (E2C(R2) guidance) and ``E2C(R2) Periodic Benefit-Risk Evaluation Report--Questions and Answers'' (E2C(R2) Q&A guidance). These guidances were prepared under the auspices of the International Council for Harmonisation (ICH), formerly the International Conference on Harmonisation. The E2C(R2) draft guidance, issued April 11, 2012, updated and combined two ICH guidances, ``E2C Clinical Safety Data Management: Periodic Safety Update Reports for Marketed Drugs'' (E2C guidance) and ``Addendum to E2C Clinical Safety Data Management: Periodic Safety Update Reports for Marketed Drugs'' (addendum to the E2C guidance). The E2C(R2) guidance is intended to describe the format, content, and timing of a Periodic Benefit-Risk Evaluation Report (PBRER) for an approved drug or biologic, and it finalizes the draft guidance. The E2C(R2) Q&A guidance is a supplementary guidance that is intended to clarify key issues in the E2C(R2) guidance.

  10. E2C(R2) Periodic Benefit-Risk Evaluation Report and E2C(R2) Periodic Benefit-Risk Evaluation Report--Questions and Answers; International Council for Harmonisation; Guidances for Industry; Availability. Notice.

    PubMed

    2016-07-19

    The Food and Drug Administration (FDA or Agency) is announcing the availability of guidances for industry entitled ``E2C(R2) Periodic Benefit-Risk Evaluation'' (E2C(R2) guidance) and ``E2C(R2) Periodic Benefit-Risk Evaluation Report--Questions and Answers'' (E2C(R2) Q&A guidance). These guidances were prepared under the auspices of the International Council for Harmonisation (ICH), formerly the International Conference on Harmonisation. The E2C(R2) draft guidance, issued April 11, 2012, updated and combined two ICH guidances, ``E2C Clinical Safety Data Management: Periodic Safety Update Reports for Marketed Drugs'' (E2C guidance) and ``Addendum to E2C Clinical Safety Data Management: Periodic Safety Update Reports for Marketed Drugs'' (addendum to the E2C guidance). The E2C(R2) guidance is intended to describe the format, content, and timing of a Periodic Benefit-Risk Evaluation Report (PBRER) for an approved drug or biologic, and it finalizes the draft guidance. The E2C(R2) Q&A guidance is a supplementary guidance that is intended to clarify key issues in the E2C(R2) guidance. PMID:27459749

  11. pSa causes oncogenic suppression of Agrobacterium by inhibiting VirE2 protein export.

    PubMed

    Lee, L Y; Gelvin, S B; Kado, C I

    1999-01-01

    When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the osa gene of the plasmid pSa can suppress crown gall tumorigenesis incited by Agrobacterium tumefaciens. Neither T-DNA processing nor vir (virulence) gene induction is affected by the presence of osa in the bacterium. We used Arabidopsis thaliana root segments and tobacco leaf discs to demonstrate that Osa inhibits A. tumefaciens from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable beta-glucuronidase (GUS) expression. When A. tumefaciens contained osa, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of Nicotiana benthamiana by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus. The extracellular complementation of an A. tumefaciens virE2 mutant (the T-DNA donor strain) by an A. tumefaciens strain lacking T-DNA but containing a wild-type virE2 gene (the VirE2 donor strain) was blocked when osa was present in the VirE2 donor strain, but not when osa was present in the T-DNA donor strain. These data indicate that osa inhibits VirE2 protein, but not T-DNA export from A. tumefaciens. These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium. The successful infection of Datura stramonium plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an A. tumefaciens virE2 mutant carrying osa confirmed that oncogenic suppression by osa does not occur by blocking T-DNA transfer. Overexpression of virB9, virB10, and virB11 in A. tumefaciens did not overcome oncogenic suppression by osa. The finding that the expression of the osa gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by osa may differ from that of the IncQ plasmid RSF

  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. E2F1-Mediated Induction of NFYB Attenuates Apoptosis via Joint Regulation of a Pro-Survival Transcriptional Program

    PubMed Central

    Jiang, Xiaolei; Nevins, Joseph Roy

    2015-01-01

    The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors in mediating the specificity of E2F function. Here we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance. PMID:26039627

  14. E2F7 regulates transcription and maturation of multiple microRNAs to restrain cell proliferation

    PubMed Central

    Mitxelena, Jone; Apraiz, Aintzane; Vallejo-Rodríguez, Jon; Malumbres, Marcos; Zubiaga, Ana M.

    2016-01-01

    E2F transcription factors (E2F1-8) are known to coordinately regulate the expression of a plethora of target genes, including those coding for microRNAs (miRNAs), to control cell cycle progression. Recent work has described the atypical E2F factor E2F7 as a transcriptional repressor of cell cycle-related protein-coding genes. However, the contribution of E2F7 to miRNA gene expression during the cell cycle has not been defined. We have performed a genome-wide RNA sequencing analysis to identify E2F7-regulated miRNAs and show that E2F7 plays as a major role in the negative regulation of a set of miRNAs that promote cellular proliferation. We provide mechanistic evidence for an interplay between E2F7 and the canonical E2F factors E2F1-3 in the regulation of multiple miRNAs. We show that miR-25, -26a, -27b, -92a and -7 expression is controlled at the transcriptional level by the antagonistic activity of E2F7 and E2F1-3. By contrast, let-7 miRNA expression is controlled indirectly through a novel E2F/c-MYC/LIN28B axis, whereby E2F7 and E2F1-3 modulate c-MYC and LIN28B levels to impact let-7 miRNA processing and maturation. Taken together, our data uncover a new regulatory network involving transcriptional and post-transcriptional mechanisms controlled by E2F7 to restrain cell cycle progression through repression of proliferation-promoting miRNAs. PMID:26961310

  15. Loss of E2F1 Extends Survival and Accelerates Oral Tumor Growth in HPV-Positive Mice.

    PubMed

    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 pathways-cell 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.

  16. 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

    2010-07-01

    ... Permitted Tolerance for Conducting Radiative Tests E Table E-2 to Subpart E of Part 53 Protection of... Reference Methods and Class I and Class II Equivalent Methods for PM2.5 or PM10â2.5 Pt. 53, Subpt. E, Table E-2 Table E-2 to Subpart E of Part 53—Spectral Energy Distribution and Permitted Tolerance...

  17. Inhibition of endothelial cell activation by bHLH protein E2-2 and its impairment of angiogenesis.

    PubMed

    Tanaka, Aya; Itoh, Fumiko; Nishiyama, Koichi; Takezawa, Toshiaki; Kurihara, Hiroki; Itoh, Susumu; Kato, Mitsuyasu

    2010-05-20

    E2-2 belongs to the basic helix-loop-helix (bHLH) family of transcription factors. E2-2 associates with inhibitor of DNA binding (Id) 1, which is involved in angiogenesis. In this paper, we demonstrate that E2-2 interacts with Id1 and provide evidence that this interaction potentiates angiogenesis. Mutational analysis revealed that the HLH domain of E2-2 is required for the interaction with Id1 and vice versa. In addition, Id1 interfered with E2-2-mediated effects on luciferase reporter activities. Interestingly, injection of E2-2-expressing adenoviruses into Matrigel plugs implanted under the skin blocked in vivo angiogenesis. In contrast, the injection of Id1-expressing adenoviruses rescued E2-2-mediated inhibition of in vivo angiogenic reaction. Consistent with the results of the Matrigel plug assay, E2-2 could inhibit endothelial cell (EC) migration, network formation, and proliferation. On the other hand, knockdown of E2-2 in ECs increased EC migration. The blockade of EC migration by E2-2 was relieved by exogenous expression of Id1. We also demonstrated that E2-2 can perturb VEGFR2 expression via inhibition of VEGFR2 promoter activity. This study suggests that E2-2 can maintain EC quiescence and that Id1 can counter this effect. PMID:20231428

  18. 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

    ... Permitted Tolerance for Conducting Radiative Tests E Table E-2 to Subpart E of Part 53 Protection of... Reference Methods and Class I and Class II Equivalent Methods for PM2.5 or PM10â2.5 Pt. 53, Subpt. E, Table E-2 Table E-2 to Subpart E of Part 53—Spectral Energy Distribution and Permitted Tolerance...

  19. 77 FR 21782 - International Conference on Harmonisation; Draft Guidance for Industry on E2C(R2) Periodic...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-11

    ... individual case safety reports to regulatory authorities, automated data mining techniques, more attention to... and combines two ICH guidances, ``E2C Clinical Safety Data Management: Periodic Safety Update Reports for Marketed Drugs'' (E2C guidance) and ``Addendum to E2C Clinical Safety Data Management:...

  20. 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.

  1. TGFbeta-mediated formation of pRb-E2F complexes in human myeloid leukemia cells.

    PubMed

    Hu, Xiao Tang

    2008-05-01

    TGFbeta 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. TGFbeta 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, TGFbeta 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 TGFbeta 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 TGFbeta-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 TGFbeta-mediated cell cycle control in human myeloid leukemia cells.

  2. 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.

  3. Influence of HPV16 E2 and its localisation on the expression of matrix metalloproteinase-9.

    PubMed

    Mühlen, Sabrina; Behren, Andreas; Iftner, Thomas; Simon, Christian

    2010-08-01

    Infection with the high-risk HPV types 16 and 18 is the major cause of cervical cancer and plays a role in the development of certain head and neck and skin cancers. We have previously demonstrated that the Early Protein 2 of the Cottontail Rabbit papillomavirus (CRPV), required for skin carcinogenesis in a rabbit model, is able to induce the expression of a matrix metalloproteinase (MMP-9); a protease known to play a key role in invasion and metastasis. However, as of now we do not understand the underlying mechanism of activation nor relevance for the human system. Here, we report that high-risk human papillomavirus HPV16 E2 similar to our previously reported results on CRPV E2 activates the human MMP-9 promoter predominantly via the MEK1-ERK1/2-AP-1-signaling pathway. In addition this activation is associated with a nuclear sub-localisation of HPV16-E2 suggesting a nuclear protein-protein or protein-DNA interaction of E2 as the underlying mechanism of activation.

  4. Sensitivity of lymphocytes to prostaglandin E2 increases in subjects over age 70.

    PubMed Central

    Goodwin, J S; Messner, R P

    1979-01-01

    We examined the sensitivity of lymphocytes from different age groups to inhibition by prostaglandin E2. Phytohemagglutinin-stimulated cultures of peripheral blood mononuclear cells from 12 healthy subjects over the age of 70 were much more sensitive to inhibition by exogenously added prostaglandin E2 than were cells from 17 young controls (ID50 congruent to 10 nM for the subjects over 70 vs. greater than 3 micronM for the young controls). The more senstivie lymphocytes from a subject over 70 were to prostaglandin E2, the lower was his or her response to phytohemagglutinin (r = 0.75, P less than 0.01). The mean responses to phytohemagglutinin of the peripheral blood mononuclear cells from the subjects over 70 were significantly depressed compared to the young controls. Addition of indomethacin, a prostaglandin synthetase inhibitor, to the cultures resulted in an increase in [3H]thymidine incorporation of 140 +/- 16% in the cells of the subjects over 70 vs. a 36 +/- 3% increase in the young controls (mean +/- SEM, P less than 0.001). The mean phytohemagglutinin response of the subjects over 70 was 40% of the control response without indomethacin. With addition of indomethacin the response of subjects over 70 rose to 72% of control. Thus, increased sinsitivity to prostaglandin E2 appears to be responsible in part for the depressed mitogen response of peripheral blood mononuclear cells from healthy subjects over 70. PMID:457862

  5. Changes in Gene Expression and Estrogen Receptor Cistrome in Mouse Liver Upon Acute E2 Treatment.

    PubMed

    Palierne, Gaëlle; Fabre, Aurélie; Solinhac, Romain; Le Péron, Christine; Avner, Stéphane; Lenfant, Françoise; Fontaine, Coralie; Salbert, Gilles; Flouriot, Gilles; Arnal, Jean-François; Métivier, Raphaël

    2016-07-01

    Transcriptional regulation by the estrogen receptor-α (ER) has been investigated mainly in breast cancer cell lines, but estrogens such as 17β-estradiol (E2) exert numerous extrareproductive effects, particularly in the liver, where E2 exhibits both protective metabolic and deleterious thrombotic actions. To analyze the direct and early transcriptional effects of estrogens in the liver, we determined the E2-sensitive transcriptome and ER cistrome in mice after acute administration of E2 or placebo. These analyses revealed the early induction of genes involved in lipid metabolism, which fits with the crucial role of ER in the prevention of liver steatosis. Characterization of the chromatin state of ER binding sites (BSs) in mice expressing or not ER demonstrated that ER is not required per se for the establishment and/or maintenance of chromatin modifications at the majority of its BSs. This is presumably a consequence of a strong overlap between ER and hepatocyte nuclear factor 4α BSs. In contrast, 40% of the BSs of the pioneer factor forkhead box protein a (Foxa2) were dependent upon ER expression, and ER expression also affected the distribution of nucleosomes harboring dimethylated lysine 4 of Histone H3 around Foxa2 BSs. We finally show that, in addition to a network of liver-specific transcription factors including CCAAT/enhancer-binding protein and hepatocyte nuclear factor 4α, ER might be required for proper Foxa2 function in this tissue.

  6. Antibegomoviral activity of the agrobacterial virulence protein VirE2.

    PubMed

    Sunitha, Sukumaran; Marian, Dolly; Hohn, Barbara; Veluthambi, Karuppannan

    2011-12-01

    Mungbean yellow mosaic geminivirus (MYMV) causes severe yellow mosaic disease in blackgram, mungbean, Frenchbean, pigeonpea, soybean and mothbean. We attempted to induce resistance against this virus using the transcriptional activator protein gene deleted in the C-terminal activation domain (TrAP-∆AD) and Agrobacterium tumefaciens virE2. MYMV is known to replicate in agroinoculated tobacco leaf discs. Three transgenic tobacco plants which harboured a truncated MYMV transcriptional activator protein gene and two tobacco plants transformed with the octopine type A. tumefaciens virE2 gene were agroinoculated with an A. tumefaciens strain which harboured the partial dimers of both DNA A and DNA B of MYMV. The level of viral DNA accumulation in leaf discs of transgenic plants correlated inversely to the level of the MYMV TrAP-∆AD transcript. Two VirE2-transgenic plants, which complemented tumorigenesis of a virE2 mutant A. tumefaciens strain, effectively reduced MYMV DNA accumulation in the leaf disc agroinoculation assay. PMID:21842234

  7. An Agrobacterium VirE2 channel for transferred-DNA transport into plant cells.

    PubMed

    Dumas, F; Duckely, M; Pelczar, P; Van Gelder, P; Hohn, B

    2001-01-16

    Transferred DNA (T-DNA) transfer from Agrobacterium tumefaciens into eukaryotic cells is the only known example of interkingdom DNA transfer. T-DNA is a single-stranded segment of Agrobacterium's tumor-inducing plasmid that enters the plant cell as a complex with the bacterial virulence proteins VirD2 and VirE2. The VirE2 protein is highly induced on contact of A. tumefaciens with a plant host and has been reported to act in late steps of transfer. One of its previously demonstrated functions is binding to the single-stranded (ss) T-DNA and protecting it from degradation. Recent experiments suggest other functions of the protein. A combination of planar lipid bilayer experiments, vesicle swelling assays, and DNA transport experiments demonstrated that VirE2 can insert itself into artificial membranes and form channels. These channels are voltage gated, anion selective, and single-stranded DNA-specific and can facilitate the efficient transport of single-stranded DNA through membranes. These experiments demonstrate a VirE2 function as a transmembrane DNA transporter, which could have applications in gene delivery systems. PMID:11149937

  8. Bovine viral diarrhea virus structural protein E2 as a complement regulatory protein.

    PubMed

    Ostachuk, Agustín

    2016-07-01

    Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, family Flaviviridae, and is one of the most widely distributed viruses in cattle worldwide. Approximately 60 % of cattle in endemic areas without control measures are infected with BVDV during their lifetime. This wide prevalence of BVDV in cattle populations results in significant economic losses. BVDV is capable of establishing persistent infections in its host due to its ability to infect fetuses, causing immune tolerance. However, this cannot explain how the virus evades the innate immune system. The objective of the present work was to test the potential activity of E2 as a complement regulatory protein. E2 glycoprotein, produced both in soluble and transmembrane forms in stable CHO-K1 cell lines, was able to reduce complement-mediated cell lysis up to 40 % and complement-mediated DNA fragmentation by 50 %, in comparison with cell lines not expressing the glycoprotein. This work provides the first evidence of E2 as a complement regulatory protein and, thus, the finding of a mechanism of immune evasion by BVDV. Furthermore, it is postulated that E2 acts as a self-associated molecular pattern (SAMP), enabling the virus to avoid being targeted by the immune system and to be recognized as self. PMID:27038454

  9. 76 FR 75774 - Targeted Populations Under Section 45D(e)(2)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-05

    ... advance notice of proposed rulemaking (ANPRM) (70 FR 29658) to seek comments from the public with respect... proposed rulemaking (NPRM) (REG-142339-05) was published in the Federal Register (73 FR 54990). Written and... Internal Revenue Service 26 CFR Part 1 RIN 1545-BE89 Targeted Populations Under Section 45D(e)(2)...

  10. 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.

  11. Ubiquitination Accomplished: E1 and E2 Enzymes Were Not Necessary.

    PubMed

    Nakasone, Mark A; Huang, Danny T

    2016-06-16

    Qiu et al. (2016) show that a mono-ADP-ribosyltransferase, SdeA, from Legionella pneumophila catalyzes ADP-ribosylation of ubiquitin, allowing SdeA to modify substrate with ubiquitin in the absence of E1 and E2 enzymes. PMID:27315555

  12. 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.

  13. 26 CFR 1.860E-2 - Tax on transfers of residual interests to certain organizations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... disqualified organization acquires the residual interest. The discount rate to be used for this present value... 26 Internal Revenue 9 2013-04-01 2013-04-01 false Tax on transfers of residual interests to... § 1.860E-2 Tax on transfers of residual interests to certain organizations. (a) Transfers...

  14. "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…

  15. Use of Prostaglandin E2 in the Management of Missed Abortion, Missed Labour, and Hydatidiform Mole

    PubMed Central

    Karim, S. M. M.

    1970-01-01

    Treatment of six cases of missed abortion and one case of hydatidiform mole with intravenous infusion of prostaglandin E2 resulted in complete abortion in all cases. Of 15 patients with missed labour, 14 were delivered successfully with similar treatment. The technique appears to be a safe, reliable, and rapid method of managing missed abortion, missed labour, and hydatidiform mole. PMID:5448780

  16. The nuclear factor κB inhibitor (E)-2-fluoro-4'-methoxystilbene inhibits firefly luciferase.

    PubMed

    Braeuning, Albert; Vetter, Silvia

    2012-12-01

    Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4'-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4'-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4'-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4'-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

  17. 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.

  18. Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization.

    PubMed

    Liao, Xun; Wang, Zuohuan; Cao, Tong; Tong, Chao; Geng, Shichao; Gu, Yuanxing; Zhou, Yingshan; Li, Xiaoliang; Fang, Weihuan

    2016-07-19

    Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies and confers protection against CSFV infection. There are three hypervariable antigenic regions (HAR1, HAR2 and HAR3) of E2 that are different between the group 1 vaccine C-strain and group 2 clinical isolates. This study was aimed to characterize the antigenic epitope region recognized by monoclonal antibody 4F4 (mAb-4F4) that is present in the group 2 field isolate HZ1-08, but not in the C-strain, and examine its impact on neutralization titers when antisera from different recombinant viruses were cross-examined. Indirect ELISA with C-strain E2-based chimeric proteins carrying the three HAR regions showed that the mAb-4F4 bound to HAR1 from HZ1-08 E2, but not to HAR2 or HAR3, indicating that the specific epitope is located in the HAR1 region. Of the 6 major residues differences between C-strain and field isolates, Glu713 in the HAR1 region of strain HZ1-08 is critical for mAb-4F4 binding either at the recombinant protein level or using intact recombinant viruses carrying single mutations. C-strain-based recombinant viruses carrying the most antigenic part of E2 or HAR1 from strain HZ1-08 remained non-pathogenic to pigs and induced good antibody responses. By cross-neutralization assay, we observed that the anti-C-strain serum lost most of its neutralization capacity to RecC-HZ-E2 and QZ-14 (subgroup 2.1d field isolate in 2014), and vice versa. More importantly, the RecC-HAR1 virus remained competent in neutralizing ReC-HZ-E2 and QZ-14 strains without compromising the neutralization capability to the recombinant C-strain. Thus, we propose that chimeric C-strain carrying the HAR1 region of field isolates is a good vaccine candidate for classical swine fever.

  19. Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization.

    PubMed

    Liao, Xun; Wang, Zuohuan; Cao, Tong; Tong, Chao; Geng, Shichao; Gu, Yuanxing; Zhou, Yingshan; Li, Xiaoliang; Fang, Weihuan

    2016-07-19

    Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies and confers protection against CSFV infection. There are three hypervariable antigenic regions (HAR1, HAR2 and HAR3) of E2 that are different between the group 1 vaccine C-strain and group 2 clinical isolates. This study was aimed to characterize the antigenic epitope region recognized by monoclonal antibody 4F4 (mAb-4F4) that is present in the group 2 field isolate HZ1-08, but not in the C-strain, and examine its impact on neutralization titers when antisera from different recombinant viruses were cross-examined. Indirect ELISA with C-strain E2-based chimeric proteins carrying the three HAR regions showed that the mAb-4F4 bound to HAR1 from HZ1-08 E2, but not to HAR2 or HAR3, indicating that the specific epitope is located in the HAR1 region. Of the 6 major residues differences between C-strain and field isolates, Glu713 in the HAR1 region of strain HZ1-08 is critical for mAb-4F4 binding either at the recombinant protein level or using intact recombinant viruses carrying single mutations. C-strain-based recombinant viruses carrying the most antigenic part of E2 or HAR1 from strain HZ1-08 remained non-pathogenic to pigs and induced good antibody responses. By cross-neutralization assay, we observed that the anti-C-strain serum lost most of its neutralization capacity to RecC-HZ-E2 and QZ-14 (subgroup 2.1d field isolate in 2014), and vice versa. More importantly, the RecC-HAR1 virus remained competent in neutralizing ReC-HZ-E2 and QZ-14 strains without compromising the neutralization capability to the recombinant C-strain. Thus, we propose that chimeric C-strain carrying the HAR1 region of field isolates is a good vaccine candidate for classical swine fever. PMID:27317266

  20. A Novel E2F-like Protein Involved in Transcriptional Activation of Cyst Wall Protein Genes in Giardia lamblia*

    PubMed Central

    Su, Li-Hsin; Pan, Yu-Jiao; Huang, Yu-Chang; Cho, Chao-Cheng; Chen, Chia-Wei; Huang, Shao-Wei; Chuang, Sheng-Fung; Sun, Chin-Hung

    2011-01-01

    Giardia lamblia differentiates into resistant walled cysts for survival outside the host and transmission. During encystation, synthesis of cyst wall proteins is coordinately induced. The E2F family of transcription factors in higher eukaryotes is involved in cell cycle progression and cell differentiation. We asked whether Giardia has E2F-like genes and whether they influence gene expression during Giardia encystation. Blast searches of the Giardia genome database identified one gene (e2f1) encoding a putative E2F protein with two putative DNA-binding domains. We found that the e2f1 gene expression levels increased significantly during encystation. Epitope-tagged E2F1 was found to localize to nuclei. Recombinant E2F1 specifically bound to the thymidine kinase and cwp1–3 gene promoters. E2F1 contains several key residues for DNA binding, and mutation analysis revealed that its binding sequence is similar to those of the known E2F family proteins. The E2F1-binding sequences were positive cis-acting elements of the thymidine kinase and cwp1 promoters. We also found that E2F1 transactivated the thymidine kinase and cwp1 promoters through its binding sequences in vivo. Interestingly, E2F1 overexpression resulted in a significant increase of the levels of CWP1 protein, cwp1–3 gene mRNA, and cyst formation. We also found E2F1 can interact with Myb2, a transcription factor that coordinate up-regulates the cwp1–3 genes during encystation. Our results suggest that E2F family has been conserved during evolution and that E2F1 is an important transcription factor in regulation of the Giardia cwp genes, which are key to Giardia differentiation into cysts. PMID:21835923

  1. 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

  2. VirE2: a unique ssDNA-compacting molecular machine.

    PubMed

    Grange, Wilfried; Duckely, Myriam; Husale, Sudhir; Jacob, Susan; Engel, Andreas; Hegner, Martin

    2008-02-01

    The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein-VirE2-enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated-using single-molecule techniques-that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes. PMID:18303950

  3. VirE2: A Unique ssDNA-Compacting Molecular Machine

    PubMed Central

    Jacob, Susan; Engel, Andreas; Hegner, Martin

    2008-01-01

    The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein—VirE2—enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated—using single-molecule techniques—that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes. PMID:18303950

  4. 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.

  5. Neutralizing activities of caprine antibodies towards conserved regions of the HCV envelope glycoprotein E2

    PubMed Central

    2011-01-01

    Anti HCV vaccine is not currently available and the present antiviral therapies fail to cure approximately half of the treated HCV patients. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 and test their neutralizing activities in a step towards developing therapeutic and/or prophylactic immunogens against HCV infection. Antibodies were generated by vaccination of goats with synthetic peptides derived from HCV E2. Viral neutralizing capacity of the generated anti E2 antibodies was tested using in vitro assays. Goats immunized with E2 synthetic peptides termed p412 [a.a 412-419], p430 [a.a 430-447] and p517 [a.a 517-531] generated high titers of antibody responses 2 to 4.5 fold higher than comparable titers of antibodies to the same epitopes in chronic HCV patients. In post infection experiments of native HCV into cultured Huh7.5 cells anti p412 and anti p 517 were proven to be neutralizing to HCV genotype 4a from patients' sera (87.5% and 75% respectively). On the contrary anti p430 exhibited weak viral neutralization capacity on the same samples (31.25%). Furthermore Ab mixes containing anti p430 exhibited reduced viral neutralization properties. From these experiments one could predict that neutralization by Abs towards different E2-epitopes varies considerably and success in the enrichment of neutralization epitope-specific antibodies may be accompanied by favorable results in combating HCV infection. Also, E2 conserved peptides p517 and p412 represent potential components of a candidate peptide vaccine against HCV infection. PMID:21819575

  6. Combination of neutralizing monoclonal antibodies against Hepatitis C virus E2 protein effectively blocks virus infection.

    PubMed

    Bose, Mihika; Mullick, Ranajoy; Das, Soma; Das, Saumitra; Karande, Anjali A

    2016-09-15

    Hepatitis C virus (HCV) represents a major global health threat. The envelope glycoproteins, E1-E2 of HCV play an important role in infection by binding to hepatocyte surface receptors leading to viral entry. Several regions on the E1-E2 are conserved for maintaining structural stability, despite the high mutation rate of HCV. Identification of antigenic determinants in these domains would aid in the development of anti-virals. The present study was aimed to delineate neutralizing epitopes by generating monoclonal antibodies (mAbs) to envelope proteins that can block virus binding and entry. Using HCV-like particles (HCV-LPs) corresponding to genotype 3a (prevalent in India), we obtained three mAbs specific for the E2 protein that significantly inhibited virus binding to hepatoma cells. Using overlapping protein fragments and peptides of the E2 protein, the epitopes corresponding to the mAbs were delineated. MAbs H6D3 and A10F2 recognise sequential linear epitopes, whereas, mAb E3D8 recognises a discontinuous epitope. The epitope of mAb E3D8 overlaps with the CD81 receptor-binding site and that of mAb A10F2 with the hypervariable region 2 of the E2 protein. The epitopes corresponding to these mAbs are distinct and unique. A combination of these antibodies significantly inhibited HCV binding and entry in both HCV pseudoparticle (in vitro) and HCV cell culture (ex vivo) system compared to the mAbs alone (P<0.0001). In conclusion, our findings support the potential of employing a cocktail of neutralizing mAbs in the management of HCV infection. PMID:27574733

  7. Chronic estrogen treatment in female transgenic (mRen2)27 hypertensive rats augments endothelium-derived nitric oxide release.

    PubMed

    Li, P; Ferrario, C M; Ganten, D; Brosnihan, K B

    1997-06-01

    Postmenopausal estrogen replacement therapy is associated with a reduction in cardiovascular events in women, but the mechanisms for this protection are unclear, especially in hypertensive subjects. In this study we investigated the effects of 17beta-estradiol (E2) treatment on blood pressure and endothelial function of transgenic [(mRen2)27] hypertensive and normotensive rats. Thirty female transgenic negative [Tg(-)] and hypertensive positive [Tg(+)] rats were ovariectomized and received either E2 (1.5 mg/rat, subcutaneously, for 3 weeks) or placebo. Chronic 17beta-estradiol treatment lowered mean blood pressure in both Tg hypertensive (159 +/- 4 v 145 +/- 4 mm Hg, P < .05, placebo v E2) and normotensive rats (119 +/- 4 v 108 +/- 2 mm Hg, P < .05, placebo v E2). Pressor responses to intravenous injection of phenylephrine were augmented in the Tg(+) as compared with Tg(-) rats. With chronic E2 treatment the pressor responses to phenylephrine were attenuated in both groups. Isometric tension of aortic rings was measured in vitro in organ chambers. The acetylcholine (Ach)-induced endothelium-dependent vascular relaxation was less potent in Tg(+) versus Tg(-) rats. E2 treatment significantly enhanced the Ach-induced relaxation of both Tg(+) and Tg(-) groups (ED50: 55.5 +/- 11.7 v 10.3 +/- 2.6; 23.8 +/- 6.5 v 5.1 +/- 1.2 nmol/L, placebo v E2 in Tg(+) and Tg(-), respectively). After E2 treatment the ED50 response in Tg(+) rats was no different from Tg(-) rats. However, the maximum vasodilation elicited by Ach was attenuated in Tg(+) as compared with Tg(-) rats. The calcium ionophore (A23187)-induced endothelium-dependent relaxation was less potent in Tg(+) as compared to Tg(-) rats and was enhanced by E2 treatment only in Tg(+) animals. There were no differences in the vasodilator responses elicited by sodium nitroprusside. Removal of endothelium and blockade of NO production abolished the endothelium-dependent vasodilation. The selective NO synthase inhibitor, N

  8. The anti-estrogen tamoxifen blocks the stimulatory effects of interleukin-6 on 17 beta-hydroxysteroid dehydrogenase activity in MCF-7 cells.

    PubMed

    Speirs, V; Adams, E F; White, M C

    1993-11-01

    Previous studies have revealed that human breast fibroblasts secrete the cytokine, interleukin-6 (IL-6) which stimulates the ability of MCF-7 human breast carcinoma cells to convert estrone (E1) to the biologically more active 17 beta-estradiol (E2). This is mediated by an increase in reductive 17 beta-hydroxysteroid dehydrogenase (17-HSD) activity. In the studies described here, we have extended our observations using the anti-estrogen, tamoxifen, to demonstrate that in a steady state, endogenous intracellular concentrations of E2 have no effects on reductive 17-HSD activity (E1-->E2), but are already maximally inhibitory for the oxidative reaction (E2-->E1). Increasing intracellular concentrations of E2, however, stimulated the reductive 17-HSD in a dose-dependent manner. IL-6 stimulated the reductive pathway and was synergistic with E2. IL-6 is most likely acting through an E2-dependent mechanism, since tamoxifen completely reversed the effects of E2 and IL-6 separately and in combination. These observations suggest that tamoxifen may reduce intratissular levels of E2 by directly increasing oxidative 17-HSD activity and by blocking the actions of paracrine factors such as IL-6 which increase reductive 17-HSD activity.

  9. Brd4 Is Required for E2-Mediated Transcriptional Activation but Not Genome Partitioning of All Papillomaviruses†

    PubMed Central

    McPhillips, M. G.; Oliveira, J. G.; Spindler, J. E.; Mitra, R.; McBride, A. A.

    2006-01-01

    Bromodomain protein 4 (Brd4) has been identified as the cellular binding target through which the E2 protein of bovine papillomavirus type 1 links the viral genome to mitotic chromosomes. This tethering ensures retention and efficient partitioning of genomes to daughter cells following cell division. E2 is also a regulator of viral gene expression and a replication factor, in association with the viral E1 protein. In this study, we show that E2 proteins from a wide range of papillomaviruses interact with Brd4, albeit with variations in efficiency. Moreover, disruption of the E2-Brd4 interaction abrogates the transactivation function of E2, indicating that Brd4 is required for E2-mediated transactivation of all papillomaviruses. However, the interaction of E2 and Brd4 is not required for genome partitioning of all papillomaviruses since a number of papillomavirus E2 proteins associate with mitotic chromosomes independently of Brd4 binding. Furthermore, mutations in E2 that disrupt the interaction with Brd4 do not affect the ability of these E2s to associate with chromosomes. Thus, while all papillomaviruses attach their genomes to cellular chromosomes to facilitate genome segregation, they target different cellular binding partners. In summary, the E2 proteins from many papillomaviruses, including the clinically important alpha genus human papillomaviruses, interact with Brd4 to mediate transcriptional activation function but not all depend on this interaction to efficiently associate with mitotic chromosomes. PMID:16973557

  10. pRB and E2F4 play distinct cell-intrinsic roles in fetal erythropoiesis.

    PubMed

    Zhang, Jing; Lee, Eunice Y; Liu, Yangang; Berman, Seth D; Lodish, Harvey F; Lees, Jacqueline A

    2010-01-15

    The retinoblastoma tumor suppressor protein pRB functions, at least in part, by directly binding to and modulating the activity of the E2F transcription factors. Previous studies have shown that both E2F4 and pRB play important roles in fetal erythropoiesis. Given that these two proteins interact directly we investigated the overlap of E2F4 and pRB function in this process by analyzing E2f4(-/-), conditional Rb knockout (Rb(1lox/1lox)), and compound E2f4(-/-);Rb(1lox/1lox) embryos. At E15.5 E2f4(-/-) and Rb(1lox/1lox) fetal erythroid cells display distinct abnormalities in their differentiation profiles. When cultured in vitro, both E2f4(-/-) and Rb(1lox/1lox) erythroid cells show defects in cell cycle progression. Surprisingly, analysis of cell cycle profiling suggests that E2F4 and pRB control cell cycle exit through different mechanisms. Moreover, only pRB, but not E2F4, promotes cell survival in erythroid cells. We observed an additive rather than a synergistic impact upon the erythroid defects in the compound E2f4(-/-);Rb(1lox/1lox) embryos. We further found that fetal liver macrophage development is largely normal regardless of genotype. Taken together, our results show that E2F4 and pRB play independent cell-intrinsic roles in fetal erythropoiesis.

  11. Prostaglandin synthesis and catabolism in the gastric mucosa: studies in normal rabbits and rabbits immunized with prostaglandin E2

    SciTech Connect

    Redfern, J.S.

    1988-09-01

    Antral and fundic mucosal homogenates obtained from prostaglandin E2-immunized rabbits converted 14C-arachidonic acid to prostaglandin E2, 6-keto prostaglandin F1 alpha, prostaglandin F2 alpha, and prostaglandin D2. Percentage conversion of 14C-arachidonic acid to these prostaglandin products was not significantly different in prostaglandin E2-immunized rabbits compared with control rabbits (thyroglobulin-immunized and unimmunized rabbits combined). Synthesis of 6-keto prostaglandin F1 alpha, prostaglandin E2 and 13,14-dihydro 15-keto prostaglandin E2 from endogenous arachidonic acid after vortex mixing fundic mucosal homogenates was similar in prostaglandin E2 immunized rabbits and control rabbits. Both in prostaglandin E2-immunized rabbits and controls, 3H-prostaglandin E2 was catabolized extensively by the fundic mucosa, whereas 3H-6-keto prostaglandin F1 alpha, 3H-prostaglandin F2 alpha, and 3H-prostaglandin D2 were not catabolized to any appreciable extent. The rate of catabolism of PGs was not significantly different in prostaglandin E2-immunized rabbits and control rabbits, with the exception of prostaglandin F2 alpha which was catabolized slightly more rapidly in prostaglandin E2-immunized rabbits. These results indicate that development of gastric ulcers in prostaglandin E2-immunized rabbits is not associated with an alteration in the capacity of the gastric mucosa to synthesize or catabolize prostaglandins.

  12. Brd4 is required for e2-mediated transcriptional activation but not genome partitioning of all papillomaviruses.

    PubMed

    McPhillips, M G; Oliveira, J G; Spindler, J E; Mitra, R; McBride, A A

    2006-10-01

    Bromodomain protein 4 (Brd4) has been identified as the cellular binding target through which the E2 protein of bovine papillomavirus type 1 links the viral genome to mitotic chromosomes. This tethering ensures retention and efficient partitioning of genomes to daughter cells following cell division. E2 is also a regulator of viral gene expression and a replication factor, in association with the viral E1 protein. In this study, we show that E2 proteins from a wide range of papillomaviruses interact with Brd4, albeit with variations in efficiency. Moreover, disruption of the E2-Brd4 interaction abrogates the transactivation function of E2, indicating that Brd4 is required for E2-mediated transactivation of all papillomaviruses. However, the interaction of E2 and Brd4 is not required for genome partitioning of all papillomaviruses since a number of papillomavirus E2 proteins associate with mitotic chromosomes independently of Brd4 binding. Furthermore, mutations in E2 that disrupt the interaction with Brd4 do not affect the ability of these E2s to associate with chromosomes. Thus, while all papillomaviruses attach their genomes to cellular chromosomes to facilitate genome segregation, they target different cellular binding partners. In summary, the E2 proteins from many papillomaviruses, including the clinically important alpha genus human papillomaviruses, interact with Brd4 to mediate transcriptional activation function but not all depend on this interaction to efficiently associate with mitotic chromosomes.

  13. E2F1 plays a direct role in Rb stabilization and p53-independent tumor suppression.

    PubMed

    Palacios, Gustavo; Talos, Flaminia; Nemajerova, Alice; Moll, Ute M; Petrenko, Oleksi

    2008-06-15

    To better understand the role of E2F1 in tumor formation, we analyzed spontaneous tumorigenesis in p53(-/-)E2F1(+/+) and p53(-/-)E2F1(-/-) mice. We show that the combined loss of p53 and E2F1 leads to an increased incidence of sarcomas and carcinomas compared to the loss of p53 alone. E2F1-deficient tumors show wide chromosomal variation, indicative of genomic instability. Consistent with this, p53(-/-)E2F1(-/-) primary fibroblasts have a reduced capacity to maintain genomic stability when exposed to S-phase inhibitors or genotoxic drugs. A major mechanism of E2F1's contribution to genomic integrity lies in mediating stabilization and engagement of the Rb protein.

  14. 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

  15. Dose-dependent regulation of the early promoter of human papillomavirus type 18 by the viral E2 protein.

    PubMed

    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.

  16. pRB-E2F1 complexes are resistant to adenovirus E1A-mediated disruption.

    PubMed

    Seifried, L A; Talluri, S; Cecchini, M; Julian, L M; Mymryk, J S; Dick, F A

    2008-05-01

    Disruption of pRB-E2F interactions by E1A is a key event in the adenoviral life cycle that drives expression of early viral transcription and induces cell cycle progression. This function of E1A is complicated by E2F1, an E2F family member that controls multiple processes besides proliferation, including apoptosis and DNA repair. Recently, a second interaction site in pRB that only contacts E2F1 has been discovered, allowing pRB to control proliferation separately from other E2F1-dependent activities. Based on this new insight into pRB-E2F1 regulation, we investigated how E1A affects control of E2F1 by pRB. Our data reveal that pRB-E2F1 interactions are resistant to E1A-mediated disruption. Using mutant forms of pRB that selectively force E2F1 to bind through only one of the two binding sites on pRB, we determined that E1A is unable to disrupt E2F1's unique interaction with pRB. Furthermore, analysis of pRB-E2F complexes during adenoviral infection reveals the selective maintenance of pRB-E2F1 interactions despite the presence of E1A. Our experiments also demonstrate that E2F1 functions to maintain cell viability in response to E1A expression. This suggests that adenovirus E1A's seemingly complex mechanism of disrupting pRB-E2F interactions provides selectivity in promoting viral transcription and cell cycle advancement, while maintaining cell viability.

  17. Mechanisms by Which 17β-Estradiol (E2) Suppress Neuronal cox-2 Gene Expression

    PubMed Central

    Stacey, Winfred; Bhave, Shreyas; Uht, Rosalie M.

    2016-01-01

    E2 attenuates inflammatory responses by suppressing expression of pro-inflammatory genes. Given that inflammation is increasingly being associated with neurodegenerative and psychiatric processes, we sought to elucidate mechanisms by which E2 down-regulates a component of an inflammatory response, cyclooxygenase– 2 (COX-2) expression. Although inflammatory processes in the brain are usually associated with microglia and astrocytes, we found that the COX-2 gene (cox-2) was expressed in a neuronal context, specifically in an amygdalar cell line (AR-5). Given that COX-2 has been reported to be in neurons in the brain, and that the amygdala is a site involved in neurodegenerative and neuropsychiatric processes, we investigated mechanisms by which E2 could down-regulate cox-2 expression in the AR-5 line. These cells express estrogen receptors alpha (ERα) and beta (ERβ), and as shown here cox-2. At the level of RNA, E2 and the ERβ selective ligand diarylpropionitrile (DPN) both attenuated gene expression, whereas the ERα selective ligand propyl pyrazole triol (PPT) had no effect. Neither ligand increased ERβ at the cox-2 promoter. Rather, DPN decreased promoter occupancy of NF-κB p65 and histone 4 (H4) acetylation. Treatment with the non-specific HDAC inhibitor Trichostatin A (TSA) counteracted DPN’s repressive effects on cox-2 expression. In keeping with the TSA effect, E2 and DPN increased histone deacetylase one (HDAC1) and switch-independent 3A (Sin3A) promoter occupancy. Lastly, even though E2 increased CpG methylation, DPN did not. Taken together, the pharmacological data indicate that ERβ contributes to neuronal cox-2 expression, as measured by RNA levels. Furthermore, ER ligands lead to increased recruitment of HDAC1, Sin3A and a concomitant reduction of p65 occupancy and Ac-H4 levels. None of the events, however, are associated with a significant recruitment of ERβ at the promoter. Thus, ERβ directs recruitment to the cox-2 promoter, but does so in

  18. Mechanisms by Which 17β-Estradiol (E2) Suppress Neuronal cox-2 Gene Expression.

    PubMed

    Stacey, Winfred; Bhave, Shreyas; Uht, Rosalie M

    2016-01-01

    E2 attenuates inflammatory responses by suppressing expression of pro-inflammatory genes. Given that inflammation is increasingly being associated with neurodegenerative and psychiatric processes, we sought to elucidate mechanisms by which E2 down-regulates a component of an inflammatory response, cyclooxygenase- 2 (COX-2) expression. Although inflammatory processes in the brain are usually associated with microglia and astrocytes, we found that the COX-2 gene (cox-2) was expressed in a neuronal context, specifically in an amygdalar cell line (AR-5). Given that COX-2 has been reported to be in neurons in the brain, and that the amygdala is a site involved in neurodegenerative and neuropsychiatric processes, we investigated mechanisms by which E2 could down-regulate cox-2 expression in the AR-5 line. These cells express estrogen receptors alpha (ERα) and beta (ERβ), and as shown here cox-2. At the level of RNA, E2 and the ERβ selective ligand diarylpropionitrile (DPN) both attenuated gene expression, whereas the ERα selective ligand propyl pyrazole triol (PPT) had no effect. Neither ligand increased ERβ at the cox-2 promoter. Rather, DPN decreased promoter occupancy of NF-κB p65 and histone 4 (H4) acetylation. Treatment with the non-specific HDAC inhibitor Trichostatin A (TSA) counteracted DPN's repressive effects on cox-2 expression. In keeping with the TSA effect, E2 and DPN increased histone deacetylase one (HDAC1) and switch-independent 3A (Sin3A) promoter occupancy. Lastly, even though E2 increased CpG methylation, DPN did not. Taken together, the pharmacological data indicate that ERβ contributes to neuronal cox-2 expression, as measured by RNA levels. Furthermore, ER ligands lead to increased recruitment of HDAC1, Sin3A and a concomitant reduction of p65 occupancy and Ac-H4 levels. None of the events, however, are associated with a significant recruitment of ERβ at the promoter. Thus, ERβ directs recruitment to the cox-2 promoter, but does so in the

  19. 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.

  20. E2F1 and p53 Transcription Factors as Accessory Factors for Nucleotide Excision Repair

    PubMed Central

    Vélez-Cruz, Renier; Johnson, David G.

    2012-01-01

    Many of the biochemical details of nucleotide excision repair (NER) have been established using purified proteins and DNA substrates. In cells however, DNA is tightly packaged around histones and other chromatin-associated proteins, which can be an obstacle to efficient repair. Several cooperating mechanisms enhance the efficiency of NER by altering chromatin structure. Interestingly, many of the players involved in modifying chromatin at sites of DNA damage were originally identified as regulators of transcription. These include ATP-dependent chromatin remodelers, histone modifying enzymes and several transcription factors. The p53 and E2F1 transcription factors are well known for their abilities to regulate gene expression in response to DNA damage. This review will highlight the underappreciated, transcription-independent functions of p53 and E2F1 in modifying chromatin structure in response to DNA damage to promote global NER. PMID:23202967

  1. Expression and Purification of E2 Glycoprotein from Insect Cells (Sf9) for Use in Serology.

    PubMed

    Chua, Chong Long; Sam, I-Ching; Chan, Yoke Fun

    2016-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which poses a major threat to global public health. Definitive CHIKV diagnosis is crucial, especially in distinguishing the disease from dengue virus, which co-circulates in endemic areas and shares the same mosquito vectors. Laboratory diagnosis is mainly based on serological or molecular approaches. The E2 glycoprotein is a good candidate for serological diagnosis since it is the immunodominant antigen during the course of infection, and reacts with seropositive CHIKV sera. In this chapter, we describe the generation of stable clone Sf9 (Spodoptera frugiperda) cells expressing secreted, soluble, and native recombinant CHIKV E2 glycoprotein. We use direct plasmid expression in insect cells, rather than the traditional technique of generating recombinant baculovirus. This recombinant protein is useful for serological diagnosis of CHIKV infection. PMID:27233260

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. Electrophile-Modified Lipoic Derivatives of PDC-E2 Elicits Anti-mitochondrial Antibody Reactivity

    PubMed Central

    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.

    2011-01-01

    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 antimitochondrial 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 lipoyl1oiety 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

  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. 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.

  9. Draft Genome Sequence of Brazilian Escherichia coli Uropathogenic Strain E2

    PubMed Central

    Pieta, Luiza; Prichula, Janira; Sambrano, Gustavo E.; Soares, Renata; Caierão, Juliana; Frazzon, Jeverson; d’Azevedo, Pedro A.

    2016-01-01

    Escherichia coli is a common pathogen recovered from cystitis infections. In this report, we announce the draft genome sequence of strain E2 isolated from the urine specimen from a female patient in South Brazil. The genome assembly has 5,081,209 bp, a G+C content of 50.57%, and virulence factors associated with both enteroaggregative and uropathogenic E. coli strains. PMID:27795253

  10. Oral E2 prostaglandins affect endocrine cell populations in the gastric antrum of the rat.

    PubMed

    Uribe, A; Grimelius, L; Theodorsson, L E; Riis-Angelo, L; Johansson, C

    1989-04-01

    The aim of the present study was to investigate antral endocrine cell populations and tissue and circulating hormone levels following a 4-week oral regimen with prostaglandin E2 (25, 250 and 5000 micrograms/kg-1 b.i.d.) or a stable methyl analogue (5 and 50 micrograms kg-1 b.i.d.). Epithelial hyperplasia of the gastric antrum was observed with the highest dose of prostaglandin E2 and both doses of the analogue, as evaluated by stereological methods and conventional cell count. The treatments significantly affected the endocrine cell population. Somatostatin-immunoreactive cells were increased in proportion to the increased epithelial cellularity and plasma levels of somatostatin were increased in parallel. The tissue content of somatostatin-like immunoreactivity differed according to the extraction procedure used, and was significantly higher than controls in specimens extracted in neutral water. In the neutral extracts an immunoreactive somatostatin of unidentified molecular structure dominated quantitatively over somatostatin 14 and 28, which were the major components in acetic acid extracts. The serotonin-immunoreactive cell population was also significantly increased by natural prostaglandin E2 and the analogue but the gastrin cell population was not significantly affected by treatments. Accordingly, no significant changes were observed in tissue or plasma gastrin levels. It is concluded that the epithelial hyperplasia of the antral epithelia produced by E2 prostaglandins is associated with selective changes of endocrine cell populations. The changes were proportional to the increases of epithelial cellularity and required quantitative determination of the total antral volume to be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. 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

  12. DNA-damage-responsive acetylation of pRb regulates binding to E2F-1

    PubMed Central

    Markham, Douglas; Munro, Shonagh; Soloway, Judith; O'Connor, Darran P; La Thangue, Nicholas B

    2006-01-01

    The pRb (retinoblastoma protein) tumour suppressor protein has a crucial role in regulating the G1- to S-phase transition, and its phosphorylation by cyclin-dependent kinases is an established and important mechanism in controlling pRb activity. In addition, the targeted acetylation of lysine (K) residues 873/874 in the carboxy-terminal region of pRb located within a cyclin-dependent kinase-docking site hinders pRb phosphorylation and thereby retains pRb in an active state of growth suppression. Here, we report that the acetylation of pRb K873/874 occurs in response to DNA damage and that acetylation regulates the interaction between the C-terminal E2F-1-specific domain of pRb and E2F-1. These results define a new role for pRb acetylation in the DNA damage signalling pathway, and suggest that the interaction between pRb and E2F-1 is controlled by DNA-damage-dependent acetylation of pRb. PMID:16374512

  13. 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. PMID:27242526

  14. E2-2 Dependent Plasmacytoid Dendritic Cells Control Autoimmune Diabetes.

    PubMed

    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.

  15. 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

  16. 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.

  17. Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness

    PubMed Central

    Raimundo, Nuno; Song, Lei; Shutt, Timothy E.; McKay, Sharen E.; Cotney, Justin; Guan, Min-Xin; Gilliland, Thomas C.; Hohuan, David; Santos-Sacchi, Joseph; Shadel, Gerald S.

    2012-01-01

    SUMMARY Mitochondrial dysfunction causes poorly understood tissue-specific pathology stemming from primary defects in respiration, coupled with altered reactive oxygen species (ROS), metabolic signaling and apoptosis. The A1555G mtDNA mutation that causes maternally inherited deafness disrupts mitochondrial ribosome function, in part, via increased methylation of the mitochondrial 12S rRNA by the methyltransferase mtTFB1. In patient-derived A1555G cells, we show that 12S rRNA hyper-methylation causes ROS-dependent activation of AMP kinase and the pro-apoptotic nuclear transcription factor E2F1. This retrograde mitochondrial-stress relay is operative in vivo as transgenic-mtTFB1 mice exhibit enhanced 12S rRNA methylation in multiple tissues, increased E2F1 and apoptosis in the stria vascularis and spiral ganglion neurons of the inner ear, and progressive E2F1-dependent hearing loss. This transgenic-mtTFB1 mouse mitochondrial disease model provides a robust platform for deciphering the complex tissue-specificity of human mitochondrial-based disorders, as well as the precise pathogenic mechanism of maternally inherited deafness and its exacerbation by environmental factors. PMID:22341444

  18. 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

  19. 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; Pérez García-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.

  20. 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.

  1. 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.

  2. Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway.

    PubMed

    Yunus, Ali A; Lima, Christopher D

    2006-06-01

    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. PMID:16732283

  3. 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.

  4. 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.

  5. E2 Regulates Epigenetic Signature on Neuroglobin Enhancer-Promoter in Neuronal Cells

    PubMed Central

    Guglielmotto, Michela; Reineri, Stefania; Iannello, Andrea; Ferrero, Giulio; Vanzan, Ludovica; Miano, Valentina; Ricci, Laura; Tamagno, Elena; De Bortoli, Michele; Cutrupi, Santina

    2016-01-01

    Estrogens are neuroprotective factors in several neurological diseases. Neuroglobin (NGB) is one of the estrogen target genes involved in neuroprotection, but little is known about its transcriptional regulation. Estrogen genomic pathway in gene expression regulation is mediated by estrogen receptors (ERα and ERβ) that bind to specific regulatory genomic regions. We focused our attention on 17β-estradiol (E2)-induced NGB expression in human differentiated neuronal cell lines (SK-N-BE and NT-2). Previously, using bioinformatics analysis we identified a putative enhancer in the first intron of NGB locus. Therefore, we observed that E2 increased the enrichment of the H3K4me3 epigenetic marks at the promoter and of the H3K4me1 and H3K27Ac at the intron enhancer. In these NGB regulatory regions, we found estrogen receptor alpha (ERα) binding suggesting that ERα may mediate chromatin remodeling to induce NGB expression upon E2 treatment. Altogether our data show that NGB expression is regulated by ERα binding on genomic regulatory regions supporting hormone therapy applications for the neuroprotection against neurodegenerative diseases. PMID:27313512

  6. Neddylation requires glycyl-tRNA synthetase to protect activated E2.

    PubMed

    Mo, Zhongying; Zhang, Qian; Liu, Ze; Lauer, Janelle; Shi, Yi; Sun, Litao; Griffin, Patrick R; Yang, Xiang-Lei

    2016-08-01

    Neddylation is a post-translational modification that controls the cell cycle and proliferation by conjugating the ubiquitin-like protein NEDD8 to specific targets. Here we report that glycyl-tRNA synthetase (GlyRS), an essential enzyme in protein synthesis, also plays a critical role in neddylation. In human cells, knockdown of GlyRS, but not knockdown of a different tRNA synthetase, decreased the global level of neddylation and caused cell-cycle abnormality. This function of GlyRS is achieved through direct interactions with multiple components of the neddylation pathway, including NEDD8, E1, and E2 (Ubc12). Using various structural and functional approaches, we show that GlyRS binds the APPBP1 subunit of E1 and captures and protects activated E2 (NEDD8-conjugated Ubc12) before the activated E2 reaches a downstream target. Therefore, GlyRS functions as a chaperone that critically supports neddylation. This function is probably conserved in all eukaryotic GlyRS enzymes and may contribute to the strong association of GlyRS with cancer progression. PMID:27348078

  7. 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.

  8. Identification of ligands that target the HCV-E2 binding site on CD81.

    PubMed

    Olaby, Reem Al; Azzazy, Hassan M; Harris, Rodney; Chromy, Brett; Vielmetter, Jost; Balhorn, Rod

    2013-04-01

    Hepatitis C is a global health problem. While many drug companies have active R&D efforts to develop new drugs for treating Hepatitis C virus (HCV), most target the viral enzymes. The HCV glycoprotein E2 has been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. This paper describes the use of AutoDock to identify ligand binding sites on the large extracellular loop of the open conformation of CD81 and to perform virtual screening runs to identify sets of small molecule ligands predicted to bind to two of these sites. The best sites selected by AutoLigand were located in regions identified by mutational studies to be the site of E2 binding. Thirty-six ligands predicted by AutoDock to bind to these sites were subsequently tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 26 out of 36 (72 %) of the ligands bound in vitro to the recombinant CD81-LEL protein. Competition experiments performed using dual polarization interferometry showed that one of the ligands predicted to bind to the large cleft between the C and D helices was also effective in blocking E2 binding to CD81-LEL.

  9. 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.

  10. Multiple E2F-Induced MicroRNAs Prevent Replicative Stress in Response to Mitogenic Signaling▿ †

    PubMed Central

    Bueno, María J.; Gómez de Cedrón, Marta; Laresgoiti, Usua; Fernández-Piqueras, José; Zubiaga, Ana M.; Malumbres, Marcos

    2010-01-01

    Transcription of microRNAs (miRNAs) is thought to be regulated similarly to that of protein-coding genes. However, how miRNAs are regulated during the cell division cycle is not well understood. We have analyzed the transcription profiles of miRNAs in response to mitogenic stimulation in primary fibroblasts. About 33% of the miRNAs expressed in these cells are induced upon exit from quiescence. Many of these miRNAs are specifically induced by E2F1 or E2F3 during the G1/S transition and are repressed in E2F1/3-knockout cells. At least four miRNA clusters, let-7a-d, let-7i, mir-15b-16-2, and mir-106b-25, are direct targets of E2F1 and E2F3 during G1/S and are repressed in E2F1/3-null cells. Interestingly, these miRNAs do not contribute to E2F-dependent entry into S phase but rather inhibit the G1/S transition by targeting multiple cell cycle regulators and E2F targets. In fact, E2F1 expression results in a significant increase in S-phase entry and DNA damage in the absence of these microRNAs. Thus, E2F-induced miRNAs contribute to limiting the cellular responses to E2F activation, thus preventing replicative stress. Given the known function of E2F of inducing other oncogenic miRNAs, control of miRNAs by E2F is likely to play multiple roles in cell proliferation and in proliferative diseases such as cancer. PMID:20404092

  11. Human Herpesvirus 6A (HHV-6A) and HHV-6B Alter E2F1/Rb Pathways and E2F1 Localization and Cause Cell Cycle Arrest in Infected T Cells▿

    PubMed Central

    Mlechkovich, Guy; Frenkel, Niza

    2007-01-01

    E2F transcription factors play pivotal roles in controlling the expression of genes involved in cell viability as well as genes involved in cell death. E2F1 is an important constituent of this protein family, which thus far contains eight members. The interaction of E2F1 with its major regulator, retinoblastoma protein (Rb), has been studied extensively in the past two decades, concentrating on the role of E2F1 in transcriptional regulation and the role of Rb in cell replication and cancer formation. Additionally, the effect of viral infections on E2F1/Rb interactions has been analyzed for different viruses, concentrating on cell division, which is essential for viral replication. In the present study, we monitored E2F1-Rb interactions during human herpesvirus 6A (HHV-6A) and HHV-6B infections of SupT1 T cells. The results have shown the following dramatic alterations in E2F1-Rb pathways compared to the pathways of parallel mock-infected control cultures. (i) The E2F1 levels were elevated during viral infections. (ii) The cellular localization of E2F1 was dramatically altered, and it was found to accumulate both in the cytoplasmic and nuclear fractions, as opposed to the strict nuclear localization seen in the mock-infected cells. (iii) Although E2F1 expression was elevated, two exemplary target genes, cyclin E and MCM5, were not upregulated. (iv) The Rb protein was dephosphorylated early postinfection, a trait that also occurred with UV-inactivated virus. (v) Infection was associated with significant reduction of E2F1/Rb complexing. (vi) HHV-6 infections were accompanied by cell cycle arrest. The altered E2F1-Rb interactions and functions might contribute to the observed cell cycle arrest. PMID:17913805

  12. 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.

  13. 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

  14. Involvement of atypical transcription factor E2F8 in the polyploidization during mouse and human decidualization

    PubMed Central

    Qi, Qian-Rong; Zhao, Xu-Yu; Zuo, Ru-Juan; Wang, Tong-Song; Gu, Xiao-Wei; Liu, Ji-Long; Yang, Zeng-Ming

    2015-01-01

    Polyploid decidual cells are specifically differentiated cells during mouse uterine decidualization. However, little is known about the regulatory mechanism and physiological significance of polyploidization in pregnancy. Here we report a novel role of E2F8 in the polyploidization of decidual cells in mice. E2F8 is highly expressed in decidual cells and regulated by progesterone through HB-EGF/EGFR/ERK/STAT3 signaling pathway. E2F8 transcriptionally suppresses CDK1, thus triggering the polyploidization of decidual cells. E2F8-mediated polyploidization is a response to stresses which are accompanied by decidualization. Interestingly, polyploidization is not detected during human decidualization with the down-regulation of E2F8, indicating differential expression of E2F8 may lead to the difference of decidual cell polyploidization between mice and humans. PMID:25892397

  15. 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.

  16. Papillomavirus E2 Proteins and the Host Brd4 Protein Associate with Transcriptionally Active Cellular Chromatin▿ †

    PubMed Central

    Jang, Moon Kyoo; Kwon, Deukwoo; McBride, Alison A.

    2009-01-01

    The interaction of papillomavirus E2 proteins with cellular Brd4 protein is important for transcriptional regulation of viral genes and partitioning of viral genomes. Bovine papillomavirus type 1 (BPV-1) E2 binds cellular chromatin in complex with Brd4 in both mitotic and interphase cells. To identify specific sites of E2 interaction on cellular chromatin, a genome-wide chromatin immunoprecipitation-on-chip analysis was carried out using human promoter sequences. Both E2 and Brd4 were found bound to most transcriptionally active promoters in C33A cells. These promoters were also bound by RNA polymerase II and were modified by histone H3 acetylation and K4 trimethylation, all indicators of active transcription. E2 binding strongly correlated with Brd4 and RNA polymerase II occupancy and H3K4me3 modification at all human promoters, indicating that E2 bound to active promoters. E2 binding did not correlate with the presence of consensus E2 binding sites in the promoters. Furthermore, the mRNA levels of E2-bound cellular genes were not significantly changed by E2 expression. Thus, the papillomavirus E2 proteins bind to transcriptionally active cellular genes but do not change their activity. We propose that this may be a way for the virus to ensure that the viral genome is retained in transcriptionally active regions of the nucleus to escape silencing. Therefore, E2-mediated tethering of viral genomes to host chromatin has multiple roles: to partition the viral genome to daughter cells, to ensure that the genomes are retained in the nucleus, and to make certain that the genomes are retained in functionally active nuclear domains. PMID:19129460

  17. [Production of miniantibodies to Agrobacterium tumefaciens VirE2 virulence protein by the method of phage display].

    PubMed

    Velikov, V A; Ermoshina, O S; Volokhina, I V; Chumakov, M I

    2006-01-01

    The scFv miniantibodies to the recombinant protein VirE2 from Agrobacterium tumefaciens were obtained by the method of phage display. The miniantibodies were purified and tested using timmunodot method for binding to a recombinant protein from Escherichia coli and to the native protein VirE2 from A. tumefaciens. The functional activity of the miniantibodies was comparable to the activity of mouse polyclonal antibodies against the VirE2 protein. PMID:16512606

  18. 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.

  19. Disruption of pubertal onset by exogenous testosterone and estrogen in two species of rodents.

    PubMed

    Pak, Toni R; Lynch, G Robert; Ziegler, D Matthew; Lunden, Jason B; Tsai, Pei-San

    2003-01-01

    The administration of adult physiological levels of testosterone (T) and 17beta-estradiol (E(2)) to male Siberian hamsters was previously shown to delay the onset of puberty. To examine whether this is a response common to other rodents, we investigated whether exogenous steroids also alter the onset of puberty in Syrian hamsters and mice. Juvenile male Syrian hamsters and mice were implanted with Silastic capsules containing T, E(2), or cholesterol control. After 15 days, plasma, pituitaries, and testes were processed for histological analysis or measurements of gonadotropins and circulating steroid hormones. T and E(2) implants reduced testis mass and gonadotropin stores in both species and arrested spermatogenesis in Syrian hamsters. In contrast, spermatogenesis in mice was unaffected by T and only modestly affected by E(2). Although E(2) inhibited circulating follicle-stimulating hormone (FSH) in both species, T inhibited circulating FSH in mice only. Overall, our results demonstrate that the hypothalamic-pituitary-gonadal axis of each rodent species responds uniquely to T and E(2) during the pubertal transition. Despite the highly varied effects of T and E(2) in these two species, the ability of steroid hormones to disrupt the onset of puberty appears to be a feature common to many rodents.

  20. Glycoprotein E2 of classical swine fever virus expressed by baculovirus induces the protective immune responses in rabbits.

    PubMed

    Zhang, Huawei; Li, Xiangmin; Peng, Guiqing; Tang, Chenkai; Zhu, Shixuan; Qian, Suhong; Xu, Jinfang; Qian, Ping

    2014-11-20

    Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious and devastating disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. Several CSFV genotypes, including 1.1, 2.1, 2.2, and 2.3, have been identified in Mainland China. The glycoprotein E2 of genotypes 1.1 and 2.1 was expressed by using a baculovirus system and tested for its protective immunity in rabbits to develop novel CSF vaccines that elicit a broad immune response. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with E2 of genotypes 1.1 (CSFV-1.1E2), 2.1 (CSFV-2.1E2), or their combination (CSFV-1.1 + 2.1E2). A commercial CSF vaccine (C-strain) and phosphate-buffered saline (PBS) were used as positive or negative controls, respectively. All animals were challenged with CSFV C-strain at 4 weeks and then boosted with the same dose. All rabbits inoculated with CSFV-1.1E2, CSFV-2.1E2, and CSFV-1.1 + 2.1E2 elicited high levels of ELISA antibody, neutralizing antibody, and lymphocyte proliferative responses to CSFV. The rabbits inoculated with CSFV-1.1E2 and CSFV-1.1 + 2.1E2 received complete protection against CSFV C-strain. Two of the four rabbits vaccinated with CSFV-2.1E2 were completely protected. These results demonstrate that CSFV-1.1E2 and CSFV-1.1 + 2.1E2 not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits. Therefore, CSFV-1.1E2 and CSFV-1.1 + 2.1E2 are promising candidate subunit vaccines against CSF.

  1. E2F1 Hinders Skin Wound Healing by Repressing Vascular Endothelial Growth Factor (VEGF) Expression, Neovascularization, and Macrophage Recruitment

    PubMed Central

    Zeng, Ning; Wang, Haiping; Deng, Pei; Xu, Yi; Feng, Youping; Zeng, Hong; Yang, Hongxia; Hou, Kai; Wang, Andrew; Parthasarathy, Keshav; Goyal, Samaksh; Qin, Gangjian; Wu, Min

    2016-01-01

    Background Refractory surface of wound and dermal chronic ulcer are largely attributed to poor neovascularization. We have previously shown that E2F1 suppresses VEGF expression in the ischemic heart, and that genetic deletion of E2F1 leads to better cardiac recovery. However, whether E2F1 has a role in dermal wound healing is currently not known. Methods and Results Skin wounds were surgically induced in E2F1-null (E2F1–/–) mice and WT littermates. E2F1–/– displayed an accelerated wound healing including wound closure, dermal thickening and collagen deposition, which was associated with an increased endothelial cell proliferation and greater vessel density in the border zone of the wound. Furthermore, more macrophages were recruited to the skin lesions and the level of VEGF expression was markedly higher in E2F1–/– than in WT mice. Conclusions E2F1 hinders skin wound healing by suppressing VEGF expression, neovascularization, and macrophage recruitment. Strategies that target E2F1 may enhance wound healing. PMID:27490344

  2. Involvement of the CDK2-E2F1 pathway in cisplatin cytotoxicity in vitro and in vivo.

    PubMed

    Yu, Fang; Megyesi, Judit; Safirstein, Robert L; Price, Peter M

    2007-07-01

    E2F1 is a key regulator that links cell cycle progression and cell death. E2F1 activity is controlled by Cdk2-cyclin complexes via several mechanisms, such as phosphorylation of retinoblastoma protein (pRb) to release E2F1, direct phosphorylation, and stable physical interaction. We have demonstrated that cisplatin cytotoxicity depends on Cdk2 activity, and Cdk2 inhibition protects kidney cells from cisplatin-induced cell death in vitro and in vivo. Now we show that E2F1 is an important downstream effector of Cdk2 that accumulates in mouse kidneys and in cultured mouse proximal tubular cells (TKPTS) after cisplatin exposure by a Cdk2-dependent mechanism. Direct inhibition of E2F1 by transduction with adenoviruses expressing an E2F1-binding protein (TopBP1) protected TKPTS cells from cisplatin-induced apoptosis, whereas overexpression of E2F1 caused cell death. Moreover, E2F1 knockout mice were markedly protected against cisplatin nephrotoxicity by both functional and histological criteria. Collectively, cisplatin-induced cell death is dependent on Cdk2 activity, which is at least partly through the Cdk2-E2F1 pathway both in vitro and in vivo.

  3. Expression of the surface glycoprotein E2 of Bovine viral diarrhea virus by recombinant vesicular stomatitis virus.

    PubMed

    Köhl, Wiebke; Gröne, Andrea; Moennig, Volker; Herrler, Georg

    2007-01-01

    This study analysed the transport behaviour of the glycoprotein E2 of Bovine viral diarrhea virus (BVDV) expressed from recombinant vesicular stomatitis virus (rVSV). E2 protein was found to be retained at an intracellular compartment. A chimeric protein containing the membrane anchor and cytoplasmic tail of the VSV G protein, E2-G(MT), was transported to the cell surface. Only the latter protein was incorporated into rVSV particles in significant amounts. A soluble form of E2 lacking the membrane anchor, E2(MTdel), appeared to be affected in conformational stability. In contrast to both membrane-anchored forms of E2, expression of the soluble form was detectable only by immunofluorescence microscopy but not by Western blotting. These results are in agreement with reports of intracellular retention of the E2 protein due to a retention signal in the membrane anchor. However, in another analysis of E2 expressed from rVSV, E2 protein was reported to be transported to the cell surface and incorporated into VSV particles [Grigera, P. R., Marzocca, M. P., Capozzo, A. V. E., Buonocore, L., Donis, R. O. & Rose, J. K. (2000). Virus Res 69, 3-15]. Reasons for these contradictory results are discussed. PMID:17170448

  4. Berberine, an isoquinoline alkaloid, inhibits melanoma cancer cell migration by reducing the expressions of cyclooxygenase-2, prostaglandin E2 and prostaglandin E2 receptors

    PubMed Central

    Singh, Tripti; Vaid, Mudit; Katiyar, Nandan; Sharma, Samriti; Katiyar, Santosh K.

    2011-01-01

    Melanoma is the leading cause of death from skin disease due, in large part, to its propensity to metastasize. We have examined the effect of berberine, an isoquinoline alkaloid, on human melanoma cancer cell migration and the molecular mechanisms underlying these effects using melanoma cell lines, A375 and Hs294. Using an in vitro cell migration assay, we show that over expression of cyclooxygenase (COX)-2, its metabolite prostaglandin E2 (PGE2) and PGE2 receptors promote the migration of cells. We found that treatment of A375 and Hs294 cells with berberine resulted in concentration-dependent inhibition of migration of these cells, which was associated with a reduction in the levels of COX-2, PGE2 and PGE2 receptors (EP2 and EP4). Treatment of cells with celecoxib, a COX-2 inhibitor, or transient transfection of cells with COX-2 small interfering RNA, also inhibited cell migration. Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of COX-2 or PGE2, enhanced cell migration, whereas berberine inhibited TPA- or PGE2-promoted cell migration. Berberine reduced the basal levels as well as PGE2-stimulated expression levels of EP2 and EP4. Treatment of the cells with the EP4 agonist stimulated cell migration and berberine blocked EP4 agonist-induced cell migration activity. Moreover, berberine inhibited the activation of nuclear factor-kappa B (NF-κB), an upstream regulator of COX-2, in A375 cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, inhibited cell migration. Together, these results indicate for the first time that berberine inhibits melanoma cell migration, an essential step in invasion and metastasis, by inhibition of COX-2, PGE2 and PGE2 receptors. PMID:20974686

  5. 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.

  6. 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

  7. Carbohydrate Structure of Sindbis Virus Glycoprotein E2 from Virus Grown in Hamster and Chicken Cells

    PubMed Central

    Burke, David; Keegstra, Kenneth

    1979-01-01

    Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N-acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two residues of sialic acid, whereas glycopeptides S2 and S3 contain 1 and 0 residues of sialic acid, respectively. The smallest glycopeptide, S4, contains only N-acetyglucosamine and mannose, and is also similar to mannose-rich oligosaccharides found on other glycoproteins. Each of the complex glycopeptides (S1, S2, or S3) from virus grown in BHK cells is indistinguishable from the corresponding glycopeptides derived from virus grown in chicken cells. Glycopeptide S4 is also very similar in size, composition, and sugar linkages from virus derived from the two hosts. These results suggest that chicken cells and BHK cells have similar glycosylation mechanisms and glycosylate Sindbis glycoprotein E2 in nearly identical ways. PMID:430605

  8. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

    PubMed Central

    Qiu, Jiazhang; Sheedlo, Michael J.; Yu, Kaiwen; Tan, Yunhao; Nakayasu, Ernesto S.; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

    2016-01-01

    Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. 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 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or as enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalyzed by these enzymes is energized by NAD which activates ubiquitin by the formation of ADP-ribosylated ubiquitin (ADPR-Ub). These results establish that ubiquitination can be catalyzed by a single enzyme whose activity does not require ATP. PMID:27049943

  9. The Spt-Ada-Gcn5-acetyltransferase complex interaction motif of E2a is essential for a subset of transcriptional and oncogenic properties of E2a-Pbx1.

    PubMed

    Scheele, Jürgen S; Kolanczyk, Mateusz; Gantert, Melanie; Zemojtel, Tomasz; Dorn, Annette; Sykes, David B; Sykes, David P; Möbest, Dietrich C C; Kamps, Mark P; Räpple, Daniel; Duchniewicz, Marlena

    2009-05-01

    The oncogene E2a-Pbx1 is formed by the t(1;19) translocation, which joins the N-terminal transactivation domain of E2a with the C-terminal homeodomain of PBX1. The goal of this work was to elucidate the mechanisms by which E2a-Pbx1 can lead to deregulated target gene expression. For reporter constructs it was shown that E2a-Pbx1 can activate transcription through homodimer elements (TGATTGAT) or through heterodimer elements with Hox proteins (e.g. TGATTAAT). We show a novel mechanism by which E2a-Pbx1 activates transcription of EF-9 using a promoter in intron 1 of the EF-9 gene, resulting in an aminoterminal truncated transcript. Our results indicate that the LDFS motif of E2a is essential for the transactivation of EF-9, but dispensable for transactivation of fibroblast growth factor 15. The E2a LDFS motif was also essential for proliferation of NIH3T3 fibroblasts but was dispensable for the E2a-Pbx1-induced differentiation arrest of myeloid progenitors.

  10. Interband B (E2) ratios in the rigid triaxial model, a review

    NASA Astrophysics Data System (ADS)

    Gupta, J. B.; Sharma, S.

    1989-01-01

    Uptodate accurate extensive data on γ-g B(E2) ratios for even-even rare-earth nuclei is compared with the predictions of the rigid triaxial model of collective rotation to search for a correlation between the nuclear structure variation with Z, N and the γ0 parameter of the model. The internal consistency in the predictions of the model is investigated and the spectral features vis-a-vis the γ-soft and the γ-rigid potential are discussed.

  11. 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

  12. 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

  13. Future climate change under RCP emission scenarios with GISS ModelE2

    DOE PAGES

    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

  14. Measurement of E2 transition strengths in {sup 32,34}Mg

    SciTech Connect

    Church, J.A.; Campbell, C.M.; Dinca, D.-C.; Glasmacher, T.; Olliver, H.; Perry, B.C.; Yurkewicz, K.L.; Enders, J.; Gade, A.; Hu, Z.; Mueller, W.F.; Janssens, R.V.F.; Riley, L.A.

    2005-11-01

    The degree of collectivity in the neutron-rich nuclei {sup 32}Mg and {sup 34}Mg has been determined via intermediate-energy Coulomb excitation in inverse kinematics. Measured energies of the first excited 2{sup +} states and reduced electric quadrupole transition probabilities B(E2;0{sub g.s.}{sup +}{yields}2{sub 1}{sup +}) are presented for {sup 32}Mg and {sup 34}Mg. The results agree with previous measurements and confirm the placement of both nuclei within the ''island of inversion.''.

  15. The influence of pre-induction vaginal prostaglandin E2 gel upon subsequent labour.

    PubMed

    Mackenzie, I Z; Embrey, M P

    1978-09-01

    Eight hundred and three patients with singleton viable pregnancies and fetal cephalic presentation were given prostaglandin E2 in viscous gel by intravaginal instillation the evening before planned surgical induction. When the cervix was 'ripe', surgical induction was avoided in 65.9 per cent of primigravidae and 87.5 per cent of multigravidae; the administration of epidural analgesia was less frequent, the rate of spontaneous vaginal delivery greater, the Caesarean section rate lower, and the state of the newborn at delivery better than in those patients who required surgical induction. Side effects attributable to the prostaglandin gel were rare as were complications.

  16. 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.

  17. A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E2 synthase

    PubMed Central

    Brock, Joseph S.; Hamberg, Mats; Balagunaseelan, Navisraj; Goodman, Michael; Morgenstern, Ralf; Strandback, Emilia; Samuelsson, Bengt; Rinaldo-Matthis, Agnes; Haeggström, Jesper Z.

    2016-01-01

    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 human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126–Gln, Asp-49–Asn, and Arg-126–Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents. PMID:26755582

  18. Global Carbon Cycle Modeling in GISS ModelE2 GCM

    NASA Astrophysics Data System (ADS)

    Aleinov, I. D.; Kiang, N. Y.; Romanou, A.; Romanski, J.

    2014-12-01

    Consistent and accurate modeling of the Global Carbon Cycle remains one of the main challenges for the Earth System Models. NASA Goddard Institute for Space Studies (GISS) ModelE2 General Circulation Model (GCM) was recently equipped with a complete Global Carbon Cycle algorithm, consisting of three integrated components: Ent Terrestrial Biosphere Model (Ent TBM), Ocean Biogeochemistry Module and atmospheric CO2 tracer. Ent TBM provides CO2 fluxes from the land surface to the atmosphere. Its biophysics utilizes the well-known photosynthesis functions of Farqhuar, von Caemmerer, and Berry and Farqhuar and von Caemmerer, and stomatal conductance of Ball and Berry. Its phenology is based on temperature, drought, and radiation fluxes, and growth is controlled via allocation of carbon from labile carbohydrate reserve storage to different plant components. Soil biogeochemistry is based on the Carnegie-Ames-Stanford (CASA) model of Potter et al. Ocean biogeochemistry module (the NASA Ocean Biogeochemistry Model, NOBM), computes prognostic distributions for biotic and abiotic fields that influence the air-sea flux of CO2 and the deep ocean carbon transport and storage. Atmospheric CO2 is advected with a quadratic upstream algorithm implemented in atmospheric part of ModelE2. Here we present the results for pre-industrial equilibrium and modern transient simulations and provide comparison to available observations. We also discuss the process of validation and tuning of particular algorithms used in the model.

  19. A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

    PubMed

    Mulder, Monique P C; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C O; Schulman, Brenda A; Komander, David; Neefjes, Jacques; El Oualid, Farid; Ovaa, Huib

    2016-07-01

    Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades. PMID:27182664

  20. 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 human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents.

  1. Domain structure and reorientation in CoF e2O4

    NASA Astrophysics Data System (ADS)

    Abes, M.; Koops, C. T.; Hrkac, S. B.; McCord, J.; Urs, N. O.; Wolff, N.; Kienle, L.; Ren, W. J.; Bouchenoire, L.; Murphy, B. M.; Magnussen, O. M.

    2016-05-01

    The microscopic processes underlying magnetostriction in ferrites were studied for the case of CoF e2O4 single crystals by high-resolution in situ x-ray diffraction and complementary magnetic microscopy techniques. The data support the reports of Yang and Ren [Phys. Rev. B 77, 014407 (2008), 10.1103/PhysRevB.77.014407] that magnetostriction in these materials originates from the switching of crystallographic domains, similar to ferroelastic or ferroelectric domain switching, and reveals the presence of two coexisting tetragonal spinel structures, corresponding to domains of high and of low strain. The latter alternate in the crystal, separated by 90° domain boundaries, and can be explained by the effect of internal stress emerging during the transition into the ferrimagnetic phase. During magnetization of the sample two structural transitions are observed: a conversion of the transversal into axial domains at 1.95 kOe and a growth of the high-strain domains at the cost of the low-strain axial domains at 2.8 kOe. These microscopic changes are in good agreement with the macroscopic magnetization and magnetostriction behavior of CoF e2O4 .

  2. 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.

  3. E2 transition probabilities for decays of isomers observed in neutron-rich odd Sn isotopes

    DOE PAGES

    Iskra, Ł. W.; Broda, R.; Janssens, R. V.F.; Wrzesiński, J.; Chiara, C. J.; Carpenter, M. P.; Fornal, B.; Hoteling, N.; Kondev, F. G.; Królas, W.; et al

    2015-01-01

    High-spin states were investigated with gamma coincidence techniques in neutron-rich Sn isotopes produced in fission processes following ⁴⁸Ca + ²⁰⁸Pb, ⁴⁸Ca + ²³⁸U, and ⁶⁴Ni + ²³⁸U reactions. By exploiting delayed and cross-coincidence techniques, level schemes have been delineated in odd ¹¹⁹⁻¹²⁵Sn isotopes. Particular attention was paid to the occurrence of 19/2⁺ and 23/2⁺ isomeric states for which the available information has now been significantly extended. Reduced transition probabilities, B(E2), extracted from the measured half-lives and the established details of the isomeric decays exhibit a striking regularity. This behavior was compared with the previously observed regularity of the B(E2) amplitudesmore » for the seniority ν = 2 and 3, 10⁺ and 27/2⁻ isomers in even- and odd-Sn isotopes, respectively.« less

  4. E2 transition probabilities for decays of isomers observed in neutron-rich odd Sn isotopes

    SciTech Connect

    Iskra, Ł. W.; Broda, R.; Janssens, R. V.F.; Wrzesiński, J.; Chiara, C. J.; Carpenter, M. P.; Fornal, B.; Hoteling, N.; Kondev, F. G.; Królas, W.; Lauritsen, T.; Pawłat, T.; Seweryniak, D.; Stefanescu, I.; Walters, W. B.; Zhu, S.

    2015-01-01

    High-spin states were investigated with gamma coincidence techniques in neutron-rich Sn isotopes produced in fission processes following ⁴⁸Ca + ²⁰⁸Pb, ⁴⁸Ca + ²³⁸U, and ⁶⁴Ni + ²³⁸U reactions. By exploiting delayed and cross-coincidence techniques, level schemes have been delineated in odd ¹¹⁹⁻¹²⁵Sn isotopes. Particular attention was paid to the occurrence of 19/2⁺ and 23/2⁺ isomeric states for which the available information has now been significantly extended. Reduced transition probabilities, B(E2), extracted from the measured half-lives and the established details of the isomeric decays exhibit a striking regularity. This behavior was compared with the previously observed regularity of the B(E2) amplitudes for the seniority ν = 2 and 3, 10⁺ and 27/2⁻ isomers in even- and odd-Sn isotopes, respectively.

  5. The identification of prostaglandins E2, F2α and A2 from rabbit kidney medulla

    PubMed Central

    Lee, J. B.; Crowshaw, K.; Takman, B. H.; Attrep, Katherine A.; Gougoutas, J. Z.

    1967-01-01

    Rabbit kidney medulla (10kg.) was homogenized in 5mm-disodium hydrogen phosphate and deproteinized with ethanol, and the concentrated supernatant solution was extracted at pH8 with light petroleum and at pH2 with chloroform. The acidic lipids present in the chloroform phase were separated on silicic acid columns into three biologically active fractions. The first fraction contained only vasodepressor activity; the second fraction contained both vasodepressor and non-vascular-smooth-muscle-stimulating activity; the third fraction contained both vasopressor and non-vascular-smooth-muscle-stimulating activity. Purification of each fraction by reversed-phase partition and thick-layer chromatography yielded three pure acids. Thin-layer chromatographic, spectroscopic and mass-spectral analysis of the acids and their methyl esters established their structures as prostaglandins E2, F2α and A2. Evidence is presented demonstrating that part or all of the prostaglandin A2 is formed during the isolation procedures from endogenous prostaglandin E2. PMID:16742553

  6. New insights of asteroid 4179 Toutatis using China Chang'e-2 close flyby optical measurements

    SciTech Connect

    Bu, Yanlong; Tang, Geshi; Cao, Jianfeng; Hu, Songjie; Yang, Cheng; Liu, Chuankai; Di, KaiChang; Xu, Bin; Liu, Bin; Fa, Wenzhe; Hu, Tianjiang; Ding, Chibiao E-mail: tanggeshi@bacc.org.cn

    2015-01-01

    The mysteries of near-Earth asteroid 4179 Toutatis have been more comprehensively unveiled by analyzing the optical images taken during the Chang'e-2 flyby in 2012. Compared with previous works, this paper concentrates on the photogrammetric relation between the Chang'e-2 spacecraft and Toutatis and the imaging shadow effect during the flyby. Accurate models of imaging and optical measurements are developed to study Toutatis's dimensions and rotational state at the time of imaging. As the illumination study shows, the shadowed region perpendicular to the long axis accounts for 27.78% of the Toutatis images, while the long axis of the body is fully captured. With a compensation on the shadow effect, the optical measurements reveal that Toutatis's long axis is 4354 ± 56 m, the maximum length is 4391 ± 56 m, and the spatial orientation described with the angles of direction cosine during the flyby is (126.°13 ± 0.°29, 122.°98 ± 0.°21, 126.°63 ± 0.°46). Furthermore, a new triaxial ellipsoid of 4354 × 1835 × 2216 m and a volume of 7.5158 km{sup 3} are proposed based on the previous Toutatis shape model. The effectiveness of the proposed method is validated, since typical features such as the neck and endpoints agree well with the results simultaneously observed by the ground radar. Moreover, it also potentially provides a feasible approach to precisely calculate the spin period of Toutatis.

  7. New Insights of Asteroid 4179 Toutatis Using China Chang'e-2 Close Flyby Optical Measurements

    NASA Astrophysics Data System (ADS)

    Bu, Yanlong; Tang, Geshi; Di, KaiChang; Fa, Wenzhe; Hu, Tianjiang; Ding, Chibiao; Xu, Bin; Liu, Bin; Cao, Jianfeng; Hu, Songjie; Yang, Cheng; Liu, Chuankai

    2015-01-01

    The mysteries of