Journal articles on the topic 'E1 and E2'
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Mu, Yu, Birke Andrea Tews, Christine Luttermann, and Gregor Meyers. "Interaction of Pestiviral E1 and E2 Sequences in Dimer Formation and Intracellular Retention." International Journal of Molecular Sciences 22, no. 14 (July 6, 2021): 7285. http://dx.doi.org/10.3390/ijms22147285.
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Pestiviruses contain three envelope proteins: Erns, E1, and E2. Expression of HA-tagged E1 or mutants thereof showed that E1 forms homodimers and -trimers. C123 and, to a lesser extent, C171, affected the oligomerization of E1 with a double mutant C123S/C171S preventing oligomerization completely. E1 also establishes disulfide linked heterodimers with E2, which are crucial for the recovery of infectious viruses. Co-expression analyses with the HA-tagged E1 wt/E1 mutants and E2 wt/E2 mutants demonstrated that C123 in E1 and C295 in E2 are the critical sites for E1/E2 heterodimer formation. Introduction of mutations preventing E1/E2 heterodimer formation into the full-length infectious clone of BVDV CP7 prevented the recovery of infectious viruses, proving that C123 in E1 and C295 in E2 play an essential role in the BVDV life cycle, and further support the conclusion that heterodimer formation is the crucial step. Interestingly, we found that the retention signal of E1 is mandatory for intracellular localization of the heterodimer, so that absence of the E1 retention signal directs the heterodimer to the cell surface even though the E2 retention signal is still present. The covalent linkage between E1 and E2 plays an essential role for this process.
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Majid, Ayaz M., Heather Ezelle, Sangeeta Shah, and Glen N. Barber. "Evaluating Replication-Defective Vesicular Stomatitis Virus as a Vaccine Vehicle." Journal of Virology 80, no. 14 (July 15, 2006): 6993–7008. http://dx.doi.org/10.1128/jvi.00365-06.
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ABSTRACT We have generated replication-competent (VSV-C/E1/E2) and nonpropagating (VSVΔG-C/E1/E2) vesicular stomatitis virus (VSV) contiguously expressing the structural proteins of hepatitis C virus (HCV; core [C] and glycoproteins E1 and E2) and report on their immunogenicity in murine models. VSV-C/E1/E2 and VSVΔG-C/E1/E2 expressed high levels of HCV C, E1, and E2, which were authentically posttranslationally processed. Both VSV-expressed HCV E1-E2 glycoproteins were found to form noncovalently linked heterodimers and appeared to be correctly folded, as confirmed by coimmunoprecipitation analysis using conformationally sensitive anti-HCV-E2 monoclonal antibodies (MAbs). Intravenous or intraperitoneal immunization of BALB/c mice with VSV-C/E1/E2 or VSVΔG-C/E1/E2 resulted in significant and surprisingly comparable HCV core or E2 antibody responses compared to those of control mice. In addition, both virus types generated HCV C-, E1-, or E2-specific gamma interferon (IFN-γ)-producing CD8+ T cells, as determined by enzyme-linked immunospot (ELISPOT) analysis. Mice immunized with VSVΔG-C/E1/E2 were also protected against the formation of tumors expressing HCV E2 (CT26-hghE2t) and exhibited CT26-hghE2t-specific IFN-γ-producing and E2-specific CD8+ T-cell activity. Finally, recombinant vaccinia virus (vvHCV.S) expressing the HCV structural proteins replicated at significantly lower levels when inoculated into mice immunized with VSV-C/E1/E2 or VSVΔG-C/E1/E2, but not with control viruses. Our data therefore illustrate that potentially safer replication-defective VSV can be successfully engineered to express high levels of antigenically authentic HCV glycoproteins. In addition, this strategy may therefore serve in effective vaccine and immunotherapy-based approaches to the treatment of HCV-related disease.
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Kerbey, A. L., and P. J. Randle. "Pyruvate dehydrogenase kinase activity of pig heart pyruvate dehydrogenase (E1 component of pyruvate dehydrogenase complex)." Biochemical Journal 231, no. 3 (November 1, 1985): 523–29. http://dx.doi.org/10.1042/bj2310523.
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The pyruvate dehydrogenase (E1) and acetyltransferase (E2) components of pig heart and ox kidney pyruvate dehydrogenase (PDH) complex were separated and purified. The E1 component was phosphorylated (alpha-chain) and inactivated by MgATP. Phosphorylation was mainly confined to site 1. Addition of E2 accelerated phosphorylation of all three sites in E1 alpha and inactivation of E1. On the basis of histone H1 phosphorylation, E2 is presumed to contain PDH kinase, which was removed (greater than 98%) by treatment with p-hydroxymercuriphenylsulphonate. Stimulation of ATP-dependent inactivation of E1 by E2 was independent of histone H1 kinase activity of E2. The effect of E2 is attributed to conformational change(s) induced in E1 and/or E1-associated PDH kinase. PDH kinase activity associated with E1 could not be separated from it be gel filtration or DEAE-cellulose chromatography. Subunits of PDH kinase were not detected on sodium dodecyl sulphate/polyacrylamide gels of E1 or E2, presumably because of low concentration. The activity of pig heart PDH complex was increased by E2, but not by E1, indicating that E2 is rate-limiting in the holocomplex reaction. ATP-dependent inactivation of PDH complex was accelerated by E1 or by phosphorylated E1 plus associated PDH kinase, but not by E2 plus presumed PDH kinase. It is suggested that a substantial proportion of PDH kinase may accompany E1 when PDH complex is dissociated into its component enzymes. The possibility that E1 may possess intrinsic PDH kinase activity is considered unlikely, but may not have been fully excluded.
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Crago, Elizabeth A., Paula R. Sherwood, Catherine Bender, Jeffrey Balzer, Dianxu Ren, and Samuel M. Poloyac. "Plasma Estrogen Levels Are Associated With Severity of Injury and Outcomes After Aneurysmal Subarachnoid Hemorrhage." Biological Research For Nursing 17, no. 5 (December 29, 2014): 558–66. http://dx.doi.org/10.1177/1099800414561632.
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Background:Biochemical mediators alter cerebral perfusion and have been implicated in delayed cerebral ischemia (DCI) and poor outcomes after aneurysmal subarachnoid hemorrhage (aSAH). Estrogens (estrone [E1] and estradiol [E2]) are mediators with neuroprotective properties that could play a role in DCI. This study explored associations between plasma estrogen levels and outcomes following aSAH.Methods:Plasma samples from 1–4, 4–6, and 7–10 days after hemorrhage from 99 adult aSAH patients were analyzed for estrogen levels using liquid chromatography tandem mass spectrometry. DCI was operationalized as radiographic/ultrasonic evidence of impaired cerebral blood flow accompanied by neurological deterioration. Outcomes were assessed using the Modified Rankin Scale at 3 and 12 months after hemorrhage. Statistical analysis included correlation, regression, and group-based trajectory.Results:Higher E1 and E2 levels were associated with higher Hunt and Hess grade (E1, p = .01; E2, p = .03), the presence of DCI (E1, p = .02; E2, p = .02), and poor 3-month outcomes (E1, p = .002; E2, p = .002). Trajectory analysis identified distinct populations over time for E1 (61% E1 high) and E2 (68% E2 high). Patients in higher trajectory groups had higher Fisher grades (E1, p = .008; E2, p = .01), more frequent DCI (E1, p = .04; E2, p = .08), and worse 3-month outcomes (E1, p = .01; E2, p = .004) than low groups.Conclusions:These results provide the first clinical evidence that plasma E1 and E2 concentrations are associated with severity of injury and outcomes after aSAH.
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Liu, Bing, L. Maria Lois, and David Reverter. "Structural insights into SUMO E1–E2 interactions in Arabidopsis uncovers a distinctive platform for securing SUMO conjugation specificity across evolution." Biochemical Journal 476, no. 14 (July 31, 2019): 2127–39. http://dx.doi.org/10.1042/bcj20190232.
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Abstract SUMOylation of proteins involves the concerted action of the E1-activating enzyme, E2-conjugating enzyme and E3-ligases. An essential discrimination step in the SUMOylation pathway corresponds to the initial interaction between E1 ubiquitin-fold domain (UFD) and E2 enzymes. Although E2 orthologs possess high sequence identity, the E2 binding region of the UFD domains has diverged across evolution. Moreover, in reciprocal in vitro conjugation reactions Arabidopsis E1 and E2 SCE1 fail to interact efficiently with cognate human E2 Ubc9 and E1 partners, respectively. To gain more insights into the properties of this interface in evolutionary distant organisms, we solved the crystal structure of SUMO E2 SCE1 and its complex with E1 UFD in Arabidopsis. In addition to a few common structural determinants, the interface between the E1 UFD and E2 in Arabidopsis is distinct compared with human and yeast, in particular by the presence of a longer α-helix in the Arabidopsis UFD domain. Despite the variability of E1 UFD domains in these surfaces, they establish specific interactions with highly conserved surfaces of their cognate E2 enzymes. Functional analysis of the different E2 interface residues between human and Arabidopsis revealed Val37 (Met36 in human), as a determinant that provides specificity in the E1–E2 recognition in plants.
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Zou, Nianxiang, Jen-Sing Liu, Shu-Ru Kuo, Thomas R. Broker, and Louise T. Chow. "The Carboxyl-Terminal Region of the Human Papillomavirus Type 16 E1 Protein Determines E2 Protein Specificity during DNA Replication." Journal of Virology 72, no. 4 (April 1, 1998): 3436–41. http://dx.doi.org/10.1128/jvi.72.4.3436-3441.1998.
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ABSTRACT The mechanism of DNA replication is conserved among papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit with different efficiencies, indicating a certain degree of specificity in E1-E2 interactions. We report that, in the assays of our study, the human papillomavirus type 11 (HPV-11) E1 protein functioned with the HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no detectable activity with the HPV-11 E2 protein. Taking advantage of this distinction, we used chimeric E1 proteins to delineate the E1 protein domains responsible for this specificity. Hybrids containing HPV-16 E1 amino-terminal residues up to residue 365 efficiently replicated either viral origin in the presence of either E2 protein. The reciprocal hybrids containing amino-terminal HPV-11 sequences exhibited a high activity with HPV-16 E2 but no activity with HPV-11 E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues from either E1 had an intermediate property, but both collaborated more efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras with a junction in the putative ATPase domain showed little or no activity with either E2 protein. We conclude that the E1 protein consists of distinct structural and functional domains, with the carboxyl-terminal 284 residues of the HPV-16 E1 protein being the primary determinant for E2 specificity during replication, and that chimeric exchanges in or bordering the ATPase domain inactivate the protein.
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Cocquerel, Laurence, Jean-Christophe Meunier, André Pillez, Czeslaw Wychowski, and Jean Dubuisson. "A Retention Signal Necessary and Sufficient for Endoplasmic Reticulum Localization Maps to the Transmembrane Domain of Hepatitis C Virus Glycoprotein E2." Journal of Virology 72, no. 3 (March 1, 1998): 2183–91. http://dx.doi.org/10.1128/jvi.72.3.2183-2191.1998.
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ABSTRACT The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which is retained in the endoplasmic reticulum (ER). To identify whether E1 and/or E2 contains an ER-targeting signal potentially involved in ER retention of the E1-E2 complex, these proteins were expressed alone and their intracellular localization was studied. Due to misfolding of E1 in the absence of E2, no conclusion on the localization of its native form could be drawn from the expression of E1 alone. E2 expressed in the absence of E1 was shown to be retained in the ER similarly to E1-E2 complex. Chimeric proteins in which E2 domains were exchanged with corresponding domains of a protein normally transported to the plasma membrane (CD4) were constructed to identify the sequence responsible for its ER retention. The transmembrane domain (TMD) of E2 (C-terminal 29 amino acids) was shown to be sufficient for retention of the ectodomain of CD4 in the ER compartment. Replacement of the E2 TMD by the anchor signal of CD4 or a glycosyl phosphatidylinositol (GPI) moiety led to its expression on the cell surface. In addition, replacement of the E2 TMD by the anchor signal of CD4 or a GPI moiety abolished the formation of E1-E2 complexes. Together, these results suggest that, besides having a role as a membrane anchor, the TMD of E2 is involved in both complex formation and intracellular localization.
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Merola, Marcello, Michela Brazzoli, Fabienne Cocchiarella, Jens M. Heile, Ari Helenius, Amy J. Weiner, Michael Houghton, and Sergio Abrignani. "Folding of Hepatitis C Virus E1 Glycoprotein in a Cell-Free System." Journal of Virology 75, no. 22 (November 15, 2001): 11205–17. http://dx.doi.org/10.1128/jvi.75.22.11205-11217.2001.
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ABSTRACT The hepatitis C virus (HCV) envelope proteins, E1 and E2, form noncovalent heterodimers and are leading candidate antigens for a vaccine against HCV. Studies in mammalian cell expression systems have focused primarily on E2 and its folding, whereas knowledge of E1 folding remains fragmentary. We used a cell-free in vitro translation system to study E1 folding and asked whether the flanking proteins, Core and E2, influence this process. We translated the polyprotein precursor, in which the Core is N-terminal to E1, and E2 is C-terminal, and found that when the core protein was present, oxidation of E1 was a slow, E2-independent process. The half-time for E1 oxidation was about 5 h in the presence or absence of E2. In contrast with previous reports, analysis of three constructs of different lengths revealed that the E2 glycoprotein undergoes slow oxidation as well. Unfolded or partially folded E1 bound to the endoplasmic reticulum chaperones calnexin and (with lower efficiency) calreticulin, whereas no binding to BiP/GRP78 or GRP94 could be detected. Release from calnexin and calreticulin was used to assess formation of mature E1. When E1 was expressed in the absence of Core and E2, its oxidation was impaired. We conclude that E1 folding is a process that is affected not only by E2, as previously shown, but also by the Core. The folding of viral proteins can thus depend on complex interactions between neighboring proteins within the polyprotein precursor.
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de Mes, T. Z. D., K. Kujawa-Roeleveld, G. Zeeman, and G. Lettinga. "Fate of oestrogens during anaerobic blackwater treatment with micro-aerobic post-treatment." Water Science and Technology 56, no. 5 (September 1, 2007): 15–23. http://dx.doi.org/10.2166/wst.2007.552.
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The fate of oestrone (E1), 17β-oestradiol (E2) and 17α-ethynyloestradiol (EE2) was investigated in a concentrated blackwater treatment system consisting of an UASB septic tank, with micro-aerobic post-treatment. In UASB septic tank effluent a (natural) total concentration of 4.02 μg/L E1 and 18.69 μg/L E2, comprising the sum of conjugated (>70% for E1 and >80% for E2) and unconjugated forms, was measured. During post-treatment the unconjugated oestrogens were removed to below 1 μg/L. A percentage of 77% of the measured unconjugated E1 and 82% of E2 was associated with particles >1.2 μm in the final effluent implying high sorption affinity of both compounds. When spiking the UASB septic tank effluent with E1, E2, EE2 and the sulphate conjugate of E2, removal in the micro-aerobic post-treatment was >99% for both E2 and EE2 and 83% for E1. The lower removal value for E1 was a result of (slow) deconjugation during the treatment, and in the final effluent still 40% of E1 and 99% of E2 was present in conjugated form. The latter was the result of incomplete deconjugation of the spiked E2(3S) in the post-treatment system.
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Zhang, Xinyue, Tingting Wu, Huiwen Wen, Wenwen Song, Cailong Xu, Tianfu Han, Shi Sun, and Cunxiang Wu. "Allelic Variation of Soybean Maturity Genes E1–E4 in the Huang-Huai-Hai River Valley and the Northwest China." Agriculture 11, no. 6 (May 22, 2021): 478. http://dx.doi.org/10.3390/agriculture11060478.
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Soybean is planted in a wide span of the world, and flowering and maturity time is an important trait determining soybean yield formation and adaptation. Maturity loci E1, E2, E3 and E4 were frequently reported as the most influential genetic loci for soybean flowering and maturity. To understand the allelic variation and assess the phenological traits of cultivars with different E allelic combinations in natural environments, 251 cultivars of maturity group (MG) I–V were field tested in 42 locations across four sub-regions in the Huang-Huai-Hai and Northwest region of China and genotyped with KASP markers for E1–E4 loci. The results indicated that mutant alleles were only found in the E1 and E2 locus, all of the cultivars carried functional alleles in the E3 and E4 loci in this area, with the frequency of mutant allele to be higher in early maturity groups (MGs) than late MGs. Among nine E allelic combinations in this area, one photoperiodic insensitive mutation in E2 loci (E1/e2-ns/E3-Ha/E4 and E1/e2-ns/E3-Mi/E4) made up the largest proportion (25.10 and 18.33%), while two photoperiodic insensitive mutations in both E1 and E2 loci (e1-as/e2-ns/E3-Ha/E4) (1.20%) occupied the lowest proportion in this panel. The major combinations of E locus for MGI, MGII and MG III in this area were E1/E2-dl/E3-Mi/E4, E1/e2-ns/E3-Mi/E4 and E1/e2-ns/E3-Ha/E4, respectively. Cultivars carrying e1-as/e2-ns/E3-Ha/E4 genotype flowered earliest (34 days) on average, 7.6 days earlier than the latest-flowering E haplotype (E1/e2-ns/E3-Ha/E4). This study provided an opportunity to detect the E allelic combinations in the Huang-Huai-Hai River Valley and the Northwest China, which would facilitate the improvement of soybean adaptation in the future.
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Knight, Ronald L., Kimberly L. W. Schultz, Rebekah J. Kent, Meera Venkatesan, and Diane E. Griffin. "Role of N-Linked Glycosylation for Sindbis Virus Infection and Replication in Vertebrate and Invertebrate Systems." Journal of Virology 83, no. 11 (March 18, 2009): 5640–47. http://dx.doi.org/10.1128/jvi.02427-08.
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ABSTRACT Each Sindbis virus (SINV) surface glycoprotein has two sites for N-linked glycosylation (E1 positions 139 and 245 [E1-139 and E1-245] and E2 positions 196 and 318 [E2-196 and E2-318]). Studies of SINV strain TE12 mutants with each site eliminated identified the locations of carbohydrates by cryo-electron microscopy (S. V. Pletnev et al., Cell 105:127-136, 2001). In the current study, the effects of altered glycosylation on virion infectivity, growth in cells of vertebrates and invertebrates, heparin binding, virulence in mice, and replication in mosquitoes were assessed. Particle-to-PFU ratios for E1-139 and E2-196 mutant strains were similar to that for TE12, but this ratio for the E1-245 mutant was 100-fold lower than that for TE12. Elimination of either E2 glycosylation site increased virus binding to heparin and increased replication in BHK cells. Elimination of either E1 glycosylation site had no effect on heparin binding but resulted in an approximately 10-fold decrease in virus yield from BHK cells compared to the TE12 amount. No differences in pE2 processing were detected. E2-196 and E2-318 mutants were more virulent in mice after intracerebral inoculation, while E1-139 and E1-245 mutants were less virulent. The E1-245 mutant showed impaired replication in C7/10 mosquito cells and in Culex quinquefasciatus after intrathoracic inoculation. We conclude that the increased replication and virulence of E2-196 and E2-318 mutants are primarily due to increased efficiency of binding to heparan sulfate on mammalian cells. Lack of glycosylation at E1-139 or E1-245 impairs replication in vertebrate cells, while E1-245 also severely affects replication in invertebrate cells.
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Yang, Decheng, Dorothy Hwang, Zhiyong Qiu, and Shirley Gillam. "Effects of Mutations in the Rubella Virus E1 Glycoprotein on E1-E2 Interaction and Membrane Fusion Activity." Journal of Virology 72, no. 11 (November 1, 1998): 8747–55. http://dx.doi.org/10.1128/jvi.72.11.8747-8755.1998.
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ABSTRACT Rubella virus (RV) virions contain two glycosylated membrane proteins, E1 and E2, that exist as a heterodimer and form the viral spike complexes on the virion surface. Formation of an E1-E2 heterodimer is required for transport of E1 out of the endoplasmic reticulum lumen to the Golgi apparatus and plasma membrane. To investigate the nature of the E1-E2 interaction, we have introduced mutations in the internal hydrophobic region (residues 81 to 109) of E1. Substitution of serine at Cys82 (mutant C82S) or deletion of this hydrophobic domain (mutant dt) of E1 resulted in a disruption of the E1 conformation that ultimately affected E1-E2 heterodimer formation and cell surface expression of both E1 and E2. Substitution of either aspartic acid at Gly93 (G93D) or glycine at Pro104 (P104G) was found to impair neither E1-E2 heterodimer formation nor the transport of E1 and E2 to the cell surface. Fusion of RV-infected cells is induced by a brief treatment at a pH below 6.0. To test whether this internal hydrophobic domain is involved in the membrane fusion activity of RV, transformed BHK cell lines expressing either wild-type or mutant spike proteins were exposed to an acidic pH and polykaryon formation was measured. No fusion activity was observed in the C82S, dt, and G93D mutants; however, the wild type and the P104G mutant exhibited fusogenic activities, with greater than 60% and 20 to 40% of the cells being fused, respectively, at pH 4.8. These results suggest that it is likely that the region of E1 between amino acids 81 and 109 is involved in the membrane fusion activity of RV and that it may be important for the interaction of that protein with E2 to form the E1-E2 heterodimer.
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Masterson, Philip J., Margaret A. Stanley, Alan P. Lewis, and Michael A. Romanos. "A C-Terminal Helicase Domain of the Human Papillomavirus E1 Protein Binds E2 and the DNA Polymerase α-Primase p68 Subunit." Journal of Virology 72, no. 9 (September 1, 1998): 7407–19. http://dx.doi.org/10.1128/jvi.72.9.7407-7419.1998.
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ABSTRACT The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase α-primase (polα-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polα-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind theori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polα-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.
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Claus, Claudia, Jörg Hofmann, Klaus Überla, and U. G. Liebert. "Rubella virus pseudotypes and a cell–cell fusion assay as tools for functional analysis of the rubella virus E2 and E1 envelope glycoproteins." Journal of General Virology 87, no. 10 (October 1, 2006): 3029–37. http://dx.doi.org/10.1099/vir.0.82035-0.
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The rubivirus Rubella virus contains the two envelope glycoproteins E2 and E1 as a heterodimeric spike complex embedded in its lipid envelope. The functions of both proteins, especially of E2, in the process of viral entry are still not entirely understood. In order to dissect E2 and E1 entry functions from post-entry steps, pseudotypes of lentiviral vectors based on Simian immunodeficiency virus were used. C-terminally modified E2 and E1 variants successfully pseudotyped lentiviral vector particles. This is the first report to show that not only E1, but also E2, is able to mediate infectious viral entry. Furthermore, a cell–cell fusion assay was used to further clarify membrane-fusion activities of E2 and E1 as one of the early steps of infection. It was demonstrated that the capsid protein, when coexpressed in cis, enhances the degree of E2- and E1-mediated cell–cell fusion.
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Chen, Grace, and Arne Stenlund. "Characterization of the DNA-Binding Domain of the Bovine Papillomavirus Replication Initiator E1." Journal of Virology 72, no. 4 (April 1, 1998): 2567–76. http://dx.doi.org/10.1128/jvi.72.4.2567-2576.1998.
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ABSTRACT The bovine papillomavirus replication initiator protein E1 is an origin of replication (ori)-binding protein absolutely required for viral DNA replication. In the presence of the viral transcription factor E2, E1 binds to the ori and initiates DNA replication. To understand how the E1 initiator recognizes theori and how E2 assists in this process, we have expressed and purified a 166-amino-acid fragment which corresponds to the minimal E1 DNA-binding domain (DBD). DNA binding studies using this protein demonstrate that the E1 DBD can bind to the palindromic E1 binding site in several forms but that binding of two monomers, each recognizing one half-site of the E1 palindrome, is the predominant form. This is reminiscent of the binding of the T-antigen DBD to the SV40ori, and interestingly, the arrangement of E1 binding sites shows striking similarities to the arrangement of T-antigen binding sites in the SV40 ori even though the recognition sequences are unrelated. The E1 DBD is capable of interacting cooperatively with E2; however, the E2 DBD and not the E2 activation domain mediates this interaction. Furthermore, the E2 DBD stimulates binding of two monomers of the E1 DBD to the ori by binding cooperatively with one E1 monomer. Finally, we show that our results concerning the DNA-binding properties of the E1 DBD can be extended to full-length E1.
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Davis, Susan R., Alejandra Martinez-Garcia, Penelope J. Robinson, David J. Handelsman, Reena Desai, Rory Wolfe, and Robin J. Bell. "Estrone Is a Strong Predictor of Circulating Estradiol in Women Age 70 Years and Older." Journal of Clinical Endocrinology & Metabolism 105, no. 9 (July 2, 2020): e3348-e3354. http://dx.doi.org/10.1210/clinem/dgaa429.
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Abstract Importance After menopause, estradiol (E2) is predominately an intracrine hormone circulating in very low serum concentrations. Objective The objective of this work is to examine determinants of E2 concentrations in women beyond age 70 years. Design and Setting A cross-sectional, community-based study was conducted. Participants A total of 5325 women participated, with a mean age of 75.1 years (± 4.2 years) and not using any sex steroid, antiandrogen/estrogen, glucocorticoid, or antiglycemic therapy. Main Outcome Measures Sex steroids were measured by liquid chromatography–tandem mass spectrometry. Values below the limit of detection (LOD; E2 11 pmol/L [3 pg/mL] were assigned a value of LOD/√2 to estimate total E2. Results E2 and estrone (E1) were below the LOD in 66.1% and 0.9% of women, respectively. The median (interdecile ranges) for E1 and detectable E2 were 181.2 pmol/L (range, 88.7-347.6 pmol/L) and 22.0 pmol/L (range, 11.0-58.7 pmol/L). Women with undetectable E2 vs detectable E2 were older (median age 74.1 years vs 73.8, P = .02), leaner (median body mass index [BMI] 26.8 kg/m2 vs 28.5, P < .001), and had lower E1, testosterone and DHEA concentrations (P < .001). A linear regression model, including age, BMI, E1, and testosterone, explained 20.9% of the variation in total E2, but explained only an additional 1.2% of variation over E1 alone. E1 and testosterone made significant contributions (r2 = 0.162, P < .001) in a model for the subset of women with detectable E2. Conclusions Our findings support E1 as a principal circulating estrogen and demonstrate a robust association between E1 and E2 concentrations in postmenopausal women. Taken together with prior evidence for associations between E1 and health outcomes, E1 should be included in studies examining associations between estrogen levels and health outcomes in postmenopausal women.
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Ujjainkar, Vaibhav V. "Study of Environmental Influence on Genetic Diversity Estimates (D2) of Parental Lines in Upland Cotton (Gossypium hirsutum L.)." International Journal of Current Microbiology and Applied Sciences 11, no. 7 (July 10, 2022): 212–22. http://dx.doi.org/10.20546/ijcmas.2022.1107.026.
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The hereditary parenthood of genepool plays important role in crop improvement. D2 statistics is a powerful estimating the magnitude of genetic diversity. Comparative study was conducted to estimate the genetic distances among ten parental lines of upland cotton in two different sets of environments indicated the varying pattern of genetic distances along with varying pattern of dendograms revealed the role of environmental factors controlling expression of quantitative characters and subsequent magnitude of genetic diversity estimates. Clustering based on D2 analysis provides a statistical tool for selecting the diverse parents for hybridization program. The D2 distances matrix calculated for Environment (E1), Environment (E2) and average (E1 & E2) shown mean genetic distances of 15.38, 14.83 and 14.15 respectively. Comparing D2 matrices among E1, E2 and average of environments (E1 & E2) revealed the significant effect of environment on genetic distances with mean values 0.56 (E1 vs E2), 0.82 (E1 vs Avg [E1 & E2]) and 0.62 (E2 vs Avg.[E1 & E2]). The average deviation of 0.82 was estimated for genetic diversity estimates over the environments. The recent techniques of molecular techniques (MAS) should be used for precise estimation of genetic diversity followed by clustering pattern for effective selection of diverse parents for exploitation of phenomenon of heterosis.
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Khaksari, Mohammad, Zahra Soltani, Nader Shahrokhi, Gholamreza Moshtaghi, and Gholamreza Asadikaram. "The role of estrogen and progesterone, administered alone and in combination, in modulating cytokine concentration following traumatic brain injury." Canadian Journal of Physiology and Pharmacology 89, no. 1 (January 2011): 31–40. http://dx.doi.org/10.1139/y10-103.
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Cytokines play an important role in the pathophysiology of traumatic brain injury (TBI). This study was designed to determine the effects of administering progesterone (P) and estrogen (E), alone and in combination, on brain water content, blood–brain barrier (BBB) disturbance, and brain level of cytokines following diffuse TBI. Ovariectomized rats were divided into 9 groups, treated with vehicle, E1, E2, P1, P2, E1+P1, E1+P2, E2+P1, and E2+P2. Levels of BBB disruption (5 h), cytokines, and water content (24 h) were evaluated after TBI induced by the Marmarou method. Physiological (E1 and P1) and pharmacological (E2 and P2) doses of estrogen and progesterone were administered 30 min after TBI. Water content in the E1+P2-treated group was higher than in the E1-treated group. The inhibitory effect of E2 on water content was reduced by adding progesterone. The inhibitory effect of E1 and E2 on Evans blue content was reduced by treatment with E1+P1 and E2+P2, respectively. The brain level of IL-1β was reduced in E1 and E2, after TBI. In the E2+P2-treated group, this level was higher than in the E2-treated group. The brain level of TGF-β was also elevated by the administration of progesterone and estrogen alone, and reduced when the hormones were administered in combination. In conclusion, a combined administration of progesterone and estrogen inhibited the decreasing effects of administration of progesterone and estrogen alone on water content and BBB disruption that mediated to change the proinflammatory cytokines.
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Frederiksen, Hanne, Trine Holm Johannsen, Stine Ehlern Andersen, Jakob Albrethsen, Selma Kløve Landersoe, Jørgen Holm Petersen, Anders Nyboe Andersen, et al. "Sex-specific Estrogen Levels and Reference Intervals from Infancy to Late Adulthood Determined by LC-MS/MS." Journal of Clinical Endocrinology & Metabolism 105, no. 3 (November 13, 2019): 754–68. http://dx.doi.org/10.1210/clinem/dgz196.
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Abstract Context The lack of sensitive and robust analytical methods has hindered the reliable quantification of estrogen metabolites in subjects with low concentrations. Objective To establish sex-specific reference ranges for estrone (E1) and estradiol (E2) throughout life and to evaluate sex-differences using the state-of-the-art liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of E1, E2, and estriol (E3). Design LC-MS/MS method development and construction of estrogen reference ranges. Settings Population-based cross-sectional cohorts from the greater Copenhagen and Aarhus areas. Participants Healthy participants aged 3 months to 61 years (n = 1838). Results An isotope diluted LC-MS/MS method was developed and validated for measurements of serum E1, E2, and E3. Limits of detections (LODs) were 3 pmol/L (E1), 4 pmol/L (E2), and 12 pmol/L (E3), respectively. This sensitive method made it possible to differentiate between male and female concentration levels of E1 and E2 in children. In girls, E2 levels ranged from <LOD to 100 pmol/L during mini-puberty, whereas it was ≤20 pmol/L during childhood. E1 and E2 increased with age and pubertal breast stage and varied during the menstrual cycle; E1 was lower than E2 in girls and premenopausal women, and higher than E2 in postmenopausal women. In boys, E1 and E2 increased with age and pubertal stage, whereas little changes with age were observed in men. High E3 concentrations were confirmed in pregnant women. Conclusion Reference ranges of simultaneous quantification of E1 and E2 by this novel specific and highly sensitive LC-MS/MS method provide an invaluable tool in clinical practice and in future research studies.
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Olesen, Christina Holmboe, Elias H. Augestad, Fulvia Troise, Jens Bukh, and Jannick Prentoe. "In vitro adaptation and characterization of attenuated hypervariable region 1 swap chimeras of hepatitis C virus." PLOS Pathogens 17, no. 7 (July 19, 2021): e1009720. http://dx.doi.org/10.1371/journal.ppat.1009720.
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Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions.
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Kim, Nayoung, and Sungwook Chun. "Association between the serum estrone-to-estradiol ratio and parameters related to glucose metabolism and insulin resistance in women with polycystic ovary syndrome." Clinical and Experimental Reproductive Medicine 48, no. 4 (December 1, 2021): 374–79. http://dx.doi.org/10.5653/cerm.2021.04553.
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Objective: We aimed to evaluate associations between the ratio of serum estrone (E1) to estradiol (E2) and parameters related to serum glucose metabolism and insulin resistance in women with polycystic ovary syndrome (PCOS). Methods: In total, 133 women between the ages of 18 and 33 diagnosed with PCOS were enrolled in this study. All participants with PCOS underwent blood tests to determine hormonal and biochemical metabolic parameters and a standard 2-hour 75-g oral glucose tolerance test. They were divided into two groups according to the serum E1-to-E2 ratio: group 1 (E1/E2 ratio <2.0) and group 2 (E1/E2 ratio ≥2.0). Results: In the comparative analysis, the waist-to-hip ratio (WHR) was the only clinical variable that was significantly different between the two groups. Patients with a higher E1/E2 ratio showed higher fasting insulin levels, homeostasis model for insulin resistance, and postprandial glucose levels at 2 hours (PPG2). In a correlation analysis, only PPG2 was significantly related to the serum E1/E2 ratio. However, after controlling for the confounding effects of body mass index (BMI) and WHR, fasting glucose was also significantly correlated with the serum E1/E2 ratio. Conclusion: Women with PCOS with a higher serum E1/E2 ratio were found to be more likely to show higher fasting insulin and postprandial glucose levels. Significant correlations were found between the serum E1/E2 ratio and both fasting and postprandial serum glucose levels after adjusting for BMI and WHR in women with PCOS.
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Deng, Wentao, Ge Jin, Biing-Yuan Lin, Brian A. Van Tine, Thomas R. Broker, and Louise T. Chow. "mRNA Splicing Regulates Human Papillomavirus Type 11 E1 Protein Production and DNA Replication." Journal of Virology 77, no. 19 (October 1, 2003): 10213–26. http://dx.doi.org/10.1128/jvi.77.19.10213-10226.2003.
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ABSTRACT The papillomavirus replicative helicase E1 and the origin recognition protein E2 are required for efficient viral DNA replication. We fused the green fluorescent protein (GFP) to the human papillomavirus type 11 E1 protein either in a plasmid with the E1 coding region alone (nucleotides [nt] 832 to 2781) (pGFP-11E1) or in a plasmid containing both the E1 and E2 regions (nt 2723 to 3826) and the viral origin of replication (ori) (p11Rc). The former supported transient replication of an ori plasmid, whereas the latter was a self-contained replicon. Unexpectedly, these plasmids produced predominantly a cytoplasmic variant GFP or a GFP-E1^E4 protein, respectively. The majority of the mRNAs had an intragenic or intergenic splice from nt 847 to nt 2622 or from nt 847 to nt 3325, corresponding to the E2 or E1^E4 messages. pGFP-11E1dm and p11Rc-E1dm, mutated at the splice donor site, abolished these splices and increased GFP-E1 protein expression. Three novel, alternatively spliced, putative E2 mRNAs were generated in higher abundance from the mutated replicon than from the wild type. Relative to pGFP-11E1, low levels of pGFP-11E1dm supported more efficient replication, but high levels had a negative effect. In contrast, elevated E2 levels always increased replication. Despite abundant GFP-E1 protein, p11Rc-E1dm replicated less efficiently than the wild type. Collectively, these observations show that the E1/E2 ratio is as important as the E1 and E2 concentrations in determining the replication efficiency. These findings suggest that alternative mRNA splicing could provide a mechanism to regulate E1 and E2 protein expression and DNA replication during different stages of the virus life cycle.
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Olsen, Shaun K., and Christopher D. Lima. "Structure of a Ubiquitin E1-E2 Complex: Insights to E1-E2 Thioester Transfer." Molecular Cell 49, no. 5 (March 2013): 884–96. http://dx.doi.org/10.1016/j.molcel.2013.01.013.
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Cao, Liang, Wei Wang, Wenchao Sun, Jinyong Zhang, Jicheng Han, Changzhan Xie, Zhuo Ha, et al. "Construction and Evaluation of Recombinant Adenovirus Candidate Vaccines for Chikungunya Virus." Viruses 14, no. 8 (August 15, 2022): 1779. http://dx.doi.org/10.3390/v14081779.
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Chikungunya virus (CHIKV) is a mosquito-borne virus. The emergence of CHIKV infection has raised global concern, and there is a growing need to develop safe and effective vaccines. Here, adenovirus 5 was used as the vaccine vector to construct recombinant adenoviruses expressing CHIKV E2, E1, and E2-6K-E1, respectively. And then the immunogenicity and protective efficiency against CHIKV were evaluated in BALB/c mice. Compared to the ad-wt control group, all three vaccines elicited significant humoral and cellar immune responses. The levels of neutralizing antibodies in the rAd-CHIKV-E2-6K-E1 and rAd-CHIKV-E2 groups both reached 1:256, which were 3.2 times higher than those in the rAd-CHIKV-E1 group. Furthermore, the levels of lymphocyte proliferation in rAd-CHIKV-E2-6K-E1 group were the highest. Besides, the concentrations of IFN-γ and IL-4 in mice immunized with rAd-CHIKV-E2-6K-E1 were 1.37 and 1.20 times higher than those in ad-wt immunized mice, respectively. After the challenge, mice in the rAd-CHIKV-E2-6K-E1 and rAd-CHIKV-E2 groups lost 2% of their body weight compared with 5% in the ad-wt control group. And low viral loads were detected in the heart, kidney, and blood of mice immunized with rAd-CHIKV-E2-6K-E1 and rAd-CHIKV-E2 at 3–5 dpc, which decreased by 0.4–0.7 orders of magnitude compared with the ad-wt control. Overall, these data suggest that the recombinant adenovirus is a potential candidate vaccine against CHIKV.
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Berto, Dirlei Antonio, Francisco Stefano Wechsler, and Cristiana Chrysóstomo Noronha. "Exigências de treonina de leitões dos 7 aos 12 e dos 12 aos 23 kg." Revista Brasileira de Zootecnia 31, no. 3 (June 2002): 1176–83. http://dx.doi.org/10.1590/s1516-35982002000500014.
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Foram realizados dois experimentos (E) com 204 leitões Large White (E1: dos 7,23 aos 12,32 kg; e E2: dos 12,64 aos 23,81 kg). O delineamento experimental foi de blocos ao acaso com quatro níveis de treonina na ração (E1: 0,80; 0,87; 0,93 e 0,99 %; e E2: 0,69; 0,74; 0,80 e 0,85 %); oito (E1) e nove (E2) repetições para o consumo diário de ração (CDR), ganho diário de peso (GDP), ganho diário de peso ajustado (GDPA) e conversão alimentar (CA); e cinco repetições para uréia plasmática (U). Não se observaram diferenças no CDR e GDP (P>0,10). Verificaram-se efeitos quadráticos da treonina no GDPA do E1 (P=0,086) e E2 (P=0,052), na CA do E2 (P=0,035) e na U do E1 (P=0,002), bem como efeito linear negativo na CA do E1 (P=0,030) e U do E2 (P=0,044). O nível de 0,89% de treonina minimizou o teor plasmático de uréia e o de 0,94% maximizou o ganho diário de peso ajustado no E1, enquanto no E2 0,76% de treonina na ração maximizou o ganho diário de peso ajustado e minimizou a conversão alimentar.
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Caldon, C. Elizabeth, C. Marcelo Sergio, Judith Schütte, Marijke N. Boersma, Robert L. Sutherland, Jason S. Carroll, and Elizabeth A. Musgrove. "Estrogen Regulation of Cyclin E2 Requires Cyclin D1 but Not c-Myc." Molecular and Cellular Biology 29, no. 17 (June 29, 2009): 4623–39. http://dx.doi.org/10.1128/mcb.00269-09.
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ABSTRACT During estrogen-induced proliferation, c-Myc and cyclin D1 initiate independent pathways that activate cyclin E1-Cdk2 by sequestration and/or downregulation of the CDK inhibitor p21Waf1/Cip1, without significant increases in cyclin E1 protein levels. In contrast, cyclin E2 undergoes a marked increase in expression, which occurs within 9 to 12 h of estrogen treatment of antiestrogen-pretreated MCF-7 breast cancer cells. Both E cyclins are important to estrogen action, as small interfering RNA (siRNA)-mediated knockdown of either cyclin E1 or cyclin E2 attenuated estrogen-mediated proliferation. Inducible expression of cyclin D1 upregulated cyclin E2, while siRNA-mediated knockdown of cyclin D1 attenuated estrogen effects on cyclin E2. However, manipulation of c-Myc levels did not profoundly affect cyclin E2. Cyclin E2 induction by estrogen was accompanied by recruitment of E2F1 to the cyclin E1 and E2 promoters, and cyclin D1 induction was sufficient for E2F1 recruitment. siRNA-mediated knockdown of the chromatin remodelling factor CHD8 prevented cyclin E2 upregulation. Together, these data indicate that cyclin E2-Cdk2 activation by estrogen occurs via E2F- and CHD8-mediated transcription of cyclin E2 downstream of cyclin D1. This contrasts with the predominant regulation of cyclin E1-Cdk2 activity via CDK inhibitor association downstream of both c-Myc and cyclin D1 and indicates that cyclins E1 and E2 are not always coordinately regulated.
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Hung, Hao-Shen, Kuei-Jyum C. Yeh, Chi-Ying Hsieh, and Ting-Chien Chen. "Occurrence and Degradation of Free and Conjugated Estrogens in a River Receiving Feedlot Animal Discharge." Applied Sciences 12, no. 23 (November 23, 2022): 11961. http://dx.doi.org/10.3390/app122311961.
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This study analyzed concentrations of 17β-estradiol (E2), estrone (E1), estriol (E3), 17α-ethynylestradiol (EE2), diethylstilbestrol (DES), 17β-estradiol-3-sulfate (E2-3S), estrone-3-sulfate (E1-3S), 17β-estradiol-3-glucuronide (E2-3G), and estrone-3-glucuronide (E1-3G) in river water, received from intensive feedlot operations wastewater in WuLo Creek, Taiwan. Moreover, the estrogen degradation in situ was analyzed. The average concentrations were 54.15 ± 31.42, 9.71 ± 6.42 and 3.55 ± 2.41 ng/L for E1, E2 and E3, respectively. The concentrations and order were similar to the polluted river and higher than most rivers’ concentrations. The conjugated estrogen concentrations ranged from ND to 13.2 ng/L (E1-3S), ND to 10.4 ng/L (E2-3S), ND to 10.0 ng/L (E1-3G), and ND to 3.6 ng/L (E2-3G), and the detection rates were 76%, 71%, 56%, and 15%, respectively. In the present study, the high detection rates of conjugate estrogen were more elevated than the water receiving STP effluent, suggesting that the source was the river water close to the animal wastewater discharge. In the degradation test, the DES concentrations slightly declined after 24 h, but E1-3G did not significantly change over time (p > 0.05). The degradation of free estrogen occurred during the first 12 h period, and residual concentration was not further decreased after 24 h. In the environment, E1 had higher concentrations than E2 and E3, suggesting that E1 was more resistant to degradation than E2 and E3 at low concentrations. However, the degradation test in the present study suggested that E1 rapidly degraded at high ambient concentrations due to the high degradation constant.
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Slavicek, James M., and Holly J. R. Popham. "The Lymantria dispar Nucleopolyhedrovirus Enhancins Are Components of Occlusion-Derived Virus." Journal of Virology 79, no. 16 (August 15, 2005): 10578–88. http://dx.doi.org/10.1128/jvi.79.16.10578-10588.2005.
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ABSTRACT Enhancins are metalloproteinases, first identified in granuloviruses, that can enhance nucleopolyhedrovirus (NPV) potency. We had previously identified two enhancin genes (E1 and E2) in the Lymantria dispar multinucleocapsid NPV (LdMNPV) and showed that both were functional. For this study, we have extended our analysis of LdMNPV enhancin genes through an immunocytochemical analysis of E1 and E2 expression and localization. E1 and E2 peptide antibodies recognized proteins of ∼84 kDa and 90 kDa, respectively, on Western blots of extracts from L. dispar 652Y cells infected with wild-type virus. The 84- and 90-kDa proteins were first detected at 48 h postinfection (p.i.) and were present through 96 h p.i. E1 and E2 peptide antibodies detected E1 and E2 in polyhedron extracts, and the antibodies were shown to be specific for E1 and E2, respectively, through the use of recombinant virus strains lacking the enhancin genes. E1 and E2 were further localized to occlusion-derived virus (ODV). The enhancins were not found in budded virus. Immunoelectron microscopy indicated that E1 and E2 were present in ODV envelopes and possibly in nucleocapsids. Fractionation studies with several detergents suggested that the enhancins were present in ODV envelopes in association with nucleocapsids. In contrast, enhancins in granuloviruses are located within the granulin matrix. The presence of LdMNPV enhancins within ODV provides a position for the proteins to interact directly on the peritrophic membrane as ODV traverses this host defense barrier.
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Benítez, Carlos, and Manuel Fernández. "Norms in product spaces which preserve approximation properties." Proceedings of the Royal Society of Edinburgh: Section A Mathematics 105, no. 1 (1987): 199–203. http://dx.doi.org/10.1017/s0308210500022034.
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SynopsisLet E1 and E2 be real normed linear spaces such that the dimension of any of them is at least 2. We prove that the norms in E1 × E2 which verify a simple property of monotonicity with regard to the initial norms in E1 and E2 are the only norms in E1 × E2 which preserve best linear approximations, in the sense that ifyk ∊ Lk is best approximation to xk from the linear subspace Lk, (k = 1,2), then (y1, y2) is best approximation to (x1, x2) from L1 × L2.
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Jannah, Dewi Wardatul, Anik Maunatin, and Akyunul Jannah. "Identifikasi dan Uji Toksisitas terhadap Larva Udang (Artemia salina L.) Ekstrak Bekatul Menggunakan Variasi Jenis Pelarut dan Lama Ekstraksi." ALCHEMY 8, no. 2 (October 30, 2020): 16–23. http://dx.doi.org/10.18860/al.v8i2.11512.
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Rice bran was extracted using ultrasonic method with varied solvents (ethanol, ethyl acetate and n-hexane) and extraction times (20, 25 and 30 minutes). The aim of this study was to determine toxicity value of rice bran extract with variation of solvents and extraction times. Rice bran extracts were tested its toxicity by BSLT method and its secondary metabolite by phytochemical test with reagents. Mortality data of shrimp larvae was analyzed by probit to determine LC50 values. Yield of ethanolic extract of rice bran for E1-20, E1-25 and E1-30 samples was 18.159, 19.132 and 18.280%, respectively. Rice bran extracts by ethyl acetate solvent (E2) and n-hexane (E3) gave different yields such as 8.302% (E2-20), 7.282% (E2-25), 9.18% (E2-30), 7.815% (E3-20), 7.125% (E3-25), and 7.279% (E3-30). All of rice bran extracts contained flavonoids, steroids and triterpenoids. LC50 values in ethanolic extract of rice bran for E1-20, E1-25 dan E1-30 samples were 613.258, 673.210 and 2217.255 ppm, respectively. Toxicity (LC50) values of ethyl acetate extract of rice bran were 1161.298 ppm (E2-20), 1170.774 ppm (E2-25) and 701.532 ppm (E2-30), while toxicity (LC50) values of n-hexane extract of rice bran were 592.901 ppm (E3-20), 617.425 ppm (E3-25) and 695.198 ppm (E3-30). Keywords: Rice bran, secondary metabolite, toxicity, ultrasonic method Bekatul diekstrak menggunakan metode ultrasonik dengan variasi pelarut (etanol, etil asetat dan n-heksana) dan lama ekstraksi (20, 25 dan 30 menit). Tujuan penelitian ini adalah mengetahui nilai toksisitas ekstrak bekatul dengan variasi pelarut dan lama ekstraksi. Ekstrak bekatul diuji kemampuan toksisitas terhadap larva udang dengan metode BSLT dan diuji fitokimia dengan reagen. Data kematian larva udang dianalisis dengan analisis probit untuk menentukan nilai LC50. Rendemen ekstrak etanol bekatul yang diperoleh pada sampel E1-20, E1-25 dan E1-30 masing-masing sebesar 18,159; 19,132; dan 18,280%. Adapun rendemen ekstrak etil asetat dan n-heksana bekatul sebesar 8,302% (E2-20), 7,282% (E2-25), 9,18% (E2-30), 7,815% (E3-20), 7,125% (E3-25) dan 7,279% (E3-30). Senyawa metabolit sekunder yang terkandung pada masing-masing ekstrak adalah flavonoid, steroid dan triterpenoid. Nilai LC50 ekstrak etanol pada masing-masing sampel E1-20, E1-25 dan E1-30 adalah 613,258; 673,210 dan 2217,255 ppm. Ekstrak etil asetat bekatul pada sampel E2-20, E2-25 dan E2-30 memberikan nilai toksisitas dengan LC50 sebesar 1161,398; 1170,774 dan 701,532 ppm, berturut-turut. Nilai toksisitas (LC50) ekstrak n-heksana pada sampel E3-20, E3-25 dan E3-30 adalah 592,901; 617,425 dan 695,198 ppm, berturut-turut. Kata kunci: Bekatul, metabolit sekunder, toksisitas, metode ultrasonik
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Woytek, Kelly J., Dhandapani Rangasamy, Cynthia Bazaldua-Hernandez, Mike West, and Van G. Wilson. "Effects of mutations within two hydrophilic regions of the bovine papillomavirus type 1 E1 DNA-binding domain on E1–E2 interaction." Journal of General Virology 82, no. 10 (October 1, 2001): 2341–51. http://dx.doi.org/10.1099/0022-1317-82-10-2341.
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The interaction between papillomavirus E1 and E2 proteins is essential for viral genome replication. Using both in vivo and in vitro assays to evaluate the regions of the two proteins necessary for the E1–E2 interaction, three independent interactions were identified for bovine papillomavirus E1: the N terminus of E1 (E1N, residues 1–311) interacts with the E2 transactivation domain (E2TAD) and the E2 DNA-binding domain (E2DBD) and the C terminus of E1 (E1C, residues 315–605) interacts with E2. Nine mutations within E1N were evaluated for their effects on E2 interaction. Five mutations eliminated interaction with the E2TAD; four of these were located within two previously identified conserved, hydrophilic regions, HR1 and HR3. Since HR1 and HR3 residues appear to comprise the origin of replication recognition element for E1, simultaneous interaction with the E2TAD during initiation complex formation would seem unlikely. Consistent with this inference is the fact that three of the five mutants defective for E2TAD binding exhibited wild-type levels of replication. The replication-positive phenotype of these mutants suggests that the E1N–E2TAD interaction is not essential for replication function and is probably involved in some other E1–E2 function, such as regulating transcription. Only one of the five mutations defective for E2TAD binding also prevented E2DBD interaction, indicating that the regions of E1N that interact with the E2TAD and the E2DBD are not identical. The ability of E1N to cooperatively interact with E2 bound to E2-binding site (E2BS) 11 versus E2BS12 was also examined, and cooperative binding was only observed when E2 was bound to E2BS12.
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Liao, Qiu Hui, Xiao Xun Zhang, and Qin Chao Ruan. "Effects of Polymer-Filler Properties on the Warpage of Injection Molded Automobile Door." Advanced Materials Research 239-242 (May 2011): 2511–14. http://dx.doi.org/10.4028/www.scientific.net/amr.239-242.2511.
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The accurate prediction of warpage of injection molded parts is important to achieve successful mold design with high precision. In this study, effects of polymer-filler properties, such as filler aspect ratio (L/D), filler modulus parallel to major axis (E1) and filler modulus perpendicular to major axis (E2), on warpage displacement of automobile door were studied quantitatively by experimental investigation and numerical simulation. The numerical results are in good agreement with the experimental measurements. It is also found that: (1) the thermal displacement decreases as E1 and E2 decrease, (2) the PVT displacement is not influenced by change of L/D, E1 and E2, (3) the orientation effect displacement is neglected small when L/D=1 and E1= E2, and it also increases as L/D and E1/E2 increase.
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Sandrin, Virginie, Pierre Boulanger, Francois Penin, Christelle Granier, François-Loïc Cosset, and Birke Bartosch. "Assembly of functional hepatitis C virus glycoproteins on infectious pseudoparticles occurs intracellularly and requires concomitant incorporation of E1 and E2 glycoproteins." Journal of General Virology 86, no. 12 (December 1, 2005): 3189–99. http://dx.doi.org/10.1099/vir.0.81428-0.
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Hepatitis C virus (HCV) E1 and E2 envelope glycoproteins (GPs) displayed on retroviral cores (HCVpp) are a powerful and highly versatile model system to investigate wild-type HCV entry. To further characterize this model system, the cellular site of HCVpp assembly and the respective roles of the HCV GPs in this process were investigated. By using a combination of biochemical methods with confocal and electron microscopic techniques, it was shown that, in cells producing HCVpp, both E1 and E2 colocalized with retroviral core proteins intracellularly, presumably in multivesicular bodies, but not at the cell surface. When E1 and E2 were expressed individually with retroviral core proteins, only E2 colocalized with and was incorporated on retroviral cores. Conversely, the colocalization of E1 with retroviral core proteins and its efficient incorporation occurred only upon co-expression of E2. Moreover, HCVpp infectivity correlated strictly with the presence of both E1 and E2 on retroviral cores. Altogether, these results confirm that the E1E2 heterodimer constitutes the prebudding form of functional HCV GPs and, more specifically, show that dimerization with E2 is a prerequisite for efficient E1 incorporation onto particles.
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Ezelle, Heather J., Dubravka Markovic, and Glen N. Barber. "Generation of Hepatitis C Virus-Like Particles by Use of a Recombinant Vesicular Stomatitis Virus Vector." Journal of Virology 76, no. 23 (December 1, 2002): 12325–34. http://dx.doi.org/10.1128/jvi.76.23.12325-12334.2002.
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ABSTRACT Hepatitis C virus (HCV), a major etiologic agent of hepatocellular carcinoma, presently infects approximately 400 million people worldwide, making the development of protective measures against HCV infection a key objective. Here we have generated a recombinant vesicular stomatitis virus (VSV), which expresses the HCV structural proteins, by inserting the contiguous Core, E1, and E2 coding region of HCV into the VSV genome. Recombinant VSV expressing HCV Core, E1, and E2 (VSV-HCV-C/E1/E2) grew to high titers in vitro and efficiently expressed the incorporated HCV gene product, which became fully processed into the individual HCV structural proteins. Biochemical and biophysical analysis indicated that the HCV Core, E1, and E2 proteins assembled to form HCV-like particles (HCV-LPs) possessing properties similar to the ultrastructural properties of HCV virions. Mice immunized with VSV-HCV-C/E1/E2 generated cell-mediated immune responses to all of the HCV structural proteins, and humoral responses, particularly to E2, were also readily evident. Our data collectively indicate that engineered VSVs expressing HCV Core, E1, and E2 and/or HCV-LPs represent useful tools in vaccine and immunotherapeutic strategies designed to address HCV infection.
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Rothstein, Susan Deborah. "Secondary predication and aspectual structure." ZAS Papers in Linguistics 17 (January 1, 2000): 241–64. http://dx.doi.org/10.21248/zaspil.17.2000.49.
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This paper presents an analysis of secondary predicates as aspectual modifiers and secondary predication as a summing operation which sums the denotation of the matrix verb and the secondary predicate. I argue that, as opposed to the summing peration involved in simple conjunction, there is a constraint on secondary predication; in the 0 case of depictives, the event introduced by the matrix verb must be PART-OF the event introduced by the secondary predicate, where e1 is PART-OF e2 if the running time of e1 is contained in the running time of e2 and if e1 and e2 share a grammatical argument. I argue resultative predication differs from depictive predication in that the PART-OF constraint holds in resultative constructions between the event which is the culmination of e1 and e2: formally, while depictive predication introduces the statement PART-OF(e1,e2), resultative predication introduces the statement PART-OF(cul(e1),e2). I show that this is all that is necessary to explain the well-known properties of resultative predication.
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Titolo, Steve, Alex Pelletier, Frédéric Sauvé, Karine Brault, Elizabeth Wardrop, Peter W. White, Anthony Amin, Michael G. Cordingley, and Jacques Archambault. "Role of the ATP-Binding Domain of the Human Papillomavirus Type 11 E1 Helicase in E2-Dependent Binding to the Origin." Journal of Virology 73, no. 7 (July 1, 1999): 5282–93. http://dx.doi.org/10.1128/jvi.73.7.5282-5293.1999.
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ABSTRACT Replication of the genome of human papillomaviruses (HPV) is initiated by the recruitment of the viral E1 helicase to the origin of DNA replication by the viral E2 protein, which binds specifically to the origin. We determined, for HPV type 11 (HPV-11), that the C-terminal 296 amino acids of E1 are sufficient for interaction with the transactivation domain of E2 in the yeast two-hybrid system and in vitro. This region of E1 encompasses the ATP-binding domain. Here we have examined the role of this ATP-binding domain, and of ATP, on E2-dependent binding of E1 to the origin. Several amino acid substitutions in the phosphate-binding loop (P loop), which is implicated in binding the triphosphate moiety of ATP, abolished E2 binding, indicating that the structural integrity of this domain is essential for the interaction. The structural constraints imposed on the E1 P loop may differ between HPV-11 and bovine papillomavirus type 1 (BPV-1), since the P479S substitution that inactivates BPV-1 E1 is tolerated in the HPV-11 enzyme. Other substitutions in the E1 P loop, or in two other conserved motifs of the ATP-binding domain, were tolerated, indicating that ATP binding is not essential for interaction with E2. Nevertheless, ATP-Mg stimulated the E2-dependent binding of E1 to the origin in vitro. This stimulation was maximal at the physiological temperature (37°C) and did not require ATP hydrolysis. In contrast, ATP-Mg did not stimulate the E2-dependent binding to the origin of an E1 protein containing only the C-terminal domain (353 to 649) or that of mutant E1 proteins with alterations in the DNA-binding domain. These results are discussed in light of a model in which the E1 ATP-binding domain is required for formation of the E2-binding surface and can, upon the binding of ATP, facilitate and/or stabilize the interaction of E1 with the origin.
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Jiang, W. Q., V. Wendel-Hansen, A. Lundkvist, N. Ringertz, G. Klein, and A. Rosen. "Intranuclear distribution of Epstein-Barr virus-encoded nuclear antigens EBNA-1, -2, -3 and -5." Journal of Cell Science 99, no. 3 (July 1, 1991): 497–502. http://dx.doi.org/10.1242/jcs.99.3.497.
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Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) express at least seven virally encoded proteins. Their functional role, and their relationships to each other and to normal nuclear constituents are virtually unknown. As the first step towards a topographical study, the intranuclear distribution of EBV-encoded nuclear antigens EBNA-1, -2, -3 and -5 (abbreviated E1, E2 etc.) was examined in EBV-transformed LCLs by immunofluorescence and digital image analysis of fluorescence patterns. E1-E3 showed a finely granular distribution. The E2 patterns were virtually identical when comparing indirect staining using an E2-specific mouse monoclonal antibody with anticomplement immunofluorescence using a human antibody, rendered monospecific to E2 by absorption. The E1/E2 patterns showed 32% overlap and the E2/E3 10% overlap in the high overlap category (66.7-100%), while the E2/E2 comparison with two reagents showed 61% overlap in this category. This suggests that E2 and E3 largely appear in different nuclear structures, whereas E1 appears to be randomly distributed with regard to E2. The E5 pattern was radically different from that of E1, E2 and E3. The anti-E5 mouse monoclonal antibody detected 4–10 huge, globular, sharply circumscribed dots, located in dispersed chromatin areas, while the distribution of E1, E2 and E3 showed no obvious relationship to chromatin distribution. The methods described here allow a more refined topographical analysis of the EBNA protein family, mostly in relation to each other, in relation to other nuclear proteins, and with respect to specialized functional domains in interphase chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)
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Kučić Grgić, Dajana, Martina Miloloža, Vesna Ocelić Bulatović, Šime Ukić, Miroslav Slouf, and Veronika Gajdosova. "Screening the Efficacy of a Microbial Consortium of Bacteria and Fungi Isolated from Different Environmental Samples for the Degradation of LDPE/TPS Films." Separations 10, no. 2 (January 24, 2023): 79. http://dx.doi.org/10.3390/separations10020079.
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In this study, a screening of the efficacy of a microbial consortium of bacteria and fungi isolated from activated sludge, river sediment, and compost for the degradation of LDPE/TPS was performed. According to the morphological and biochemical characterization, eight bacteria, Bacillus sonorensis, Bacillus subtilis, Lysinibacillus massiliensis, Bacillus licheniformis, Bacillus indicus, Bacillus megaterium, Bacillus cereus, and Pseudomonas alcaligenes, five molds, Aspergillus sp. 1, Aspergillus sp. 2, Trichoderma sp., Rhizopus sp., Penicillium sp., and Alternaria sp., and a yeast, Candida parapsilosis, were identified. The first experiment E1 was inoculated with microorganisms isolated from activated sludge and river sediment, and E2 with microorganisms isolated from compost. In both experiments, different types of polymeric materials, low density polyethylene (E1-1 and E2-1), thermoplastic starch (E1-2 and E2-2), low density polyethylene + thermoplastic starch (E1-3 and E2-3), low density polyethylene + thermoplastic starch + styrene-ethylene-styrene (E1-4 and E2-4) were added. The obtained results, weight loss, SEM, and FTIR analysis showed that the microorganisms in both experiments were able to degrade polymeric materials. The mixed culture of microorganisms in experiments E1-2 and E2-2 completely degraded TPS (thermoplastic starch). The percent weight losses of LDPE, LDPE+20% TPS, and LDPE+20% TPS+SEBS in experiment E1 were 3.3184%, 14.1152%, and 16.0062% and in experiment E2 were 3.9625%, 20.4520% and 21.9277%, respectively. SEM microscopy shows that the samples with a LDPE matrix exhibited moderate surface degradation and negligible oxidative degradation under the given conditions. FTIR/ATR data demonstrate that degradation was more intense in E2 than in E1.
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de Mes, T. Z. D., K. Kujawa-Roeleveld, G. Zeeman, and G. Lettinga. "Anaerobic biodegradation of estrogens—hard to digest." Water Science and Technology 57, no. 8 (April 1, 2008): 1177–82. http://dx.doi.org/10.2166/wst.2008.102.
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Although many publications are available on the fate of estrone (E1), 17β-estradiol (E2) and 17α-ethynylestradiol (EE2) during aerobic wastewater treatment, little is published on their fate under strictly anaerobic conditions. Present research investigated the digestibility of E1 and EE2, using digested pig manure, granular UASB sludge, UASB-septic tank sludge and activated sludge as inocula. Besides, actual concentrations were measured in a UASB septic tank treating black water. Under anaerobic conditions E1 is reduced to E2 but the extent of this reduction depends on type of inoculum. No significant loss of the sum of E1 and E2 and of EE2 was observed. Adsorption was responsible for a 32–35% loss of E1 and E2 from the liquid phase in the UASB septic tank and the effluent still contained considerable concentrations of respectively 4.02 μg/l and 18.79 μg/l for E1 and E2 with a large fraction present in conjugated form. No EE2 was detected in the UASB effluent
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Onda, K., Y. Nakamura, C. Takatoh, A. Miya, and Y. Katsu. "The behavior of estrogenic substances in the biological treatment process of sewage." Water Science and Technology 47, no. 9 (May 1, 2003): 109–16. http://dx.doi.org/10.2166/wst.2003.0504.
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A study was conducted for about one year on the fate and behavior of estrogens, namely 17β-estradiol (E2), estrone (E1), and estriol (E3) in an activated sludge process of a pilot scale plant supplied with domestic sewage. A simultaneous analytical method for these three substances using LC-MS/MS was developed and applied to sewage samples. The average removal of E2 was 94.7%, while that of E3 was 96.9%. In contrast, the average removal of E1 was relatively low at 69.2% with a maximum concentration of 55.4 ng/L detected in the treated water. The theoretical values of estrogenic activity calculated from the concentrations of each natural estrogen in treated water were found to correlate with the values of estrogenic activity measured by a yeast estrogen screening assay. The effect of E2 on estrogenic activity in influent was found to be high, while that of E1 in treated water was considerably higher. In batch treatment tests on E2, E2 turned into E1 immediately after being charged. After three hours of aeration, the values of both E1 and E2 were around threshold limits. It was determined from this that E1 and E2 were substances that could be degraded by biological treatment. As the removal of E2 was found to be sufficiently high at times, optimization of operational conditions based on E1 removal should be important for reducing estrogenic activity in treated water.
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Voitenleitner, Christian, and Michael Botchan. "E1 Protein of Bovine Papillomavirus Type 1 Interferes with E2 Protein-Mediated Tethering of the Viral DNA to Mitotic Chromosomes." Journal of Virology 76, no. 7 (April 1, 2002): 3440–51. http://dx.doi.org/10.1128/jvi.76.7.3440-3451.2002.
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ABSTRACT Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal plasmids. It is therefore of vital importance for viruses to ensure nuclear retention and proper segregation of their viral DNA. The bovine papillomavirus (BPV) E2 enhancer protein plays a key role in these processes by tethering the viral DNA to the host cell chromosomes. Viral genomes that harbor phosphorylation mutations in the E2 gene are transformation defective, and for these mutant genomes, neither the viral DNA nor the E2 protein is detected on mitotic chromosomes, while other key functions of E2 in transcription and replication were wild type. Moreover, secondary mutations in both the E2 and E1 proteins lead to suppression of the phosphorylation mutant phenotype and resulted in reattachment of the viral DNA and the E2 protein onto mitotic chromosomes, suggesting that E1 also plays a role in viral genome partitioning. The E1 protein was cytologically always excluded from mitotic chromatin, either as a suppressor allele or as the wild type. In the absence of other viral proteins, an E2 protein containing alanine substitutions for phosphorylation substrates in the hinge region (E2-A4) was detected as wild-type on mitotic chromosomes. However, when wild-type E1 protein levels were increased in cells expressing either the A4 mutant E2 proteins or wild-type E2, the E2-A4 protein was much more sensitive to chromosomal dislocation than was the wild-type protein. In contrast, suppressor alleles of E1 were not capable of such abrogation of E2 binding (A4 or wild-type) to chromosomes. These results suggest that wild-type E1 can be a negative regulator of the chromosomal attachment of E2.
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Lim, Daniel A., Manfred Gossen, Chris W. Lehman, and Michael R. Botchan. "Competition for DNA Binding Sites between the Short and Long Forms of E2 Dimers Underlies Repression in Bovine Papillomavirus Type 1 DNA Replication Control." Journal of Virology 72, no. 3 (March 1, 1998): 1931–40. http://dx.doi.org/10.1128/jvi.72.3.1931-1940.1998.
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ABSTRACT Papillomaviruses establish a long-term latency in vivo by maintaining their genomes as nuclear plasmids in proliferating cells. Bovine papillomavirus type 1 encodes two proteins required for viral DNA replication: the helicase E1 and the positive regulator E2. The homodimeric E2 is known to cooperatively bind to DNA with E1 to form a preinitiation complex at the origin of DNA replication. The virus also codes for two short forms of E2 that can repress viral functions when overexpressed, and at least one copy of the repressor is required for stable plasmid maintenance in transformed cells. Employing a tetracycline-regulated system to control E1 and E2 production from integrated loci, we show that the short form of E2 negatively regulates DNA replication. We also found that the short form could repress replication in a cell-free replication system and that the repression requires the DNA binding domain of the protein. In contrast, heterodimers of the short and long forms were activators and, by footprint analysis, were shown to be as potent as homodimeric E2 in loading E1 to its cognate site. DNA binding studies show that when E1 levels are low and are dependent upon E2 for occupancy of the origin site, the repressor can block E1-DNA interactions. We conclude that DNA replication modulation results from competition between the different forms of E2 for DNA binding. Given that heterodimers are active and that the repressor form of E2 shows little cooperativity with E1 for DNA binding, this protein is a weak repressor.
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Hubert, Walter G., and Laimonis A. Laimins. "Human Papillomavirus Type 31 Replication Modes during the Early Phases of the Viral Life Cycle Depend on Transcriptional and Posttranscriptional Regulation of E1 and E2 Expression." Journal of Virology 76, no. 5 (March 1, 2002): 2263–73. http://dx.doi.org/10.1128/jvi.76.5.2263-2273.2002.
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ABSTRACT The E1 and E2 proteins are both required for papillomavirus DNA replication, and replication efficiency is controlled by the abundance of these factors. In human papillomaviruses (HPVs), the regulation of E1 and E2 expression and its effect on viral replication are not well understood. In particular, it is not known if E1 and E2 modulate their own expression and how posttranscriptional mechanisms may affect the levels of the replication proteins. Previous studies have implicated splicing within the E6 open reading frame (ORF) as being important for modulating replication of HPV type 31 (HPV31) through altered expression of E1 and E2. To analyze the function of the E6 intron in viral replication more specifically, we examined the effects of E6 splicing mutations in the context of entire viral genomes in transient assays. HPV31 genomes which had mutations in the splice donor site (E6SD) or the splice acceptor site (E6SA), a deletion of the intron (E6ID), or substituted heterologous intron sequences (E6IS) were constructed. Compared to wild-type (wt) HPV31, pHPV31-E6SD, -E6SA, and -E6IS replicated inefficiently while pHPV31-E6ID replicated at an intermediate level. Cotransfection of the E6 mutant genomes with an E1 expression vector strongly activated their replication levels, indicating that efficient expression of E1 requires E6 internal splicing. In contrast, replication was activated only moderately with an E2 expression vector. Replacing the wt E6 intron in HPV31 with a heterologous intron from simian virus 40 (E6SR2) resulted in replication levels similar to that of the wt in the absence of expression vectors, suggesting that mRNA splicing upstream of the E1 ORF is important for high-level replication. To examine the effects of E6 intron splicing on E1 and E2 expression directly, we constructed reporter DNAs in which the luciferase coding sequences were fused in frame to the E1 (E1Luc) or E2 (E2Luc) gene. Reporter activities were then analyzed in transient assays with cotransfected E1 or E2 expression vectors. Both reporters were moderately activated by E1 in a dose-dependent manner. In addition, E1Luc was activated by low doses of E2 but was repressed at high doses. In contrast, E2 had little effect on E2Luc activity. These data indicate that E1 expression and that of E2 are interdependent and regulated differentially. When the E6 splicing mutations were analyzed in both reporter backgrounds, only E1Luc activities correlated with splicing competence in the E6 ORF. These findings support the hypothesis that the E6 intron primarily regulates expression of E1. Finally, in long-term replication assays, none of the E6 mutant genomes could be stably maintained. However, cotransfection of the E6 splicing mutant genomes with pHPV31-E7NS, which contains a nonsense mutation in the E7 coding sequence, restored stable replication of some mutants. Our observations indicate that E1 expression and that of E2 are differentially regulated at multiple levels and that efficient expression of E1 is required for transient and stable viral replication. These regulatory mechanisms likely act to control HPV copy number during the various phases of the viral life cycle.
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Gauson, Elaine J., Mary M. Donaldson, Edward S. Dornan, Xu Wang, Molly Bristol, Jason M. Bodily, and Iain M. Morgan. "Evidence Supporting a Role for TopBP1 and Brd4 in the Initiation but Not Continuation of Human Papillomavirus 16 E1/E2-Mediated DNA Replication." Journal of Virology 89, no. 9 (February 18, 2015): 4980–91. http://dx.doi.org/10.1128/jvi.00335-15.
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ABSTRACTTo replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination.IMPORTANCEHuman papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted.
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Prentoe, Jannick, Christoph M. Janitzek, Rodrigo Velázquez-Moctezuma, Louise Goksøyr, Rebecca W. Olsen, Margherita Fanalista, Elias H. Augestad, et al. "Antigenic and immunogenic evaluation of permutations of soluble hepatitis C virus envelope protein E2 and E1 antigens." PLOS ONE 16, no. 7 (July 30, 2021): e0255336. http://dx.doi.org/10.1371/journal.pone.0255336.
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Yearly, about 1.5 million people become chronically infected with hepatitis C virus (HCV) and for the 71 million with chronic HCV infection about 400,000 die from related morbidities, including liver cirrhosis and cancer. Effective treatments exist, but challenges including cost-of-treatment and wide-spread undiagnosed infection, necessitates the development of vaccines. Vaccines should induce neutralizing antibodies (NAbs) against the HCV envelope (E) transmembrane glycoprotein 2, E2, which partly depends on its interaction partner, E1, for folding. Here, we generated three soluble HCV envelope protein antigens with the transmembrane regions deleted (i.e., fused peptide backbones), termed sE1E2 (E1 followed by E2), sE2E1 (E2 followed by E1), and sE21E (E2 followed by inverted E1). The E1 inversion for sE21E positions C-terminal residues of E1 near C-terminal residues of E2, which is in analogy to how they likely interact in native E1/E2 complexes. Probing conformational E2 epitope binding using HCV patient-derived human monoclonal antibodies, we show that sE21E was superior to sE2E1, which was consistently superior to sE1E2. This correlated with improved induction of NAbs by sE21E compared with sE2E1 and especially compared with sE1E2 in female BALB/c mouse immunizations. The deletion of the 27 N-terminal amino acids of E2, termed hypervariable region 1 (HVR1), conferred slight increases in antigenicity for sE2E1 and sE21E, but severely impaired induction of antibodies able to neutralize in vitro viruses retaining HVR1. Finally, comparing sE21E with sE2 in mouse immunizations, we show similar induction of heterologous NAbs. In summary, we find that C-terminal E2 fusion of E1 or 1E is superior to N-terminal fusion, both in terms of antigenicity and the induction of heterologous NAbs. This has relevance when designing HCV E1E2 vaccine antigens.
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Hartley, Kelly A., and Kenneth A. Alexander. "Human TATA Binding Protein Inhibits Human Papillomavirus Type 11 DNA Replication by Antagonizing E1-E2 Protein Complex Formation on the Viral Origin of Replication." Journal of Virology 76, no. 10 (May 15, 2002): 5014–23. http://dx.doi.org/10.1128/jvi.76.10.5014-5023.2002.
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ABSTRACT The human papillomavirus (HPV) protein E2 possesses dual roles in the viral life cycle. By interacting directly with host transcription factors in basal keratinocytes, E2 promotes viral transcription. As keratinocyte differentiation progresses, E2 associates with the viral helicase, E1, to activate vegetative viral DNA replication. How E2's major role switches from transcription to replication during keratinocyte differentiation is not understood, but the presence of a TATA site near the viral origin of replication led us to hypothesize that TATA-binding protein (TBP) could affect HPV replication. Here we show that the C-terminal domain of TBP (TBPc) is a potent inhibitor of E2-stimulated HPV DNA replication in vitro (50% inhibitory concentration = 0.56 nM). Increasing the E1 concentration could not overcome TBPc inhibition in replication assays, indicating that TBPc is a noncompetitive inhibitor of E1 binding. While direct E2-TBPc association could be demonstrated, this interaction could not fully account for the mechanism of TBPc-mediated inhibition of viral replication. Because E2 supports sequence-specific binding of E1 to the viral ori, we proposed that TBPc antagonizes E1-ori association indirectly through inhibition of E2-DNA binding. Indeed, TBPc potently antagonized E2 binding to DNA in the absence (Ki = 0.5 ± 0.1 nM) and presence (Ki = 0.6 ± 0.3 nM) of E1. Since E2 and TBPc cannot be coadjacent on viral sequences, direct DNA-binding competition between TBPc and E2 was responsible for replication inhibition. Given the ability of TBPc to inhibit HPV DNA replication in vitro and data indicating that TBPc antagonized E2-ori association, we propose that transcription factors regulate HPV DNA replication as well as viral transcription.
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Kasukawa, Hiroaki, Peter M. Howley, and John D. Benson. "A Fifteen-Amino-Acid Peptide Inhibits Human Papillomavirus E1-E2 Interaction and Human Papillomavirus DNA Replication In Vitro." Journal of Virology 72, no. 10 (October 1, 1998): 8166–73. http://dx.doi.org/10.1128/jvi.72.10.8166-8173.1998.
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ABSTRACT Mutation of the conserved glutamic acid residue at position 39 of human papillomavirus type 16 (HPV-16) E2 to alanine (E39A) disrupts its E1 interaction activity and its replication function in transient replication assays but does not affect E2 transcriptional activation. This E39A mutation also disrupts replication activity of HPV-16 E2 in HPV-16 in vitro DNA replication. On this basis, we designed 23- and 15-amino-acid peptides derived from HPV-16 E2 sequences flanking the E39 residue and tested the ability of these peptides to inhibit interaction between HPV-16 E1 and E2 in vitro. The inhibitory activity of these peptides was specific, since analogous peptides in which alanine was substituted for the E39 residue did not inhibit interaction. The 15-amino-acid peptide E2N-WP15 was the smallest peptide tested that effectively inhibited HPV-16 E1-E2 interaction. This peptide also inhibited in vitro replication of HPV-16 DNA. The efficacy of E2N-WP15 was not exclusive to HPV-16: this peptide also inhibited interaction of HPV-11 E1 with the E2 proteins of both HPV-11 and HPV-16 and inhibited in vitro replication with these same combinations of E1 and E2 proteins. These results provide further evidence that E1-E2 interaction is required for papillomavirus DNA replication and constitute the first demonstration that inhibition of this interaction is sufficient to prevent HPV DNA replication in vitro.
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Bassett, Suzanne E., David L. Thomas, Kathleen M. Brasky, and Robert E. Lanford. "Viral Persistence, Antibody to E1 and E2, and Hypervariable Region 1 Sequence Stability in Hepatitis C Virus-Inoculated Chimpanzees." Journal of Virology 73, no. 2 (February 1, 1999): 1118–26. http://dx.doi.org/10.1128/jvi.73.2.1118-1126.1999.
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ABSTRACT The relationship of viral persistence, the immune response to hepatitis C virus (HCV) envelope proteins, and envelope sequence variability was examined in chimpanzees. Antibody reactivity to the HCV envelope proteins E1 or E2 was detected by enzyme-linked immunosorbent assay (ELISA) in more than 90% of a human serum panel. Although the ELISAs appeared to be sensitive indicators of HCV infection in human serum panels, the results of a cross-sectional study revealed that a low percentage of HCV-inoculated chimpanzees had detectable antibody to E1 (22%) and E2 (15%). Viral clearance, which was recognized in 28 (61%) of the chimpanzees, was not associated with an antibody response to E1 or E2. On the contrary, antibody to E2 was observed only in viremic chimpanzees. A longitudinal study of animals that cleared the viral infection or became chronically infected confirmed the low level of antibody to E1, E2, and the HVR-1. In 10 chronically infected animals, the sequence variation in the E2 hypervariable region (HVR-1) was minimal and did not coincide with antibody to E2 or to the HVR-1. In addition, low nucleotide and amino acid sequence variation was observed in the E1 and E2 regions from two chronically infected chimpanzees. These results suggest that mechanisms in addition to the emergence of HVR-1 antibody escape variants are involved in maintaining viral persistence. The significance of antibodies to E1 and E2 in the chimpanzee animal model is discussed.
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Csermely, P., C. Katopis, B. A. Wallace, and A. Martonosi. "The E1→E2 transition of Ca2+-transporting ATPase in sarcoplasmic reticulum occurs without major changes in secondary structure. A circular-dichroism study." Biochemical Journal 241, no. 3 (February 1, 1987): 663–69. http://dx.doi.org/10.1042/bj2410663.
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C.d. spectroscopy was used to investigate the structures of Ca2+-ATPase (Ca2+-transporting ATPase) in the E1 and E2 states in native, in fluorescein isothiocyanate (FITC)-labelled and in solubilized sarcoplasmic reticulum (SR) preparations. The E1 state was stabilized by 100 microM-Ca2+ and the E2 state by 0.5 mM-Na3 VO4 and 0.1 mM-EGTA. There were no significant differences detected in the c.d. spectra and the calculated secondary structures between the E1 and E2 states in any of the three types of preparations. The FITC-labelled SR did show the characteristic changes in FITC fluorescence on addition of Ca2+ or vanadate, indicating that the preparation was competent for E1----E2 transitions. Therefore the absence of changes in the c.d. spectra implies that the E1----E2 transition in the Ca2+-ATPase does not involve a major net rearrangement of the polypeptide backbone conformation.
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Ritter, Clare L., William F. Prigge, Mark A. Reichert, and Danuta Malejka-Giganti. "Oxidations of 17β-estradiol and estrone and their interconversions catalyzed by liver, mammary gland and mammary tumor after acute and chronic treatment of rats with indole-3-carbinol or β-naphthoflavone." Canadian Journal of Physiology and Pharmacology 79, no. 6 (June 1, 2001): 519–32. http://dx.doi.org/10.1139/y01-020.
Full textAbstract:
Altered cytochrome P450-catalyzed metabolism of 17β-estradiol (E2) and estrone (E1) in the liver and (or) extrahepatic tissues may affect estrogen-sensitive tumorigenesis. We examined the effects of oral treatments of (i) indole-3-carbinol (I3C) at 250 or 500 mg/kg or β-naphthoflavone (β-NF) at 40 mg/kg of body weight (bw)/day from 51 to 54 days of age (acute regimen), and (ii) I3C at 250 mg/kg or β-NF at 20 mg/kg bw given 3x/week from 10 to 22 weeks of age (chronic regimen) in female Sprague-Dawley rats. We determined the effects of these treatments on the P450 content and P450 (CYP)-specific activities in the liver, P450-dependent metabolism of E2 and E1 by the liver and mammary gland, and interconversion of E1 and E2 catalyzed by 17β-hydroxysteroid dehydrogenase (17β-HSD) in these tissues and malignant mammary tumors. I3C at the two levels of acute regimen elicited similar responses. Acute and chronic treatments with I3C, but not β-NF, increased P450 content ~2-fold. I3C, and to a lesser extent β-NF, increased CYP1A1 and CYP1A2 probe activities in liver up to 117- and 27- fold, respectively, and after acute regimens, that of CYP3A by ~1.8-fold. I3C also increased activity of CYP2B up to 100-fold. Overall hepatic metabolism of E2 and E1, which was ~2-fold greater at 55 than 155 days of age, was increased (~2.8-fold) by I3C with 2-, 4-, 16α-, 6α-, 6β-, and 15α-hydroxy (OH) comprising [Formula: see text]54, 3, 2, ~2, ~5, 7, and 2%, respectively, of E1 and E2 metabolites. Acute regimens of β-NF increased 2- and 15α-OH-E2 (62 and 5% of total) from E2 and 2-, 4-, and 6α-OH-E1 + 6β-OH-E1 (32, 13, and 4% of total) from E1. Mammary gland metabolized E2 to E1 and small amounts of 15α-, 4-, 16α-, 6β-, and 6α-OH-E2. After the acute IC3 regimen, E2 was also converted to 2-OH-E2. 17β-HSD-catalyzed oxidation of E2 was favored in the liver and reduction of E1 was favored in mammary gland and tumor (= 1% of hepatic activity). An increased (~2-fold) ratio of reductive to oxidative activities in malignant mammary tumors by chronic I3C regimen may stimulate tumor growth. This is the first report showing that after chronic oral regimens, the I3C-, but not β-NF-, induced changes in CYP complement led to elevated E2 and E1 metabolism. The persistent effects of increased putative carcinogenic and estrogenic 4- and 16α-OH as well as 6α- and 6β-OH-E2 and 6β-OH-E1 might counteract those of the less estrogenic 2-OH metabolites, thus accounting for the lack of suppression of mammary tumorigenesis by I3C in our previous study.Key words: estrogen metabolism, P450, 17β-hydroxysteroid dehydrogenase, indole-3-carbinol, β-naphthoflavone.