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Sample records for e2 ubiquitin-conjugating enzyme

  1. The ubiquitin-conjugating enzyme E2-EPF is overexpressed in cervical cancer and associates with tumor growth.

    PubMed

    Liang, Jing; Nishi, Hirotaka; Bian, Mei-Lu; Higuma, Chinatsu; Sasaki, Toru; Ito, Hiroe; Isaka, Keiichi

    2012-10-01

    We found that the ubiquitin-conjugating enzyme E2-EPF mRNA is highly expressed in cervical squamous cancer relative to normal tissues and its expression levels positively correlate with clinical stage. Reduction of E2-EPF protein levels by >80% using shRNA decreases the expression levels of HIF-1α, and the proliferation, invasion and tumorigenicity of SiHa, a cervical squamous cancer cell line. E2-EPF knockdown also increases the chemosensitivity to topoisomerase I inhibitor (topotecan) and II (etoposide and doxorubicin). Our results suggest that E2-EPF is associated with the growth and aggressivity of cervical tumor cells. Targeting the E2-EPF pathway may have potential clinical applications for the treatment of cervical cancer.

  2. E2 superfamily of ubiquitin-conjugating enzymes: constitutively active or activated through phosphorylation in the catalytic cleft.

    PubMed

    Valimberti, Ilaria; Tiberti, Matteo; Lambrughi, Matteo; Sarcevic, Boris; Papaleo, Elena

    2015-10-14

    Protein phosphorylation is a modification that offers a dynamic and reversible mechanism to regulate the majority of cellular processes. Numerous diseases are associated with aberrant regulation of phosphorylation-induced switches. Phosphorylation is emerging as a mechanism to modulate ubiquitination by regulating key enzymes in this pathway. The molecular mechanisms underpinning how phosphorylation regulates ubiquitinating enzymes, however, are elusive. Here, we show the high conservation of a functional site in E2 ubiquitin-conjugating enzymes. In catalytically active E2s, this site contains aspartate or a phosphorylatable serine and we refer to it as the conserved E2 serine/aspartate (CES/D) site. Molecular simulations of substrate-bound and -unbound forms of wild type, mutant and phosphorylated E2s, provide atomistic insight into the role of the CES/D residue for optimal E2 activity. Both the size and charge of the side group at the site play a central role in aligning the substrate lysine toward E2 catalytic cysteine to control ubiquitination efficiency. The CES/D site contributes to the fingerprint of the E2 superfamily. We propose that E2 enzymes can be divided into constitutively active or regulated families. E2s characterized by an aspartate at the CES/D site signify constitutively active E2s, whereas those containing a serine can be regulated by phosphorylation.

  3. RING-type E3 ligases: Master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination

    PubMed Central

    Metzger, Meredith B.; Pruneda, Jonathan N.; Klevit, Rachel E.; Weissman, Allan M.

    2013-01-01

    RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. PMID:23747565

  4. Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

    SciTech Connect

    Imura, Yoshiyuki, E-mail: imura@brs.nihon-u.ac.jp; Molho, Melissa; Chuang, Chingkai

    Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement bothmore » defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.« less

  5. Multiple E2 ubiquitin-conjugating enzymes regulate human cytomegalovirus US2-mediated immunoreceptor downregulation.

    PubMed

    van de Weijer, Michael L; Schuren, Anouk B C; van den Boomen, Dick J H; Mulder, Arend; Claas, Frans H J; Lehner, Paul J; Lebbink, Robert Jan; Wiertz, Emmanuel J H J

    2017-09-01

    Misfolded endoplasmic reticulum (ER) proteins are dislocated towards the cytosol and degraded by the ubiquitin-proteasome system in a process called ER-associated protein degradation (ERAD). During infection with human cytomegalovirus (HCMV), the viral US2 protein targets HLA class I molecules (HLA-I) for degradation via ERAD to avoid elimination by the immune system. US2-mediated degradation of HLA-I serves as a paradigm of ERAD and has facilitated the identification of TRC8 (also known as RNF139) as an E3 ubiquitin ligase. No specific E2 enzymes had previously been described for cooperation with TRC8. In this study, we used a lentiviral CRISPR/Cas9 library targeting all known human E2 enzymes to assess their involvement in US2-mediated HLA-I downregulation. We identified multiple E2 enzymes involved in this process, of which UBE2G2 was crucial for the degradation of various immunoreceptors. UBE2J2, on the other hand, counteracted US2-induced ERAD by downregulating TRC8 expression. These findings indicate the complexity of cellular quality control mechanisms, which are elegantly exploited by HCMV to elude the immune system. © 2017. Published by The Company of Biologists Ltd.

  6. Ubiquitin-conjugating enzyme E2-like gene associated to pathogen response in Concholepas concholepas: SNP identification and transcription expression.

    PubMed

    Núñez-Acuña, Gustavo; Aguilar-Espinoza, Andrea; Chávez-Mardones, Jacqueline; Gallardo-Escárate, Cristian

    2012-10-01

    Ubiquitin-conjugated E2 enzyme (UBE2) is one of the main components of the proteasome degradation cascade. Previous studies have shown an increase of expression levels in individuals challenged to some pathogen organism such as virus and bacteria. The study was to characterize the immune response of UBE2 gene in the gastropod Concholepas concholepas through expression analysis and single nucleotide polymorphisms (SNP) discovery. Hence, UBE2 was identified from a cDNA library by 454 pyrosequencing, while SNP identification and validation were performed using De novo assembly and high resolution melting analysis. Challenge trials with Vibrio anguillarum was carried out to evaluate the relative transcript abundance of UBE2 gene from two to thirty-three hours post-treatment. The results showed a partial UBE2 sequence of 889 base pair (bp) with a partial coding region of 291 bp. SNP variation (A/C) was observed at the 546th position. Individuals challenged by V. anguillarum showed an overexpression of the UBE2 gene, the expression being significantly higher in homozygous individuals (AA) than (CC) or heterozygous individuals (A/C). This study contributes useful information relating to the UBE2 gene and its association with innate immune response in marine invertebrates. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.

    PubMed

    Ramadan, Abdelaziz; Nemoto, Keiichirou; Seki, Motoaki; Shinozaki, Kazuo; Takeda, Hiroyuki; Takahashi, Hirotaka; Sawasaki, Tatsuya

    2015-11-10

    Protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which ~470 are RING-type E3s. A large proportion of the RING E3's gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, cannot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised. Using the RIKEN Arabidopsis full-length cDNA library (RAFL) with the 'split-primer' PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time. The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction

  8. The Ubiquitin-Conjugating Enzyme E2-EPF Is Overexpressed in Primary Breast Cancer and Modulates Sensitivity to Topoisomerase II Inhibition1

    PubMed Central

    Tedesco, Donato; Zhang, Jianhuan; Trinh, Lan; Lalehzadeh, Guita; Meisner, Rene; Yamaguchi, Ken D; Ruderman, Daniel L; Dinter, Harald; Zajchowski, Deborah A

    2007-01-01

    We identified the ubiquitin-conjugating enzyme E2-EPF mRNA as differentially expressed in breast tumors relative to normal tissues and performed studies to elucidate its putative role in cancer. We demonstrated that overexpression of E2-EPF protein correlated with estrogen receptor (ER) negativity in breast cancer specimens and that its expression is cell cycle-regulated, suggesting a potential function for E2-EPF in cell cycle progression. However, reduction of E2-EPF protein levels by > 80% using RNAi had no significant effects on the proliferation of HeLa cervical cancer cells or ER- MDA-MB-231 or MDA-MB-453 breast cancer cells. Because E2-EPF protein levels were elevated during the G2/M phase of the cell cycle and because E2-EPF mRNA in tumor specimens was frequently coexpressed with genes involved in cell cycle control, spindle assembly, and mitotic surveillance, the possibility that E2-EPF might have a function in the cellular response to agents that induce a G2 checkpoint or an M checkpoint was investigated. E2-EPF knockdown sensitized HeLa cells to the topoisomerase (topo) II inhibitors etoposide and doxorubicin and also increased topo IIα protein levels. These data suggest that combined administration of topo II-directed drugs and E2-EPF inhibitors may enhance their clinical effectiveness. PMID:17710163

  9. The ubiquitin-conjugating enzyme E2-EPF is overexpressed in primary breast cancer and modulates sensitivity to topoisomerase II inhibition.

    PubMed

    Tedesco, Donato; Zhang, Jianhuan; Trinh, Lan; Lalehzadeh, Guita; Meisner, Rene; Yamaguchi, Ken D; Ruderman, Daniel L; Dinter, Harald; Zajchowski, Deborah A

    2007-07-01

    We identified the ubiquitin-conjugating enzyme E2-EPF mRNA as differentially expressed in breast tumors relative to normal tissues and performed studies to elucidate its putative role in cancer. We demonstrated that overexpression of E2-EPF protein correlated with estrogen receptor (ER) negativity in breast cancer specimens and that its expression is cell cycle-regulated, suggesting a potential function for E2-EPF in cell cycle progression. However, reduction of E2-EPF protein levels by > 80% using RNAi had no significant effects on the proliferation of HeLa cervical cancer cells or ER(-) MDA-MB-231 or MDA-MB-453 breast cancer cells. Because E2-EPF protein levels were elevated during the G(2)/M phase of the cell cycle and because E2-EPF mRNA in tumor specimens was frequently coexpressed with genes involved in cell cycle control, spindle assembly, and mitotic surveillance, the possibility that E2-EPF might have a function in the cellular response to agents that induce a G(2) checkpoint or an M checkpoint was investigated. E2-EPF knockdown sensitized HeLa cells to the topoisomerase (topo) II inhibitors etoposide and doxorubicin and also increased topo IIalpha protein levels. These data suggest that combined administration of topo II-directed drugs and E2-EPF inhibitors may enhance their clinical effectiveness.

  10. E2 Ubiquitin-conjugating Enzyme, UBE2C Gene, Is Reciprocally Regulated by Wild-type and Gain-of-Function Mutant p53.

    PubMed

    Bajaj, Swati; Alam, Sk Kayum; Roy, Kumar Singha; Datta, Arindam; Nath, Somsubhra; Roychoudhury, Susanta

    2016-07-01

    Spindle assembly checkpoint governs proper chromosomal segregation during mitosis to ensure genomic stability. At the cellular level, this event is tightly regulated by UBE2C, an E2 ubiquitin-conjugating enzyme that donates ubiquitin to the anaphase-promoting complex/cyclosome. This, in turn, facilitates anaphase-onset by ubiquitin-mediated degradation of mitotic substrates. UBE2C is an important marker of chromosomal instability and has been associated with malignant growth. However, the mechanism of its regulation is largely unexplored. In this study, we report that UBE2C is transcriptionally activated by the gain-of-function (GOF) mutant p53, although it is transcriptionally repressed by wild-type p53. We showed that wild-type p53-mediated inhibition of UBE2C is p21-E2F4-dependent and GOF mutant p53-mediated transactivation of UBE2C is NF-Y-dependent. We further explored that DNA damage-induced wild-type p53 leads to spindle assembly checkpoint arrest by repressing UBE2C, whereas mutant p53 causes premature anaphase exit by increasing UBE2C expression in the presence of 5-fluorouracil. Identification of UBE2C as a target of wild-type and GOF mutant p53 further highlights the contribution of p53 in regulation of spindle assembly checkpoint. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Molecular Characterization of Plant Ubiquitin-Conjugating Enzymes Belonging to the UbcP4/E2-C/UBCx/UbcH10 Gene Family1

    PubMed Central

    Criqui, Marie Claire; de Almeida Engler, Janice; Camasses, Alain; Capron, Arnaud; Parmentier, Yves; Inzé, Dirk; Genschik, Pascal

    2002-01-01

    The anaphase promoting complex or cyclosome is the ubiquitin-ligase that targets destruction box-containing proteins for proteolysis during the cell cycle. Anaphase promoting complex or cyclosome and its activator (the fizzy and fizzy-related) proteins work together with ubiquitin-conjugating enzymes (UBCs) (E2s). One class of E2s (called E2-C) seems specifically involved in cyclin B1 degradation. Although it has recently been shown that mammalian E2-C is regulated at the protein level during the cell cycle, not much is known concerning the expression of these genes. Arabidopsis encodes two genes belonging to the E2-C gene family (called UBC19 and UBC20). We found that UBC19 is able to complement fission yeast (Schizosaccharomyces pombe) UbcP4-140 mutant, indicating that the plant protein can functionally replace its yeast ortholog for protein degradation during mitosis. In situ hybridization experiments were performed to study the expression of the E2-C genes in various tissues of plants. Their transcripts were always, but not exclusively, found in tissues active for cell division. Thus, the UBC19/20 E2s may have a key function during cell cycle, but may also be involved in ubiquitylation reactions occurring during differentiation and/or in differentiated cells. Finally, we showed that a translational fusion protein between UBC19 and green fluorescent protein localized both in the cytosol and the nucleus in stable transformed tobacco (Nicotiana tabacum cv Bright Yellow 2) cells. PMID:12427990

  12. Ubiquitin conjugating enzyme E2-N and sequestosome-1 (p62) are components of the ubiquitination process mediated by the malin-laforin E3-ubiquitin ligase complex.

    PubMed

    Sánchez-Martín, Pablo; Romá-Mateo, Carlos; Viana, Rosa; Sanz, Pascual

    2015-12-01

    Lafora disease (LD, OMIM254780, ORPHA501) is a rare neurodegenerative form of epilepsy related to mutations in two proteins: laforin, a dual specificity phosphatase, and malin, an E3-ubiquitin ligase. Both proteins form a functional complex, where laforin recruits specific substrates to be ubiquitinated by malin. However, little is known about the mechanism driving malin-laforin mediated ubiquitination of its substrates. In this work we present evidence indicating that the malin-laforin complex interacts physically and functionally with the ubiquitin conjugating enzyme E2-N (UBE2N). This binding determines the topology of the chains that the complex is able to promote in the corresponding substrates (mainly K63-linked polyubiquitin chains). In addition, we demonstrate that the malin-laforin complex interacts with the selective autophagy adaptor sequestosome-1 (p62). Binding of p62 to the malin-laforin complex allows its recognition by LC3, a component of the autophagosomal membrane. In addition, p62 enhances the ubiquitinating activity of the malin-laforin E3-ubiquitin ligase complex. These data enrich our knowledge on the mechanism of action of the malin-laforin complex as an E3-ubiquitin ligase and reinforces the role of this complex in targeting substrates toward the autophagy pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Ubiquitin in Motion: Structural Studies of the Ubiquitin-Conjugating Enzyme~Ubiquitin Conjugate

    SciTech Connect

    Pruneda, Jonathan N.; Stoll, Kate E.; Bolton, Laura J.

    2011-03-15

    Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for the transfer of Ub to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2~Ub conjugate. Therefore, full characterization of the structure and dynamics of E2~Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2~Ub conjugates of two human enzymes, UbcH5c~Ub and Ubc13~Ub,more » in solution as determined by nuclear magnetic resonance and small-angle X-ray scattering. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: the UbcH5c~Ub conjugate populates an array of extended conformations, and the population of Ubc13~Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces helix 2 of Ubc13. Finally, we propose that the varied conformations adopted by Ub represent available binding modes of the E2~Ub species and thus provide insight into the diverse E2~Ub protein interactome, particularly with regard to interaction with Ub ligases.« less

  14. A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity1[OPEN

    PubMed Central

    Connor, Richard A.

    2017-01-01

    Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart. PMID:27909045

  15. A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity.

    PubMed

    Zhou, Bangjun; Mural, Ravi V; Chen, Xuanyang; Oates, Matt E; Connor, Richard A; Martin, Gregory B; Gough, Julian; Zeng, Lirong

    2017-02-01

    Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart. © 2017 American Society of Plant Biologists. All Rights Reserved.

  16. Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes

    NASA Technical Reports Server (NTRS)

    Li, Yi-Ping; Chen, Yuling; Li, Andrew S.; Reid, Michael B.

    2003-01-01

    Reactive oxygen species (ROS) are thought to promote muscle atrophy in chronic wasting diseases, but the underlying mechanism has not been determined. Here we show that H2O2 stimulates ubiquitin conjugation to muscle proteins through transcriptional regulation of the enzymes (E2 and E3 proteins) that conjugate ubiquitin to muscle proteins. Incubation of C2C12 myotubes with 100 microM H2O2 increased the rate of 125I-labeled ubiquitin conjugation to muscle proteins in whole cell extracts. This response required at least 4-h exposure to H2O2 and persisted for at least 24 h. Preincubating myotubes with cycloheximide or actinomycin D blocked H2O2 stimulation of ubiquitin-conjugating activity, suggesting that gene transcription is required. Northern blot analyses revealed that H2O2 upregulates expression of specific E3 and E2 proteins that are thought to regulate muscle catabolism, including atrogin1/MAFbx, MuRF1, and E214k. These results suggest that ROS stimulate protein catabolism in skeletal muscle by upregulating the ubiquitin conjugation system.

  17. Characterization and expression analysis of genes encoding ubiquitin conjugating domain-containing enzymes in Carica papaya.

    PubMed

    Jue, Dengwei; Sang, Xuelian; Shu, Bo; Liu, Liqin; Wang, Yicheng; Jia, Zhiwei; Zou, Yu; Shi, Shengyou

    2017-01-01

    Ripening affects the quality and nutritional contents of fleshy fruits and is a crucial process of fruit development. Although several studies have suggested that ubiquitin-conjugating enzyme (E2s or UBC enzymes) are involved in the regulation of fruit ripening, little is known about the function of E2s in papaya (Carica papaya). In the present study, we searched the papaya genome and identified 34 putative UBC genes, which were clustered into 17 phylogenetic subgroups. We also analyzed the nucleotide sequences of the papaya UBC (CpUBC) genes and found that both exon-intron junctions and sequence motifs were highly conserved among the phylogenetic subgroups. Using real-time PCR analysis, we also found that all the CpUBC genes were expressed in roots, stems, leaves, male and female flowers, and mature fruit, although the expression of some of the genes was increased or decreased in one or several specific organs. We also found that the expression of 13 and two CpUBC genes were incresesd or decreased during one and two ripening stages, respectively. Expression analyses indicates possible E2s playing a more significant role in fruit ripening for further studies. To the best of our knowledge, this is the first reported genome-wide analysis of the papaya UBC gene family, and the results will facilitate further investigation of the roles of UBC genes in fruit ripening and will aide in the functional validation of UBC genes in papaya.

  18. Genome-Wide Identification, Phylogenetic and Expression Analyses of the Ubiquitin-Conjugating Enzyme Gene Family in Maize.

    PubMed

    Jue, Dengwei; Sang, Xuelian; Lu, Shengqiao; Dong, Chen; Zhao, Qiufang; Chen, Hongliang; Jia, Liqiang

    2015-01-01

    Ubiquitination is a post-translation modification where ubiquitin is attached to a substrate. Ubiquitin-conjugating enzymes (E2s) play a major role in the ubiquitin transfer pathway, as well as a variety of functions in plant biological processes. To date, no genome-wide characterization of this gene family has been conducted in maize (Zea mays). In the present study, a total of 75 putative ZmUBC genes have been identified and located in the maize genome. Phylogenetic analysis revealed that ZmUBC proteins could be divided into 15 subfamilies, which include 13 ubiquitin-conjugating enzymes (ZmE2s) and two independent ubiquitin-conjugating enzyme variant (UEV) groups. The predicted ZmUBC genes were distributed across 10 chromosomes at different densities. In addition, analysis of exon-intron junctions and sequence motifs in each candidate gene has revealed high levels of conservation within and between phylogenetic groups. Tissue expression analysis indicated that most ZmUBC genes were expressed in at least one of the tissues, indicating that these are involved in various physiological and developmental processes in maize. Moreover, expression profile analyses of ZmUBC genes under different stress treatments (4°C, 20% PEG6000, and 200 mM NaCl) and various expression patterns indicated that these may play crucial roles in the response of plants to stress. Genome-wide identification, chromosome organization, gene structure, evolutionary and expression analyses of ZmUBC genes have facilitated in the characterization of this gene family, as well as determined its potential involvement in growth, development, and stress responses. This study provides valuable information for better understanding the classification and putative functions of the UBC-encoding genes of maize.

  19. Genome-Wide Identification, Phylogenetic and Expression Analyses of the Ubiquitin-Conjugating Enzyme Gene Family in Maize

    PubMed Central

    Jue, Dengwei; Sang, Xuelian; Lu, Shengqiao; Dong, Chen; Zhao, Qiufang; Chen, Hongliang; Jia, Liqiang

    2015-01-01

    Background Ubiquitination is a post-translation modification where ubiquitin is attached to a substrate. Ubiquitin-conjugating enzymes (E2s) play a major role in the ubiquitin transfer pathway, as well as a variety of functions in plant biological processes. To date, no genome-wide characterization of this gene family has been conducted in maize (Zea mays). Methodology/Principal Findings In the present study, a total of 75 putative ZmUBC genes have been identified and located in the maize genome. Phylogenetic analysis revealed that ZmUBC proteins could be divided into 15 subfamilies, which include 13 ubiquitin-conjugating enzymes (ZmE2s) and two independent ubiquitin-conjugating enzyme variant (UEV) groups. The predicted ZmUBC genes were distributed across 10 chromosomes at different densities. In addition, analysis of exon-intron junctions and sequence motifs in each candidate gene has revealed high levels of conservation within and between phylogenetic groups. Tissue expression analysis indicated that most ZmUBC genes were expressed in at least one of the tissues, indicating that these are involved in various physiological and developmental processes in maize. Moreover, expression profile analyses of ZmUBC genes under different stress treatments (4°C, 20% PEG6000, and 200 mM NaCl) and various expression patterns indicated that these may play crucial roles in the response of plants to stress. Conclusions Genome-wide identification, chromosome organization, gene structure, evolutionary and expression analyses of ZmUBC genes have facilitated in the characterization of this gene family, as well as determined its potential involvement in growth, development, and stress responses. This study provides valuable information for better understanding the classification and putative functions of the UBC-encoding genes of maize. PMID:26606743

  20. Characterization and expression analysis of genes encoding ubiquitin conjugating domain-containing enzymes in Carica papaya

    PubMed Central

    Jue, Dengwei; Sang, Xuelian; Shu, Bo; Liu, Liqin; Wang, Yicheng; Jia, Zhiwei; Zou, Yu; Shi, Shengyou

    2017-01-01

    Background Ripening affects the quality and nutritional contents of fleshy fruits and is a crucial process of fruit development. Although several studies have suggested that ubiquitin-conjugating enzyme (E2s or UBC enzymes) are involved in the regulation of fruit ripening, little is known about the function of E2s in papaya (Carica papaya). Methodology/Principal findings In the present study, we searched the papaya genome and identified 34 putative UBC genes, which were clustered into 17 phylogenetic subgroups. We also analyzed the nucleotide sequences of the papaya UBC (CpUBC) genes and found that both exon-intron junctions and sequence motifs were highly conserved among the phylogenetic subgroups. Using real-time PCR analysis, we also found that all the CpUBC genes were expressed in roots, stems, leaves, male and female flowers, and mature fruit, although the expression of some of the genes was increased or decreased in one or several specific organs. We also found that the expression of 13 and two CpUBC genes were incresesd or decreased during one and two ripening stages, respectively. Expression analyses indicates possible E2s playing a more significant role in fruit ripening for further studies. Conclusions To the best of our knowledge, this is the first reported genome-wide analysis of the papaya UBC gene family, and the results will facilitate further investigation of the roles of UBC genes in fruit ripening and will aide in the functional validation of UBC genes in papaya. PMID:28231288

  1. Conformational Dynamics Modulate Activation of the Ubiquitin Conjugating Enzyme Ube2g2

    PubMed Central

    2017-01-01

    The ubiquitin conjugating enzyme Ube2g2 together with its cognate E3 ligase gp78 catalyzes the synthesis of lysine-48 polyubiquitin chains constituting signals for the proteasomal degradation of misfolded proteins in the endoplasmic reticulum. Here, we employ NMR spectroscopy in combination with single-turnover diubiquitin formation assays to examine the role of the RING domain from gp78 in the catalytic activation of Ube2g2∼Ub conjugates. We find that approximately 60% of the Ube2g2∼Ub conjugates occupy a closed conformation in the absence of gp78-RING, with the population increasing to 82% upon gp78-RING binding. As expected, strong mutations in the hydrophobic patch residues of the ∼Ub moiety result in Ube2g2∼Ub populating only open states with corresponding loss of the ubiquitin conjugation activity. Less disruptive mutations introduced into the hydrophobic patch of the ∼Ub moiety also destabilize the closed conformational state, yet the corresponding effect on the ubiquitin conjugation activity ranges from complete loss to an enhancement of the catalytic activity. These results present a picture in which Ube2g2’s active site is in a state of continual dynamic flux with the organization of the active site into a catalytically viable conformation constituting the rate-limiting step for a single ubiquitin ligation event. Ube2g2’s function as a highly specific K48-polyubiquitin chain elongator leads us to speculate that this may be a strategy by which Ube2g2 reduces the probability of nonproductive catalytic outcomes in the absence of available substrate. PMID:28884161

  2. Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock.

    PubMed

    Li, Guang-Qi; Zang, Xiao-Nan; Zhang, Xue-Cheng; Lu, Ning; Ding, Yan; Gong, Le; Chen, Wen-Chao

    2014-03-15

    To study the response of Gracilaria lemaneiformis to heat stress, two key enzymes - ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) - of the Ubiquitin/26S proteasome pathway (UPP) were studied in three strains of G. lemaneiformis-wild type, heat-tolerant cultivar 981 and heat-tolerant cultivar 07-2. The full length DNA sequence of E1 contained only one exon. The open reading frame (ORF) sequence was 981 nucleotides encoding 326 amino acids, which contained conserved ATP binding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVCAI) and the ubiquitin-activating domains (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The gene sequence of E2 contained four exons and three introns. The sum of the four exons gave an open reading frame sequence of 444 nucleotides encoding 147 amino acids, which contained a conserved ubiquitin-activating domain (GSICLDIL), ubiquitin-conjugating domains (RIYHPNIN, KVLLSICSLL, DDPLV) and ubiquitin-ligase (E3) recognition sites (KRI, YPF, WSP). Real-time-PCR analysis of transcription levels of E1 and E2 under heat shock conditions (28°C and 32°C) showed that in wild type, transcriptions of E1 and E2 were up-regulated at 28°C, while at 32°C, transcriptions of the two enzymes were below the normal level. In cultivar 981 and cultivar 07-2 of G. lemaneiformis, the transcription levels of the two enzymes were up-regulated at 32°C, and transcription level of cultivar 07-2 was even higher than that of cultivar 981. These results suggest that the UPP plays an important role in high temperature resistance of G. lemaneiformis and the bioactivity of UPP is directly related to the heat-resistant ability of G. lemaneiformis. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Modulation of TEL transcription activity by interaction with the ubiquitin-conjugating enzyme UBC9

    PubMed Central

    Chakrabarti, Subhra Ranjan; Sood, Rashmi; Ganguly, Surajit; Bohlander, Stefan; Shen, Zhiyuan; Nucifora, Giuseppina

    1999-01-01

    The E-26 transforming specific (ETS)-related gene TEL, also known as ETV6, is involved in a large number of chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. The encoded protein contains two functional domains: a helix–loop–helix (HLH) domain (also known as pointed domain) located at the N terminus and a DNA-binding domain located at the C terminus. The HLH domain is involved in protein–protein interaction with itself and other members of the ETS family of transcription factors such as FLI1. TEL is a transcription factor, and we and others have shown that it is a repressor of gene expression. To understand further the role of TEL in the cell, we have used an in vivo interaction system to identify proteins that interact with TEL. We show that a protein, UBC9, interacts specifically with TEL in vitro and in vivo. UBC9 is a member of the family of ubiquitin-conjugating enzymes. These enzymes usually are involved in proteosome-mediated degradation; however, our data suggest that interaction of TEL with UBC9 does not lead to TEL degradation. Our studies show that UBC9 binds to TEL exclusively through the HLH domain of TEL. We also show that TEL expressed as fusion to the DNA-binding domain of Gal4 completely represses a Gal4-responsive promoter, but that the coexpression of UBC9 in the same system restores the activity of the promoter. Targeted point mutation of conserved amino acids in UBC9 essential for enzymatic ubiquitination of proteins does not affect interaction nor transcriptional activity. Based on our data, we conclude that UBC9 physically interacts with TEL through the HLH domain and that the interaction leads to modulation of the transcription activity of TEL. PMID:10377438

  4. Ubiquitin-conjugating enzyme UBE2O regulates cellular clock function by promoting the degradation of the transcription factor BMAL1.

    PubMed

    Chen, Suping; Yang, Jing; Zhang, Yang; Duan, Chunyan; Liu, Qing; Huang, Zhengyun; Xu, Ying; Zhou, Liang; Xu, Guoqiang

    2018-06-05

    Dysregulation of the circadian rhythm is associated with many diseases, including diabetes, obesity, and cancer. Aryl hydrocarbon receptor nuclear translocator-like protein 1 (Arntl or Bmal1) is the only clock gene whose loss disrupts circadian locomotor behavior in constant darkness. BMAL1 levels are affected by proteasomal inhibition and by several enzymes in the ubiquitin-proteasome system, but the exact molecular mechanism remains unclear. Here, using immunoprecipitation and MS analyses, we discovered an interaction between BMAL1 and ubiquitin-conjugating enzyme E2 O (UBE2O), an E3-independent, E2-ubiquitin-conjugating enzyme (i.e. hybrid E2/E3 enzyme). Biochemical experiments with cell lines and animal tissues validated this specific interaction and uncovered that UBE2O expression reduces BMAL1 levels by promoting its ubiquitination and degradation. Moreover, UBE2O expression and UBE2O knockdown diminished and increased, respectively, BMAL1-mediated transcriptional activity, but did not affect BMAL1 gene expression. Bioluminescence experiments disclosed that UBE2O knockdown elevates the amplitude of the circadian clock in human osteosarcoma U2OS cells. Furthermore, mapping of the BMAL1-interacting domain in UBE2O and analyses of BMAL1 stability and ubiquitination revealed that the conserved region 2 (CR2) in UBE2O significantly enhances BMAL1 ubiquitination and decreases BMAL1 protein levels. A Cys-to-Ser substitution in the CR2 domain identified the critical Cys residue responsible for BMAL1 ubiquitination mediated by the CR2 domain in UBE2O. This work identifies UBE2O as a critical regulator in the ubiquitin-proteasome system, which modulates BMAL1 transcriptional activity and circadian function by promoting BMAL1 ubiquitination and degradation under normal physiological conditions. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Degradation Signals Recognized by the Ubc6p-Ubc7p Ubiquitin-Conjugating Enzyme Pair

    PubMed Central

    Gilon, Tamar; Chomsky, Orna; Kulka, Richard G.

    2000-01-01

    Proteolysis by the ubiquitin-proteasome system is highly selective. Specificity is achieved by the cooperation of diverse ubiquitin-conjugating enzymes (Ubcs or E2s) with a variety of ubiquitin ligases (E3s) and other ancillary factors. These recognize degradation signals characteristic of their target proteins. In a previous investigation, we identified signals directing the degradation of β-galactosidase and Ura3p fusion proteins via a subsidiary pathway of the ubiquitin-proteasome system involving Ubc6p and Ubc7p. This pathway has recently been shown to be essential for the degradation of misfolded and regulated proteins in the endoplasmic reticulum (ER) lumen and membrane, which are transported to the cytoplasm via the Sec61p translocon. Mutant backgrounds which prevent retrograde transport of ER proteins (hrd1/der3Δ and sec61-2) did not inhibit the degradation of the β-galactosidase and Ura3p fusions carrying Ubc6p/Ubc7p pathway signals. We therefore conclude that the ubiquitination of these fusion proteins takes place on the cytosolic face of the ER without prior transfer to the ER lumen. The contributions of different sequence elements to a 16-amino-acid-residue Ubc6p-Ubc7p-specific signal were analyzed by mutation. A patch of bulky hydrophobic residues was an essential element. In addition, positively charged residues were found to be essential. Unexpectedly, certain substitutions of bulky hydrophobic or positively charged residues with alanine created novel degradation signals, channeling the degradation of fusion proteins to an unidentified proteasomal pathway not involving Ubc6p and Ubc7p. PMID:10982838

  6. A conserved catalytic residue in the ubiquitin-conjugating enzyme family

    PubMed Central

    Wu, Pei-Ying; Hanlon, Mary; Eddins, Michael; Tsui, Colleen; Rogers, Richard S.; Jensen, Jane P.; Matunis, Michael J.; Weissman, Allan M.; Wolberger, Cynthia P.; Pickart, Cecile M.

    2003-01-01

    Ubiquitin (Ub) regulates diverse functions in eukaryotes through its attachment to other proteins. The defining step in this protein modification pathway is the attack of a substrate lysine residue on Ub bound through its C-terminus to the active site cysteine residue of a Ub-conjugating enzyme (E2) or certain Ub ligases (E3s). So far, these E2 and E3 cysteine residues are the only enzyme groups known to participate in the catalysis of conjugation. Here we show that a strictly conserved E2 asparagine residue is critical for catalysis of E2- and E2/RING E3-dependent isopeptide bond formation, but dispensable for upstream and downstream reactions of Ub thiol ester formation. In constrast, the strictly conserved histidine and proline residues immediately upstream of the asparagine are dispensable for catalysis of isopeptide bond formation. We propose that the conserved asparagine side chain stabilizes the oxyanion intermediate formed during lysine attack. The E2 asparagine is the first non-covalent catalytic group to be proposed in any Ub conjugation factor. PMID:14517261

  7. Molecular dynamics simulations reveal a new role for a conserved active site asparagine in a ubiquitin-conjugating enzyme.

    PubMed

    Wilson, R Hunter; Zamfir, Serban; Sumner, Isaiah

    2017-09-01

    The role of a highly conserved active site asparagine (N79) in the ubiquitin conjugating enzyme, Ubc13, is probed using molecular dynamics simulations. Both wild type and mutant enzymes (N79A and N79D) are studied. Contrary to a popular hypothesis, we show that it is unlikely that N79 stabilizes a reaction intermediate, but instead preferentially hydrogen bonds to a loop near the active site. This keeps the sidechain carboxylate of an aspartate in the loop (D119) near the sidechain amine of the substrate lysine. Our simulations show that this distance increases in the mutants. D119 has been hypothesized to play a variety of roles in the enzyme, including deprotonating the substrate lysine, so changing this distance can have an effect on the enzyme's efficiency. Finally, we show that it is possible for the aspartate to deprotonate the substrate even across long distances if short water wires form that connect the proton donor and acceptor. Short water wires form with greater probability in the wild type than in mutant enzymes. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. The Ubiquitin-Conjugating Enzyme Gene Family in Longan (Dimocarpus longan Lour.): Genome-Wide Identification and Gene Expression during Flower Induction and Abiotic Stress Responses.

    PubMed

    Jue, Dengwei; Sang, Xuelian; Liu, Liqin; Shu, Bo; Wang, Yicheng; Xie, Jianghui; Liu, Chengming; Shi, Shengyou

    2018-03-15

    Ubiquitin-conjugating enzymes (E2s or UBC enzymes) play vital roles in plant development and combat various biotic and abiotic stresses. Longan ( Dimocarpus longan Lour.) is an important fruit tree in the subtropical region of Southeast Asia and Australia; however the characteristics of the UBC gene family in longan remain unknown. In this study, 40 D. longan UBC genes ( DlUBCs ), which were classified into 15 groups, were identified in the longan genome. An RNA-seq based analysis showed that DlUBCs showed distinct expression in nine longan tissues. Genome-wide RNA-seq and qRT-PCR based gene expression analysis revealed that 11 DlUBCs were up- or down-regualted in the cultivar "Sijimi" (SJ), suggesting that these genes may be important for flower induction. Finally, qRT-PCR analysis showed that the mRNA levels of 13 DlUBCs under SA (salicylic acid) treatment, seven under methyl jasmonate (MeJA) treatment, 27 under heat treatment, and 16 under cold treatment were up- or down-regulated, respectively. These results indicated that the DlUBCs may play important roles in responses to abiotic stresses. Taken together, our results provide a comprehensive insight into the organization, phylogeny, and expression patterns of the longan UBC genes, and therefore contribute to the greater understanding of their biological roles in longan.

  9. Transcript Levels of Androgen Receptor Variant 7 and Ubiquitin-Conjugating Enzyme 2C in Hormone Sensitive Prostate Cancer and Castration-Resistant Prostate Cancer.

    PubMed

    Lee, Chan Ho; Ku, Ja Yoon; Ha, Jung Min; Bae, Sun Sik; Lee, Jeong Zoo; Kim, Choung-Soo; Ha, Hong Koo

    2017-01-01

    This study is designed to identify the androgen receptor variant 7 (AR-V7) status, clinical significance of AR-V7 in hormone sensitive prostate cancer (HSPC). Then, we evaluated AR-V7 and changes of its target gene, ubiquitin-conjugating enzyme E2C (UBE2C) which is an anaphase-promoting complex/cyclosome (APC/C)-specific ubiquitin-conjugating enzyme, in castration-resistant prostate cancer (CRPC) in serial tumor biopsies from patients receiving androgen deprivation therapy. We used RT-PCR and Q-PCR assay to evaluate AR-V7, androgen receptor full length (AR-FL), and UBE2C in tumor biopsies from patients with HSPC and CRPC. We examined associations between mRNA expression of AR-V7 and clinicopathologic factors. Furthermore, to identify other potential genes involved in the development of CRPC, RNA sequencing was conducted, using paired prostate cancer (PCa) tissues obtained immediately prior to treatment and at the time of therapeutic resistance. A total of 13 HSPC patients and three CRPC patients were enrolled. Neither a high Gleason score (score of 8 and 9) nor a high risk of PCa (a high risk of locally advanced PCa according to NCCN guidelines) was correlated with mRNA expression of AR-V7 in HSPC (P = 0.153 and P = 0.215). The mRNA expression of AR-FL, but not AR-V7, was significantly associated with the mRNA expression of UBE2C level in HSPC (P = 0.007). However, increased expression of AR-V7, not AR-FL, paralleled increased expression of UBE2C in the CRPC specimens (P = 0.03). AR-V7 expression status before ADT was likely related to shorter CRPC development in patients treating ADT. The result of the RNA-sequencing analysis using serial samples from the same patient before and after castration demonstrated an increased level of the PI3K regulatory subunit 1 (P = 0.018). Our study revealed the role of UBE2C as a marker of the androgen signaling pathway in PCa. Differential gene expression analysis using serial samples from the same patient

  10. Ft1, a novel gene related to ubiquitin-conjugating enzymes, is deleted in the Fused toes mouse mutation.

    PubMed

    Lesche, R; Peetz, A; van der Hoeven, F; Rüther, U

    1997-12-01

    The dominant mouse mutation Fused toes is characterized by partial syndactyly of the limbs and thymic hyperplasia. Both morphological abnormalities were shown to be related to impaired regulation of programmed cell death. Ft/Ft embryos die in midgestation showing severe malformations of fore- and midbrain as well as randomized situs. In Ft mice a large chromosomal deletion (about 300 kb) occurred after insertional mutagenesis. In this report we describe the identification of the first gene that has been mutated by Fused toes. The expression of the novel gene Ft1 is reduced in Ft/+ mice and completely absent in Ft/Ft embryos. Analysis of the Ft1 cDNA revealed an open reading frame that could code for a 32-kDa protein with similarities to ubiquitin-conjugating enzymes. Ft1 transcripts with alternative 5' UTR sequences as well as differential usage of polyadenylation sites were found. Interestingly, the 3' parts of the longest Ft1 transcripts are identical to the reverse complement of the 3'-most sequences of the Rb-related p130 gene. Both genes are transcribed in opposite directions and overlap in their 3' UTRs. Despite the close linkage, p130 expression appeared not to be affected by the Ft mutation. In wild type mice, Ft1 expression levels were found to be high in brain, kidney, and testes and detectable in all other adult organs and throughout embryonic development. Finally, we show that Ft1 is conserved among mammals and identify the human homolog.

  11. The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27{sup Kip1} protein levels

    SciTech Connect

    Butz, Nicole; Ruetz, Stephan; Natt, Francois

    2005-02-15

    Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27{sup Kip1} was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCF{sup Skp2} ubiquitin ligase has been reported to mediate p27{sup Kip1} degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27{sup Kip1}, and prevent cellular proliferation. Elevation of p27{sup Kip1} protein level is found tomore » be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27{sup Kip1} with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCF{sup Skp2} ubiquitin ligase substrate p27{sup Kip1}, but has no concomitant effect on the level of IkB{alpha} and {beta}-catenin, which are known substrates of a closely related SCF ligase.« less

  12. E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

    PubMed Central

    Hibbert, Richard G.; Huang, Anding; Boelens, Rolf; Sixma, Titia K.

    2011-01-01

    In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process. PMID:21422291

  13. Ubiquitin-conjugating enzyme complex Uev1A-Ubc13 promotes breast cancer metastasis through nuclear factor-кB mediated matrix metalloproteinase-1 gene regulation

    PubMed Central

    2014-01-01

    Introduction UEV1A encodes a ubiquitin-conjugating enzyme variant (Ubc13), which is required for Ubc13-catalyzed Lys63-linked polyubiquitination of target proteins and nuclear factor κB (NF-кB) activation. Previous reports have correlated the level of UEV1A expression with tumorigenesis; however, the detailed molecular events leading to tumors particularly breast cancer and metastasis are unclear. This study is to investigate roles of different UEV1 splicing variants, and its close homolog MMS2, in promoting tumorigenesis and metastasis in breast cancer cells. Methods We experimentally manipulated the UEV1 and MMS2 levels in MDA-MB-231 breast cancer cells and monitored their effects on cell invasion and migration, as well as tumor formation and metastasis in xenograft mice. The underlying molecular mechanisms leading to metastasis were also examined. Results It was found that overexpression of UEV1A alone, but not UEV1C or MMS2, is sufficient to induce cell invasion in vitro and metastasis in vivo. This process is mediated by NF-κB activation and requires functional Ubc13. Our experimental data establish that among NF-κB target genes, UEV1A-regulated matrix metalloproteinase-1 (MMP1) expression plays a critical role in cell invasion and metastasis. Interestingly, experimental depletion of UEV1 in MDA-MB-231 cells reduces MMP1 expression and prevents tumor formation and metastasis in a xenograft mouse model, while overexpression of MMP1 overrides the metastasis effects in UEV1-depleted cells. Conclusions These results identify UEV1A as a potential therapeutic target in the treatment of metastasic breast cancers. PMID:25022892

  14. Downregulation of Ubiquitin-conjugating Enzyme UBE2D3 Promotes Telomere Maintenance and Radioresistance of Eca-109 Human Esophageal Carcinoma Cells

    PubMed Central

    Yang, Hui; Wu, Lin; Ke, Shaobo; Wang, Wenbo; Yang, Lei; Gao, Xiaojia; Fang, Hongyan; Yu, Haijun; Zhong, Yahua; Xie, Conghua; Zhou, Fuxiang; Zhou, Yunfeng

    2016-01-01

    Ubiquitin-conjugating enzyme UBE2D3 is an important member of the ubiquitin-proteasome pathways. Our previous study showed that the expression of UBE2D3 was negatively related to human telomerase reverse transcriptase (hTERT) and radioresistance in human breast cancer cells. However, in esophageal carcinoma, the exact effects and mechanisms of UBE2D3 in radioresistance remain unclear. This study shows that UBE2D3 knockdown was associated with significant increases in radioresistance to X-rays, telomerase activity, telomere length, and telomere shelterins. UBE2D3 knockdown-mediated radioresistance was related to a decrease in the spontaneous and ionizing radiation-induced apoptosis, resulting from a decrease in the Bax/Bcl-2 ratio. Furthermore, UBE2D3 downregulation was associated with increased G1-S phase transition and prolonged IR-induced G2/M arrest through over expression of cyclin D1, decrease of CDC25A expression and promotion of the ATM/ATR-Chk1-CDC25C pathway. Moreover, UBE2D3 downregulation reduced spontaneous DNA double-strand breaks and accelerated the repair of DNA damage induced by IR. The current data thus demonstrate that UBE2D3 downregulation enhances radioresistance by increased telomere homeostasis and prolonged IR-induced G2/M arrest, but decreases the IR-induced apoptosis and the number of DNA damage foci. These results suggest that UBE2D3 might be a potential molecular target to improve radiotherapy effects in esophageal carcinoma. PMID:27326259

  15. The ubiquitin conjugating enzyme UbcH10 competes with UbcH3 for binding to the SCF complex, a ubiquitin ligase involved in cell cycle progression

    USDA-ARS?s Scientific Manuscript database

    Ubiquitylation, which regulates most biological pathways, occurs through an enzymatic cascade involving a ubiquitin (ub) activating enzyme (E1), a ub conjugating enzyme (E2) and a ub ligase (E3). UbcH3 is the E2 that interacts with SCF (Skp1/Cul1/F-box protein) complex and ubiquitylates many protein...

  16. Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity

    PubMed Central

    Kumar, Pankaj; Magala, Pearl; Geiger-Schuller, Kathryn R.; Majumdar, Ananya; Tolman, Joel R.; Wolberger, Cynthia

    2015-01-01

    Rad6 is a yeast E2 ubiquitin conjugating enzyme that monoubiquitinates histone H2B in conjunction with the E3, Bre1, but can non-specifically modify histones on its own. We determined the crystal structure of a Rad6∼Ub thioester mimic, which revealed a network of interactions in the crystal in which the ubiquitin in one conjugate contacts Rad6 in another. The region of Rad6 contacted is located on the distal face of Rad6 opposite the active site, but differs from the canonical E2 backside that mediates free ubiquitin binding and polyubiquitination activity in other E2 enzymes. We find that free ubiquitin interacts weakly with both non-canonical and canonical backside residues of Rad6 and that mutations of non-canonical residues have deleterious effects on Rad6 activity comparable to those observed to mutations in the canonical E2 backside. The effect of non-canonical backside mutations is similar in the presence and absence of Bre1, indicating that contacts with non-canonical backside residues govern the intrinsic activity of Rad6. Our findings shed light on the determinants of intrinsic Rad6 activity and reveal new ways in which contacts with an E2 backside can regulate ubiquitin conjugating activity. PMID:26286193

  17. Crystal Structure of a Ube2S-Ubiquitin Conjugate

    PubMed Central

    Lorenz, Sonja; Bhattacharyya, Moitrayee; Feiler, Christian; Rape, Michael; Kuriyan, John

    2016-01-01

    Protein ubiquitination occurs through the sequential formation and reorganization of specific protein-protein interfaces. Ubiquitin-conjugating (E2) enzymes, such as Ube2S, catalyze the formation of an isopeptide linkage between the C-terminus of a “donor” ubiquitin and a primary amino group of an “acceptor” ubiquitin molecule. This reaction involves an intermediate, in which the C-terminus of the donor ubiquitin is thioester-bound to the active site cysteine of the E2 and a functionally important interface is formed between the two proteins. A docked model of a Ube2S-donor ubiquitin complex was generated previously, based on chemical shift mapping by NMR, and predicted contacts were validated in functional studies. We now present the crystal structure of a covalent Ube2S-ubiquitin complex. The structure contains an interface between Ube2S and ubiquitin in trans that resembles the earlier model in general terms, but differs in detail. The crystallographic interface is more hydrophobic than the earlier model and is stable in molecular dynamics (MD) simulations. Remarkably, the docked Ube2S-donor complex converges readily to the configuration seen in the crystal structure in 3 out of 8 MD trajectories. Since the crystallographic interface is fully consistent with mutational effects, this indicates that the structure provides an energetically favorable representation of the functionally critical Ube2S-donor interface. PMID:26828794

  18. E2 enzyme inhibition by stabilization of a low affinity interface with ubiquitin

    PubMed Central

    St-Cyr, Daniel J.; Ziemba, Amy; Garg, Pankaj; Plamondon, Serge; Auer, Manfred; Sidhu, Sachdev; Marinier, Anne; Kleiger, Gary; Tyers, Mike; Sicheri, Frank

    2014-01-01

    Weak protein interactions between ubiquitin and the ubiquitin-proteasome system (UPS) enzymes that mediate its covalent attachment to substrates serve to position ubiquitin for optimal catalytic transfer. We show that a small molecule inhibitor of the E2 ubiquitin conjugating enzyme Cdc34A, called CC0651, acts by trapping a weak interaction between ubiquitin and the E2 donor ubiquitin binding site. A structure of the ternary CC0651-Cdc34A-ubiquitin complex reveals that the inhibitor engages a composite binding pocket formed from Cdc34A and ubiquitin. CC0651 also suppresses the spontaneous hydrolysis rate of the Cdc34A-ubiquitin thioester, without overtly affecting the interaction between Cdc34A and the RING domain subunit of the E3 enzyme. Stabilization of the numerous other weak interactions between ubiquitin and UPS enzymes by small molecules may be a feasible strategy to selectively inhibit different UPS activities. PMID:24316736

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

    SciTech Connect

    Juang, Yu-Chi; Landry, Marie-Claude; Sanches, Mario

    2012-03-26

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

  20. Activity-based probes for the ubiquitin conjugation-deconjugation machinery: new chemistries, new tools, and new insights.

    PubMed

    Hewings, David S; Flygare, John A; Bogyo, Matthew; Wertz, Ingrid E

    2017-05-01

    The reversible post-translational modification of proteins by ubiquitin and ubiquitin-like proteins regulates almost all cellular processes, by affecting protein degradation, localization, and complex formation. Deubiquitinases (DUBs) are proteases that remove ubiquitin modifications or cleave ubiquitin chains. Most DUBs are cysteine proteases, which makes them well suited for study by activity-based probes. These DUB probes report on deubiquitinase activity by reacting covalently with the active site in an enzyme-catalyzed manner. They have proven to be important tools to study DUB selectivity and proteolytic activity in different settings, to identify novel DUBs, and to characterize deubiquitinase inhibitors. Inspired by the efficacy of activity-based probes for DUBs, several groups have recently reported probes for the ubiquitin conjugation machinery (E1, E2, and E3 enzymes). Many of these enzymes, while not proteases, also posses active site cysteine residues and can be targeted by covalent probes. In this review, we will discuss how features of the probe (cysteine-reactive group, recognition element, and reporter tag) affect reactivity and suitability for certain experimental applications. We will also review the diverse applications of the current probes, and discuss the need for new probe types to study emerging aspects of ubiquitin biology. © 2017 Federation of European Biochemical Societies.

  1. How Chemical Synthesis of Ubiquitin Conjugates Helps To Understand Ubiquitin Signal Transduction.

    PubMed

    Hameed, Dharjath S; Sapmaz, Aysegul; Ovaa, Huib

    2017-03-15

    Ubiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin signaling pathways have not been completely deciphered due to the complex nature of the enzymes involved in ubiquitin conjugation and deconjugation. Hence, probes and assay reagents are important to get a better understanding of this pathway. Recently, improvements have been made in synthesis procedures of Ub derivatives. In this perspective, we explain various research reagents available and how chemical synthesis has made an important contribution to Ub research.

  2. The HIP2~Ubiquitin Conjugate Forms a Non-Compact Monomeric Thioester during Di-Ubiquitin Synthesis

    SciTech Connect

    Cook, Benjamin W.; Barber, Kathryn R.; Shilton, Brian H.

    2015-03-23

    Polyubiquitination is a post-translational event used to control the degradation of damaged or unwanted proteins by modifying the target protein with a chain of ubiquitin molecules. One potential mechanism for the assembly of polyubiquitin chains involves the dimerization of an E2 conjugating enzyme allowing conjugated ubiquitin molecules to be put into close proximity to assist reactivity. HIP2 (UBE2K) and Ubc1 (yeast homolog of UBE2K) are unique E2 conjugating enzymes that each contain a C-terminal UBA domain attached to their catalytic domains, and they have basal E3-independent polyubiquitination activity. Although the isolated enzymes are monomeric, polyubiquitin formation activity assays show thatmore » both can act as ubiquitin donors or ubiquitin acceptors when in the activated thioester conjugate suggesting dimerization of the E2-ubiquitin conjugates. Stable disulfide complexes, analytical ultracentrifugation and small angle x-ray scattering were used to show that the HIP2-Ub and Ubc1-Ub thioester complexes remain predominantly monomeric in solution. Models of the HIP2-Ub complex derived from SAXS data show the complex is not compact but instead forms an open or backbent conformation similar to UbcH5b~Ub or Ubc13~Ub where the UBA domain and covalently attached ubiquitin reside on opposite ends of the catalytic domain. Activity assays showed that full length HIP2 exhibited a five-fold increase in the formation rate of di-ubiquitin compared to a HIP2 lacking the UBA domain. This difference was not observed for Ubc1 and may be attributed to the closer proximity of the UBA domain in HIP2 to the catalytic core than for Ubc1.« less

  3. USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin

    PubMed Central

    Durcan, Thomas M; Tang, Matthew Y; Pérusse, Joëlle R; Dashti, Eman A; Aguileta, Miguel A; McLelland, Gian-Luca; Gros, Priti; Shaler, Thomas A; Faubert, Denis; Coulombe, Benoit; Fon, Edward A

    2014-01-01

    Mutations in the Park2 gene, encoding the E3 ubiquitin-ligase parkin, are responsible for a familial form of Parkinson's disease (PD). Parkin-mediated ubiquitination is critical for the efficient elimination of depolarized dysfunctional mitochondria by autophagy (mitophagy). As damaged mitochondria are a major source of toxic reactive oxygen species within the cell, this pathway is believed to be highly relevant to the pathogenesis of PD. Little is known about how parkin-mediated ubiquitination is regulated during mitophagy or about the nature of the ubiquitin conjugates involved. We report here that USP8/UBPY, a deubiquitinating enzyme not previously implicated in mitochondrial quality control, is critical for parkin-mediated mitophagy. USP8 preferentially removes non-canonical K6-linked ubiquitin chains from parkin, a process required for the efficient recruitment of parkin to depolarized mitochondria and for their subsequent elimination by mitophagy. This work uncovers a novel role for USP8-mediated deubiquitination of K6-linked ubiquitin conjugates from parkin in mitochondrial quality control. PMID:25216678

  4. The Tomato U-Box Type E3 Ligase PUB13 Acts With Group III Ubiquitin E2 Enzymes to Modulate FLS2-Mediated Immune Signaling

    PubMed Central

    Zhou, Bangjun; Zeng, Lirong

    2018-01-01

    In Arabidopsis and rice, the ubiquitin ligase PUB13-mediated protein degradation plays a significant role in plant pattern-triggered immunity (PTI) and flowering time control. The Arabidopsis PUB13 has been shown to attenuate the pattern recognition receptor FLS2-mediated immune signaling by ubiquitinating FLS2 and consequently promoting its degradation by the 26S proteasome. Nevertheless, the cognate ubiquitin-conjugating enzymes (E2) with which PUB13 acts to modulate FLS2-mediated PTI are unknown. To address this question, we investigate here the tomato (Solanum lycopersicum) homolog of PUB13, SlPUB13 by utilizing the recently characterized complete set of tomato E2s. Of the 13 groups of tomato E2s, only members in group III are found to interact and act with SlPUB13. Knocking-down of the group III E2 genes enhances callose deposition and induction of the RbohB gene in the immunity-associated, early oxidative burst after flg22 treatment. The group III E2s are also found to work with SlPUB13 to ubiquitinate FLS2 in vitro and are required for PUB13-mediated degradation of FLS2 in vivo upon flg22 treatment, suggesting an essential role for group III E2s in the modulation of FLS2-mediated immune signaling by PUB13. Additionally, another immunity-associated E3, NtCMPG1 is shown to also work specifically with members of group III E2 in the in vitro ubiquitination assay, which implies the group III E2 enzymes may cooperate with many E3 ligases to regulate different aspects of PTI. Taken together, these data corroborate the notion that group III E2 enzymes play an important role in PTI and build a foundation for further functional and mechanistic characterization of tomato PUB13.

  5. Quantitation and immunocytochemical localization of ubiquitin conjugates within rat red and white skeletal muscles

    NASA Technical Reports Server (NTRS)

    Riley, Danny A.; Bain, James L. W.; Haas, Arthur L.; Ellis, Stanley

    1988-01-01

    Solid-phase immunochemical methods were employed to probe the dynamics of ubiquitin pools within selected rat skeletal muscles. The total ubiquitin content of red muscles was greater than that of white muscles, even though the fractional conjugation was similar for both types of muscles. The specificity for conjugated ubiquitin in solid-phase applications, previously demonstrated for an affinity-purified antibody against SDS-denatured ubiquitin, was retained when used as a probe for ubiquitin-protein adducts in tissue sections. Immunohistochemical localization revealed that differences in ubiquitin pools derived from the relative content of red (oxidative) vs white (glycolytic) fibers, with the former exhibiting a higher content of ubiquitin conjugates. Subsequent immunogold labeling demonstrated statistically significant enhanced localization of ubiquitin conjugates to the Z-lines in both red and white muscle fiber types.

  6. Regulation of E2s: A Role for Additional Ubiquitin Binding Sites?

    PubMed

    Middleton, Adam J; Wright, Joshua D; Day, Catherine L

    2017-11-10

    Attachment of ubiquitin to proteins relies on a sophisticated enzyme cascade that is tightly regulated. The machinery of ubiquitylation responds to a range of signals, which remarkably includes ubiquitin itself. Thus, ubiquitin is not only the central player in the ubiquitylation cascade but also a key regulator. The ubiquitin E3 ligases provide specificity to the cascade and often bind the substrate, while the ubiquitin-conjugating enzymes (E2s) have a pivotal role in determining chain linkage and length. Interaction of ubiquitin with the E2 is important for activity, but the weak nature of these contacts has made them hard to identify and study. By reviewing available crystal structures, we identify putative ubiquitin binding sites on E2s, which may enhance E2 processivity and the assembly of chains of a defined linkage. The implications of these new sites are discussed in the context of known E2-ubiquitin interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. The E2-25K Ubiquitin-associated (UBA) Domain Aids in Polyubiquitin Chain Synthesis and Linkage Specificity

    PubMed Central

    WILSON, Randall C.; EDMONDSON, Stephen P.; FLATT, Justin W.; HELMS, Kimberli; TWIGG, Pamela D.

    2011-01-01

    E2-25K is an ubiquitin-conjugating enzyme with the ability to synthesize Lys48-linked polyubiquitin chains. E2-25K and its homologues represent the only known E2 enzymes which contain a C-terminal ubiquitin-associated (UBA) domain as well as the conserved catalytic ubiquitin-conjugating (UBC) domain. As an additional non-covalent binding surface for ubiquitin, the UBA domain must provide some functional specialization. We mapped the protein-protein interface involved in the E2-25K UBA/ubiquitin complex by solution nuclear magnetic resonance (NMR) spectroscopy and subsequently modeled the structure of the complex. Domain-domain interactions between the E2-25K catalytic UBC domain and the UBA domain do not induce significant structural changes in the UBA domain or alter the affinity of the UBA domain for ubiquitin. We determined that one of the roles of the C-terminal UBA domain, in the context of E2-25K, is to increase processivity in Lys48-linked polyubiquitin chain synthesis, possibly through increased binding to the ubiquitinated substrate. Additionally, we see evidence that the UBA domain directs specificity in polyubiquitin chain linkage. PMID:21281599

  8. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation

    PubMed Central

    Lechtenberg, Bernhard C.; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K.; Ware, Carl F.; Mace, Peter D.; Riedl, Stefan J.

    2015-01-01

    Ubiquitination is a central process affecting all facets of cellular signaling and function1. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate2,3. The RBR family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases4–6. The RBR family includes Parkin4 and HOIP, the central catalytic factor of the linear ubiquitin chain assembly complex (LUBAC)7. While structural insights into the RBR E3 ligases Parkin and HHARI in their overall autoinhibited forms are available8–13, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely enigmatic. Here we present the first structure of the fully active HOIP-RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP-RBR adopts a conformation markedly different from that of autoinhibited RBRs. HOIP-RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centers ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, surprisingly, three distinct helix–IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~Ub conjugate as well as an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP-RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

  9. Structure of the Human FANCL RING-Ube2T Complex Reveals Determinants of Cognate E3-E2 Selection

    PubMed Central

    Hodson, Charlotte; Purkiss, Andrew; Miles, Jennifer Anne; Walden, Helen

    2014-01-01

    Summary The combination of an E2 ubiquitin-conjugating enzyme with an E3 ubiquitin-ligase is essential for ubiquitin modification of a substrate. Moreover, the pairing dictates both the substrate choice and the modification type. The molecular details of generic E3-E2 interactions are well established. Nevertheless, the determinants of selective, specific E3-E2 recognition are not understood. There are ∼40 E2s and ∼600 E3s giving rise to a possible ∼24,000 E3-E2 pairs. Using the Fanconi Anemia pathway exclusive E3-E2 pair, FANCL-Ube2T, we report the atomic structure of the FANCL RING-Ube2T complex, revealing a specific and extensive network of additional electrostatic and hydrophobic interactions. Furthermore, we show that these specific interactions are required for selection of Ube2T over other E2s by FANCL. PMID:24389026

  10. Characterization and identification of ubiquitin conjugation sites with E3 ligase recognition specificities.

    PubMed

    Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Chang, Tzu-Hao; Bretaña, Neil; Lai, K; Weng, Julia; Lee, Tzong-Yi

    2015-01-01

    In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities. In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool. A case study demonstrated the effectiveness of the characterized substrate motifs for

  11. Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation

    PubMed Central

    Yunus, Ali A.; Lima, Christopher D.

    2009-01-01

    SUMO conjugation to protein substrates requires the concerted action of a dedicated E2 ubiquitin conjugation enzyme (Ubc9) and associated E3 ligases. Although Ubc9 can directly recognize and modify substrate lysine residues that occur within a consensus site for SUMO modification, E3 ligases can redirect specificity and enhance conjugation rates during SUMO conjugation in vitro and in vivo. In this chapter, we will describe methods utilized to purify SUMO conjugating enzymes and model substrates which can be used for analysis of SUMO conjugation in vitro. We will also describe methods to extract kinetic parameters during E3-dependent or E3-independent substrate conjugation. PMID:19107417

  12. p62/SQSTM1 promotes rapid ubiquitin conjugation to target proteins after endosome rupture during xenophagy.

    PubMed

    Tsuchiya, Megumi; Ogawa, Hidesato; Koujin, Takako; Mori, Chie; Osakada, Hiroko; Kobayashi, Shouhei; Hiraoka, Yasushi; Haraguchi, Tokuko

    2018-03-01

    Autophagy is a bulk degradation pathway, and selective autophagy to remove foreign entities is called xenophagy. The conjugation of ubiquitin to target pathogens is an important process in xenophagy but when and where this ubiquitination occurs remains unclear. Here, we analyzed the temporal sequence and subcellular location of ubiquitination during xenophagy using time-lapse observations, with polystyrene beads mimicking invading pathogens. Results revealed accumulation of a ubiquitination marker around the beads within 3 min after endosome rupture. Recruitment of ubiquitin to the beads was significantly delayed in p62-knockout murine embryonic fibroblast cells, and this delay was rescued by ectopic p62 expression. Ectopic expression of a phosphorylation-mimicking p62 mutated at serine residue 405 (equivalent to human serine residue 403) rescued this delay, but its unphosphorylated form did not. These results indicate that ubiquitination mainly occurs after endosome rupture and suggest that p62, specifically the phosphorylated form, promotes ubiquitin conjugation to target proteins in xenophagy.

  13. An inhibitor of ubiquitin conjugation and aggresome formation† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc01351h Click here for additional data file.

    PubMed Central

    An, Heeseon

    2015-01-01

    Proteasome inhibitors have revolutionized the treatment of multiple myeloma, and validated the therapeutic potential of the ubiquitin proteasome system (UPS). It is believed that in part, proteasome inhibitors elicit their therapeutic effect by inhibiting the degradation of misfolded proteins, which is proteotoxic and causes cell death. In spite of these successes, proteasome inhibitors are not effective against solid tumors, thus necessitating the need to explore alternative approaches. Furthermore, proteasome inhibitors lead to the formation of aggresomes that clear misfolded proteins via the autophagy–lysosome degradation pathway. Importantly, aggresome formation depends on the presence of polyubiquitin tags on misfolded proteins. We therefore hypothesized that inhibitors of ubiquitin conjugation should inhibit both degradation of misfolded proteins, and ubiquitin dependent aggresome formation, thus outlining the path forward toward more effective anticancer therapeutics. To explore the therapeutic potential of targeting the UPS to treat solid cancers, we have developed an inhibitor of ubiquitin conjugation (ABP A3) that targets ubiquitin and Nedd8 E1 enzymes, enzymes that are required to maintain the activity of the entire ubiquitin system. We have shown that ABP A3 inhibits conjugation of ubiquitin to intracellular proteins and prevents the formation of cytoprotective aggresomes in A549 lung cancer cells. Furthermore, ABP A3 induces activation of the unfolded protein response and apoptosis. Thus, similar to proteasome inhibitors MG132, bortezomib, and carfilzomib, ABP A3 can serve as a novel probe to explore the therapeutic potential of the UPS in solid and hematological malignancies. PMID:28717502

  14. Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

    NASA Technical Reports Server (NTRS)

    Solomon, V.; Baracos, V.; Sarraf, P.; Goldberg, A. L.

    1998-01-01

    The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin-proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3alpha, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3alpha-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

  15. Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

    PubMed Central

    Solomon, Vered; Baracos, Vickie; Sarraf, Pasha; Goldberg, Alfred L.

    1998-01-01

    The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin–proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3α, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3α-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway. PMID:9770532

  16. A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development*

    PubMed Central

    Franco, Maribel; Seyfried, Nicholas T.; Brand, Andrea H.; Peng, Junmin; Mayor, Ugo

    2011-01-01

    Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system. PMID:20861518

  17. The ubiquitin conjugating enzyme UbcH7, controls cell migration

    USDA-ARS?s Scientific Manuscript database

    Post translational modification by ubiquitination can target proteins for degradation, allow the interaction of proteins to form complexes or direct relocalization of proteins to different subcellular compartments. As such, ubiquitin controls a variety of essential cellular processes. Previously we ...

  18. Ubiquitin enzymes in the regulation of immune responses.

    PubMed

    Ebner, Petra; Versteeg, Gijs A; Ikeda, Fumiyo

    2017-08-01

    Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses.

  19. Ubiquitin enzymes in the regulation of immune responses

    PubMed Central

    Ebner, Petra; Versteeg, Gijs A.; Ikeda, Fumiyo

    2017-01-01

    Abstract Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses. PMID:28524749

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

    SciTech Connect

    Qiu, Jiazhang; Sheedlo, Michael J.; Yu, Kaiwen

    Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or asmore » enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. The E1/E2-independent ubiquitination catalyzed by these enzymes requires NAD but not ATP and Mg2+. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. These results establish that ubiquitination can be catalyzed by a single enzyme.« less

  1. E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis

    SciTech Connect

    Pruneda, Jonathan N.; Smith, F. Donelson; Daurie, Angela

    Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A cocrystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes anmore » active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.« less

  2. TNF-alpha increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k

    NASA Technical Reports Server (NTRS)

    Li, Yi-Ping; Lecker, Stewart H.; Chen, Yuling; Waddell, Ian D.; Goldberg, Alfred L.; Reid, Michael B.

    2003-01-01

    In some inflammatory diseases, TNF-alpha is thought to stimulate muscle catabolism via an NF-kappaB-dependent process that increases ubiquitin conjugation to muscle proteins. The transcriptional mechanism of this response has not been determined. Here we studied the potential role of UbcH2, a ubiquitin carrier protein and homologue of murine E220k. We find that UbcH2 is constitutively expressed by human skeletal and cardiac muscles, murine limb muscle, and cultured myotubes. TNF-alpha stimulates UbcH2 expression in mouse limb muscles in vivo and in cultured myotubes. The UbcH2 promoter region contains a functional NF-kappaB binding site; NF-kappaB binding to this sequence is increased by TNF-alpha stimulation. A dominant negative inhibitor of NF-kappaB activation blocks both UbcH2 up-regulation and the increase in ubiquitin-conjugating activity stimulated by TNF-alpha. In extracts from TNF-alpha-treated myotubes, ubiquitin-conjugating activity is limited by UbcH2 availability; activity is inhibited by an antiserum to UbcH2 or a dominant negative mutant of UbcH2 and is enhanced by wild-type UbcH2. Thus, UbcH2 up-regulation is a novel response to TNF-alpha/NF-kappaB signaling in skeletal muscle that appears to be essential for the increased ubiquitin conjugation induced by this cytokine.

  3. Pulmonary inflammation-induced loss and subsequent recovery of skeletal muscle mass require functional poly-ubiquitin conjugation.

    PubMed

    Ceelen, Judith J M; Schols, Annemie M W J; Thielen, Nathalie G M; Haegens, Astrid; Gray, Douglas A; Kelders, Marco C J M; de Theije, Chiel C; Langen, Ramon C J

    2018-05-02

    Pulmonary inflammation in response to respiratory infections can evoke muscle wasting. Increased activity of the ubiquitin (Ub)-proteasome system (UPS) and the autophagy lysosome pathway (ALP) have been implicated in inflammation-induced muscle atrophy. Since poly-Ub conjugation is required for UPS-mediated proteolysis and has been implicated in the ALP, we assessed the effect of impaired ubiquitin conjugation on muscle atrophy and recovery following pulmonary inflammation, and compared activation and suppression of these proteolytic systems to protein synthesis regulation. Pulmonary inflammation was induced in mice by an intratracheal instillation of LPS. Proteolysis (UPS and ALP) and synthesis signaling were examined in gastrocnemius muscle homogenates. Ub-conjugation-dependency of muscle atrophy and recovery was addressed using Ub-K48R (K48R) mice with attenuated poly-ubiquitin conjugation, and compared to UBWT control mice. Pulmonary inflammation caused a decrease in skeletal muscle mass which was accompanied by a rapid increase in expression of UPS and ALP constituents and reduction in protein synthesis signaling acutely after LPS. Muscle atrophy was attenuated in K48R mice, while ALP and protein synthesis signaling were not affected. Muscle mass recovery starting 72 h post LPS, correlated with reduced expression of UPS and ALP constituents and restoration of protein synthesis signaling. K48R mice however displayed impaired recovery of muscle mass. Pulmonary inflammation-induced muscle atrophy is in part attributable to UPS-mediated proteolysis, as activation of ALP- and suppression of protein synthesis signaling occur independently of poly-Ub conjugation during muscle atrophy. Recovery of muscle mass following pulmonary inflammation involves inverse regulation of proteolysis and protein synthesis signaling, and requires a functional poly-Ub conjugation.

  4. E2 potentializes benzo(a)pyrene-induced hepatic cytochrome P450 enzyme activities in Nile tilapia at high concentrations.

    PubMed

    Rodrigues, Aline Cristina Ferreira; Moneró, Tatiana de Oliveira; Frighetto, Rosa Toyoko Shiraishi; de Almeida, Eduardo Alves

    2015-11-01

    In the aquatic environment, biotransformation enzymes are established biomarkers for assessing PAH exposure in fish, but little is known about the effect of 17β-estradiol (E2) on these enzymes during exposure to benzo(a)pyrene (BaP). In this study, Nile tilapia (Oreochromis niloticus) were exposed for 3, 5, and 10 days to BaP (300 μg L(-1)) and E2 (5 μg L(-1)). These substances were applied isolated or mixed. In the mixture experiment, fish were analyzed pre- and postexposure in order to better understand whether preexposure to the hormone masks the responses activated by PAH or vice versa. Phase I enzymes ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-depenthylase (PROD), and benzyloxyresorufin-O-debenzylase (BROD) activities as well as the phase II enzyme glutathione S-transferase (GST) were analyzed. Isolated E2 treatment decreased EROD activity after 3 days, but this enzyme activity returned to control values after 5 and 10 days of exposure. Isolated BaP treatment significantly induced EROD activity after 3 and 5 days, and the activity returned to control levels after ten exposure days. Combined treatment (E2 + Bap) significantly increased EROD activity, both in the pre- and postexposure. This increase was even higher than in the isolated BaP treatment, suggesting a synergism between these two compounds. When E2 and BaP were used singly, they did not change BROD and PROD activities. However, combined treatment (E2 + Bap) significantly increased PROD activity. Isolated BaP treatment increased GST activity after 10 days. However, this response was not observed in the mixture treatment, suggesting that E2 suppressed the GST induction modulated by BaP. The results put together indicated that E2 altered the biotransformation pathway regarding enzymes activated by BaP in Nile tilapia.

  5. Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K.

    PubMed

    Rout, Manoj K; Lee, Brian L; Lin, Aiyang; Xiao, Wei; Spyracopoulos, Leo

    2018-05-03

    The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.

  6. Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery.

    PubMed

    Love, Kerry Routenberg; Pandya, Renuka K; Spooner, Eric; Ploegh, Hidde L

    2009-04-17

    Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubl's and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling uses chemical probes that are active-site-directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.

  7. Ubiquitin control of S phase: a new role for the ubiquitin conjugating enzyme, UbcH7

    USDA-ARS?s Scientific Manuscript database

    Events within and transitions between the phases of the eukaryotic cell cycle are tightly controlled by transcriptional and post-translational processes. Prominent among them is a profound role for the ubiquitin proteasome proteolytic pathway. The timely degradation of proteins balances the increase...

  8. Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

    PubMed

    Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

    2010-01-01

    Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

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

    PubMed Central

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

    2016-01-01

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

  10. RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

    DOE PAGES

    Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.; ...

    2016-02-22

    DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

  11. Structural model of the hUbA1-UbcH10 quaternary complex: in silico and experimental analysis of the protein-protein interactions between E1, E2 and ubiquitin.

    PubMed

    Correale, Stefania; de Paola, Ivan; Morgillo, Carmine Marco; Federico, Antonella; Zaccaro, Laura; Pallante, Pierlorenzo; Galeone, Aldo; Fusco, Alfredo; Pedone, Emilia; Luque, F Javier; Catalanotti, Bruno

    2014-01-01

    UbcH10 is a component of the Ubiquitin Conjugation Enzymes (Ubc; E2) involved in the ubiquitination cascade controlling the cell cycle progression, whereby ubiquitin, activated by E1, is transferred through E2 to the target protein with the involvement of E3 enzymes. In this work we propose the first three dimensional model of the tetrameric complex formed by the human UbA1 (E1), two ubiquitin molecules and UbcH10 (E2), leading to the transthiolation reaction. The 3D model was built up by using an experimentally guided incremental docking strategy that combined homology modeling, protein-protein docking and refinement by means of molecular dynamics simulations. The structural features of the in silico model allowed us to identify the regions that mediate the recognition between the interacting proteins, revealing the active role of the ubiquitin crosslinked to E1 in the complex formation. Finally, the role of these regions involved in the E1-E2 binding was validated by designing short peptides that specifically interfere with the binding of UbcH10, thus supporting the reliability of the proposed model and representing valuable scaffolds for the design of peptidomimetic compounds that can bind selectively to Ubcs and inhibit the ubiquitylation process in pathological disorders.

  12. Deubiquitinating enzyme regulation of the p53 pathway: A lesson from Otub1

    PubMed Central

    Sun, Xiao-Xin; Dai, Mu-Shui

    2014-01-01

    Deubiquitination has emerged as an important mechanism of p53 regulation. A number of deubiquitinating enzymes (DUBs) from the ubiquitin-specific protease family have been shown to regulate the p53-MDM2-MDMX networks. We recently reported that Otub1, a DUB from the OTU-domain containing protease family, is a novel p53 regulator. Interestingly, Otub1 abrogates p53 ubiquitination and stabilizes and activates p53 in cells independently of its deubiquitinating enzyme activity. Instead, it does so by inhibiting the MDM2 cognate ubiquitin-conjugating enzyme (E2) UbcH5. Otub1 also regulates other biological signaling through this non-canonical mechanism, suppression of E2, including the inhibition of DNA-damage-induced chromatin ubiquitination. Thus, Otub1 evolves as a unique DUB that mainly suppresses E2 to regulate substrates. Here we review the current progress made towards the understanding of the complex regulation of the p53 tumor suppressor pathway by DUBs, the biological function of Otub1 including its positive regulation of p53, and the mechanistic insights into how Otub1 suppresses E2. PMID:24920999

  13. A C2HC zinc finger is essential for the RING-E2 interaction of the ubiquitin ligase RNF125

    PubMed Central

    Bijlmakers, Marie-José; Teixeira, João M. C.; Boer, Roeland; Mayzel, Maxim; Puig-Sàrries, Pilar; Karlsson, Göran; Coll, Miquel; Pons, Miquel; Crosas, Bernat

    2016-01-01

    The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2120-128) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2120-128 region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2. PMID:27411375

  14. E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

    PubMed

    Lim, Jung Hwa; Shin, Hee Won; Chung, Kyung-Sook; Kim, Nam-Soon; Kim, Ju Hee; Jung, Hong-Ryul; Im, Dong-Soo; Jung, Cho-Rok

    Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

  15. E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

    PubMed Central

    Lim, Jung Hwa; Shin, Hee Won; Chung, Kyung-Sook; Kim, Nam-Soon; Kim, Ju Hee; Jung, Hong-Ryul; Im, Dong-Soo; Jung, Cho-Rok

    2016-01-01

    Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation. PMID:27685940

  16. Nuclear localization of pyruvate dehydrogenase complex-E2 (PDC-E2), a mitochondrial enzyme, and its role in signal transducer and activator of transcription 5 (STAT5)-dependent gene transcription.

    PubMed

    Chueh, Fu-Yu; Leong, King-Fu; Cronk, Robert J; Venkitachalam, Srividya; Pabich, Samantha; Yu, Chao-Lan

    2011-07-01

    STAT (signal transducer and activator of transcription) proteins play a critical role in cellular response to a wide variety of cytokines and growth factors by regulating specific nuclear genes. STAT-dependent gene transcription can be finely tuned through the association with co-factors in the nucleus. We showed previously that STAT5 (including 5a and 5b) specifically interacts with a mitochondrial enzyme PDC-E2 (E2 subunit of pyruvate dehydrogenase complex) in both leukemic T cells and cytokine-stimulated cells. However, the functional significance of this novel association remains largely unknown. Here we report that PDC-E2 may function as a co-activator in STAT5-dependent nuclear gene expression. Subcellular fractionation analysis revealed that a substantial amount of PDC-E2 was constitutively present in the nucleus of BaF3, an interleukin-3 (IL-3)-dependent cell line. IL-3-induced tyrosine-phosphorylated STAT5 associated with nuclear PDC-E2 in co-immunoprecipitation analysis. These findings were confirmed by confocal immunofluorescence microscopy showing constant nuclear localization of PDC-E2 and its co-localization with STAT5 after IL-3 stimulation. Similar to mitochondrial PDC-E2, nuclear PDC-E2 was lipoylated and associated with PDC-E1. Overexpression of PDC-E2 in BaF3 cells augmented IL-3-induced STAT5 activity as measured by reporter assay with consensus STAT5-binding sites. Consistent with the reporter data, PDC-E2 overexpression in BaF3 cells led to elevated mRNA levels of endogenous SOCS3 (suppressor of cytokine signaling 3) gene, a known STAT5 target. We further identified two functional STAT5-binding sites in the SOCS3 gene promoter important for its IL-3-inducibility. The observation that both cis-acting elements were essential to detect the stimulatory effect by PDC-E2 strongly supports the role of PDC-E2 in up-regulating the transactivating ability of STAT5. All together, our results reveal a novel function of PDC-E2 in the nucleus. It also

  17. Nuclear localization of pyruvate dehydrogenase complex-E2 (PDC-E2), a mitochondrial enzyme, and its role in signal transducer and activator of transcription 5 (STAT5)-dependent gene transcription

    PubMed Central

    Chueh, Fu-Yu; Leong, King-Fu; Cronk, Robert J.; Venkitachalam, Srividya; Pabich, Samantha; Yu, Chao-Lan

    2011-01-01

    STAT (signal transducer and activator of transcription) proteins play a critical role in cellular response to a wide variety of cytokines and growth factors by regulating specific nuclear genes. STAT-dependent gene transcription can be finely tuned through the association with cofactors in the nucleus. We showed previously that STAT5 (including 5a and 5b) specifically interacts with a mitochondrial enzyme PDC-E2 (E2 subunit of pyruvate dehydrogenase complex) in both leukemic T cells and cytokine-stimulated cells. However, the functional significance of this novel association remains largely unknown. Here we report that PDC-E2 may function as a co-activator in STAT5-dependent nuclear gene expression. Subcellular fractionation analysis revealed that a substantial amount of PDC-E2 was constitutively present in the nucleus of BaF3, an interleukin-3 (IL-3)-dependent cell line. IL-3-induced tyrosine-phosphorylated STAT5 associated with nuclear PDC-E2 in co-immunoprecipitation analysis. These findings were confirmed by confocal immunofluorescence microscopy showing constant nuclear localization of PDC-E2 and its co-localization with STAT5 after IL-3 stimulation. Similar to mitochondrial PDC-E2, nuclear PDC-E2 was lipoylated and associated with PDC-E1. Overexpression of PDC-E2 in BaF3 cells augmented IL-3-induced STAT5 activity as measured by reporter assay with consensus STAT5-binding sites. Consistent with the reporter data, PDC-E2 overexpression in BaF3 cells led to elevated mRNA levels of endogenous SOCS3 (suppressor of cytokine signaling 3) gene, a known STAT5 target. We further identified two functional STAT5-binding sites in the SOCS3 gene promoter important for its IL-3-inducibility. The observation that both cis-acting elements were essential to detect the stimulatory effect by PDC-E2 strongly supports the role of PDC-E2 in up-regulating the transactivating ability of STAT5. All together, our results reveal a novel function of PDC-E2 in the nucleus. It also raises

  18. SUMOylation pathway alteration coupled with downregulation of SUMO E2 enzyme at mucosal epithelium modulates inflammation in inflammatory bowel disease

    PubMed Central

    Mustfa, Salman Ahmad; Singh, Mukesh; Suhail, Aamir; Mohapatra, Gayatree; Verma, Smriti; Chakravorty, Debangana; Rana, Sarika; Rampal, Ritika; Dhar, Atika; Saha, Sudipto; Ahuja, Vineet

    2017-01-01

    Post-translational modification pathways such as SUMOylation are integral to all cellular processes and tissue homeostasis. We investigated the possible involvement of SUMOylation in the epithelial signalling in Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD). Initially in a murine model of IBD, induced by dextran–sulfate–sodium (DSS mice), we observed inflammation accompanied by a lowering of global SUMOylation of colonic epithelium. The observed SUMOylation alteration was due to a decrease in the sole SUMO E2 enzyme (Ubc9). Mass-spectrometric analysis revealed the existence of a distinct SUMOylome (SUMO-conjugated proteome) in DSS mice with alteration of key cellular regulators, including master kinase Akt1. Knocking-down of Ubc9 in epithelial cells resulted in dramatic activation of inflammatory gene expression, a phenomenon that acted via reduction in Akt1 and its SUMOylated form. Importantly, a strong decrease in Ubc9 and Akt1 was also seen in endoscopic biopsy samples (N = 66) of human CD and UC patients. Furthermore, patients with maximum disease indices were always accompanied by severely lowered Ubc9 or SUMOylated-Akt1. Mucosal tissues with severely compromised Ubc9 function displayed higher levels of pro-inflammatory cytokines and compromised wound-healing markers. Thus, our results reveal an important and previously undescribed role for the SUMOylation pathway involving Ubc9 and Akt1 in modulation of epithelial inflammatory signalling in IBD. PMID:28659381

  19. Deficiency of UBE2T, the E2 Ubiquitin Ligase Necessary for FANCD2 and FANCI Ubiquitination, Causes FA-T Subtype of Fanconi Anemia.

    PubMed

    Rickman, Kimberly A; Lach, Francis P; Abhyankar, Avinash; Donovan, Frank X; Sanborn, Erica M; Kennedy, Jennifer A; Sougnez, Carrie; Gabriel, Stacey B; Elemento, Olivier; Chandrasekharappa, Settara C; Schindler, Detlev; Auerbach, Arleen D; Smogorzewska, Agata

    2015-07-07

    Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Molecular Structure of a 9-MDa Icosahedral Pyruvate Dehydrogenase Subcomplex Containing the E2 and E3 Enzymes Using Cryoelectron Microscopy*

    PubMed Central

    Milne, Jacqueline L. S.; Wu, Xiongwu; Borgnia, Mario J.; Lengyel, Jeffrey S.; Brooks, Bernard R.; Shi, Dan; Perham, Richard N.; Subramaniam, Sriram

    2006-01-01

    The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (α2β2) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. J., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587–5598). An annular gap of ~90 Å separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of ~75 Å between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain. PMID:16308322

  1. Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats.

    PubMed

    Lecker, S H; Solomon, V; Price, S R; Kwon, Y T; Mitch, W E; Goldberg, A L

    1999-11-01

    Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of (125)I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2(14k) and E3alpha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E2(14k) inhibited this increase in ubiquitination rates. Both E2(14k) and E3alpha were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3alpha (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2(14k) and E3alpha content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2(14k) and E3alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.

  2. Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats

    NASA Technical Reports Server (NTRS)

    Lecker, S. H.; Solomon, V.; Price, S. R.; Kwon, Y. T.; Mitch, W. E.; Goldberg, A. L.

    1999-01-01

    Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of (125)I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2(14k) and E3alpha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E2(14k) inhibited this increase in ubiquitination rates. Both E2(14k) and E3alpha were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3alpha (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2(14k) and E3alpha content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2(14k) and E3alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.

  3. Evaluation of a SUMO E2 Conjugating Enzyme Involved in Resistance to Clavibacter michiganensis Subsp. michiganensis in Solanum peruvianum, Through a Tomato Mottle Virus VIGS Assay

    PubMed Central

    Esparza-Araiza, Mayra J.; Bañuelos-Hernández, Bernardo; Argüello-Astorga, Gerardo R.; Lara-Ávila, José P.; Goodwin, Paul H.; Isordia-Jasso, María I.; Castillo-Collazo, Rosalba; Rougon-Cardoso, Alejandra; Alpuche-Solís, Ángel G.

    2015-01-01

    Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker of tomato. Currently, no Solanum lycopersicum resistant varieties are commercially available, but some degree of Cmm resistance has been identified in Solanum peruvianum. Previous research showed up-regulation of a SUMO E2 conjugating enzyme (SCEI) transcript in S. peruvianum compared to S. lycopersicum following infection with Cmm. In order to test the role of SCEI in resistance to Cmm, a fragment of SCEI from S. peruvianum was cloned into a novel virus-induced gene-silencing (VIGS) vector based on the geminivirus, Tomato Mottle Virus (ToMoV). Using biolistic inoculation, the ToMoV-based VIGS vector was shown to be effective in S. peruvianum by silencing the magnesium chelatase gene, resulting in leaf bleaching. VIGS with the ToMoV_SCEI construct resulted in ~61% silencing of SCEI in leaves of S. peruvianum as determined by quantitative RT-PCR. The SCEI-silenced plants showed unilateral wilting (15 dpi) and subsequent death (20 dpi) of the entire plant after Cmm inoculation, whereas the empty vector-treated plants only showed wilting in the Cmm-inoculated leaf. The SCEI-silenced plants showed higher Cmm colonization and an average of 4.5 times more damaged tissue compared to the empty vector control plants. SCEI appears to play an important role in the innate immunity of S. peruvianum against Cmm, perhaps through the regulation of transcription factors, leading to expression of proteins involved in salicylic acid-dependent defense responses. PMID:26734014

  4. Development of a cell-based high throughput luciferase enzyme fragment complementation assay to identify nuclear-factor-e2-related transcription factor 2 activators.

    PubMed

    Xie, Wensheng; Pao, Christina; Graham, Taylor; Dul, Ed; Lu, Quinn; Sweitzer, Thomas D; Ames, Robert S; Li, Hu

    2012-12-01

    Nuclear-factor-E2-related transcription factor 2 (Nrf2) regulates a large panel of Phase II genes and plays an important role in cell survival. Nrf2 activation has been shown as preventing cigarette smoke-induced alveolar enlargement in mice. Therefore, activation of the Nrf2 protein by small-molecule activators represents an attractive therapeutic strategy that is used for chronic obstructive pulmonary disease. In this article, we describe a cell-based luciferase enzyme fragment complementation assay that identifies Nrf2 activators. This assay is based on the interaction of Nrf2 with its nuclear partner MafK or runt-related transcription factor 2 (RunX2) and is dependent on the reconstitution of a "split" luciferase. Firefly luciferase is split into two fragments, which are genetically fused to Nrf2 and MafK or RunX2, respectively. BacMam technology was used to deliver the fusion constructs into cells for expression of the tagged proteins. When the BacMam-transduced cells were treated with Nrf2 activators, the Nrf2 protein was stabilized and translocated into the nucleus where it interacted with MafK or RunX2. The interaction of Nrf2 and MafK or RunX2 brought together the two luciferase fragments that form an active luciferase. The assay was developed in a 384-well format and was optimized by titrating the BacMam concentration, transduction time, cell density, and fetal bovine serum concentration. It was further validated with known Nrf2 activators. Our data show that this assay is robust, sensitive, and amenable to high throughput screening of a large compound collection for the identification of novel Nrf2 activators.

  5. Elucidating the role of highly homologous Nicotiana benthamiana ubiquitin E2 gene family members in plant immunity through an improved virus-induced gene silencing approach.

    PubMed

    Zhou, Bangjun; Zeng, Lirong

    2017-01-01

    Virus-induced gene silencing (VIGS) has been used in many plant species as an attractive post transcriptional gene silencing (PTGS) method for studying gene function either individually or at large-scale in a high-throughput manner. However, the specificity and efficiency for knocking down members of a highly homologous gene family have remained to date a significant challenge in VIGS due to silencing of off-targets. Here we present an improved method for the selection and evaluation of gene fragments used for VIGS to specifically and efficiently knock down members of a highly homologous gene family. Using this method, we knocked down twelve and four members, respectively of group III of the gene family encoding ubiquitin-conjugating enzymes (E2) in Nicotiana benthamiana . Assays using these VIGS-treated plants revealed that the group III E2s are essential for plant development, plant immunity-associated reactive oxygen species (ROS) production, expression of the gene NbRbohB that is required for ROS production, and suppression of immunity-associated programmed cell death (PCD) by AvrPtoB, an effector protein of the bacterial pathogen Pseudomons syringae . Moreover, functional redundancy for plant development and ROS production was found to exist among members of group III E2s. We have found that employment of a gene fragment as short as approximately 70 base pairs (bp) that contains at least three mismatched nucleotides to other genes within any 21-bp sequences prevents silencing of off-target(s) in VIGS. This improved approach in the selection and evaluation of gene fragments allows for specific and efficient knocking down of highly homologous members of a gene family. Using this approach, we implicated N. benthamiana group III E2s in plant development, immunity-associated ROS production, and suppression of multiple immunity-associated PCD by AvrPtoB. We also unraveled functional redundancy among group III members in their requirement for plant development and

  6. Both conditional ablation and overexpression of E2 SUMO-conjugating enzyme (UBC9) in mouse pancreatic beta cells result in impaired beta cell function.

    PubMed

    He, Xiaoyu; Lai, Qiaohong; Chen, Cai; Li, Na; Sun, Fei; Huang, Wenting; Zhang, Shu; Yu, Qilin; Yang, Ping; Xiong, Fei; Chen, Zhishui; Gong, Quan; Ren, Boxu; Weng, Jianping; Eizirik, Décio L; Zhou, Zhiguang; Wang, Cong-Yi

    2018-04-01

    Post-translational attachment of a small ubiquitin-like modifier (SUMO) to the lysine (K) residue(s) of target proteins (SUMOylation) is an evolutionary conserved regulatory mechanism. This modification has previously been demonstrated to be implicated in the control of a remarkably versatile regulatory mechanism of cellular processes. However, the exact regulatory role and biological actions of the E2 SUMO-conjugating enzyme (UBC9)-mediated SUMOylation function in pancreatic beta cells has remained elusive. Inducible beta cell-specific Ubc9 (also known as Ube2i) knockout (KO; Ubc9 Δbeta ) and transgenic (Ubc9 Tg ) mice were employed to address the impact of SUMOylation on beta cell viability and functionality. Ubc9 deficiency or overexpression was induced at 8 weeks of age using tamoxifen. To study the mechanism involved, we closely examined the regulation of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) through SUMOylation in beta cells. Upon induction of Ubc9 deficiency, Ubc9 Δbeta islets exhibited a 3.5-fold higher accumulation of reactive oxygen species (ROS) than Ubc9 f/f control islets. Islets from Ubc9 Δbeta mice also had decreased insulin content and loss of beta cell mass after tamoxifen treatment. Specifically, at day 45 after Ubc9 deletion only 40% of beta cell mass remained in Ubc9 Δbeta mice, while 90% of beta cell mass was lost by day 75. Diabetes onset was noted in some Ubc9 Δbeta mice 8 weeks after induction of Ubc9 deficiency and all mice developed diabetes by 10 weeks following tamoxifen treatment. In contrast, Ubc9 Tg beta cells displayed an increased antioxidant ability but impaired insulin secretion. Unlike Ubc9 Δbeta mice, which spontaneously developed diabetes, Ubc9 Tg mice preserved normal non-fasting blood glucose levels without developing diabetes. It was noted that SUMOylation of NRF2 promoted its nuclear expression along with enhanced transcriptional activity, thereby preventing ROS accumulation in

  7. SerpinE2, a poor biomarker of endometrial cancer, promotes the proliferation and mobility of EC cells.

    PubMed

    Shen, Yuan; Wang, Xiaoyu; Xu, Jianping; Lu, Lin

    2017-07-04

    The SerpinE2 pathway is evolutionarily conserved and plays an important role in tumorigenesis. SerpinE2 (a small ubiquitin-related modifier), like ubiquitin, conjugates SerpinE2 proteins onto lysine residues of target proteins. SerpinE2 over-expression has been found in several tumors. Here, we detected the level of SerpinE2 in 72 samples of EC tissue using immunohistochemistry to assess the role of SerpinE2 in EC prognosis. Meanwhile, we knocked down SerpinE2 by siRNA in the HTB-111 and Ishikawa EC cell lines and analyzed the viability and mobility change using an MTT assay, an annexin V/PI apoptosis assay, a wound scratch test and a transwell assay. A Kaplan-Meier analysis indicated a negative correlation between the level of SerpinE2 and the EC prognosis. Silencing SerpinE2 induced cell apoptosis and reduced the migration ability. Our data suggest SerpinE2 works as an oncogene in EC.

  8. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  9. Environmental Technology Verification Report for Abraxis Ecologenia® 17β-Estradiol (E2) Microplate Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits

    EPA Science Inventory

    This verification test was conducted according to procedures specifiedin the Test/QA Planfor Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kis for the Quantitative Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous Phase Samples. Deviations to the...

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

    EPA Science Inventory

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

  11. Prostaglandin E2 Regulates Its Own Inactivating Enzyme, 15-PGDH, by EP2 Receptor-Mediated Cervical Cell-Specific Mechanisms

    PubMed Central

    Kishore, A. Hari; Owens, David

    2014-01-01

    Context: Prostaglandins play important roles in parturition and have been used to induce cervical ripening and labor. Prior to cervical ripening at term, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is highly expressed in the cervix and metabolizes cyclooxygenase-2-mediated increases in active prostaglandin E2 (PGE2) to inactive 15-keto PGE2. At term, 15-PGDH gene expression decreases and PGE2 accumulates, leading to cervical ripening and labor. Previously, we found that the cervical isoform of microphthalmia-associated transcription factor (MiTF-CX) serves as a progestational transcription factor that represses IL-8 and hypoxia-mediated increases in cyclooxygenase-2. Objective: We tested the hypothesis that PGE2 regulates its own inactivation through MiTF-CX. Design: We used human cervical stromal cells to investigate the regulation of 15-PGDH. Setting: This was a laboratory-based study using cells from clinical tissue samples. Main Outcome Measures: We evaluated the mechanisms by which PGE2 regulates 15-PGDH in human cervical stromal cells. Results: PGE2 repressed MiTF-CX and 15-PGDH, whereas ectopic overexpression of MiTF-CX induced 15-PGDH expression levels. Stabilization of HIF-1α by deferoxamine resulted in concomitant down-regulation of MiTF-CX and 15-PGDH. Ectopic overexpression of MiTF-CX abrogated PGE2- and deferoxamine-mediated loss of MiTF-CX and 15-PGDH. PGE2-induced loss of MiTF-CX and 15-PGDH was mediated through prostaglandin E2 receptor (EP2) receptors (PTGER2), but not cAMP. Conclusions: The 15-PGDH gene is a MiTF-CX target gene in cervical stromal cells and is down-regulated by PGE2 through EP2 receptors. The findings suggest that EP2 receptor-specific antagonists may be used as an adjunct to present clinical management for the prevention of preterm cervical ripening and preterm labor. PMID:24471568

  12. INHIBITION OF ERN1 SIGNALING ENZYME AFFECTS HYPOXIC REGULATION OF THE EXPRESSION OF E2F8, EPAS1, HOXC6, ATF3, TBX3 AND FOXF1 GENES IN U87 GLIOMA CELLS.

    PubMed

    Minchenko, O H; Tsymbal, D O; Minchenko, D O; Kovalevska, O V; Karbovskyi, L L; Bikfalvi, A

    2015-01-01

    Hypoxia as well as the endoplasmic reticulum stress are important factors of malignant tumor growth and control of the expression of genes, which regulate numerous metabolic processes and cell proliferation. Furthermore, blockade of ERN1 (endoplasmic reticulum to nucleus 1) suppresses cell proliferation and tumor growth. We studied the effect of hypoxia on the expression of genes encoding the transcription factors such as E2F8 (E2F transcription factor 8), EPAS1 (endothelial PAS domain protein 1), TBX3 (T-box 3), ATF3 (activating transcription factor 3), FOXF1 (forkhead box F), and HOXC6 (homeobox C6) in U87 glioma cells with and without ERN1 signaling enzyme function. We have established that hypoxia enhances the expression of HOXC6, E2F8, ATF3, and EPAS1 genes but does not change TBX3 and FOXF1 gene expression in glioma cells with ERNI function. At the same time, the expression level of all studied genes is strongly decreased, except for TBX3 gene, in glioma cells without ERN1 function. Moreover, the inhibition of ERN1 signaling enzyme function significantly modifies the effect of hypoxia on the expression of these transcription factor genes. removes or introduces this regulation as well as changes a direction or magnitude of hypoxic regulation. Present study demonstrates that fine-tuning of the expression of proliferation related genes depends upon hypoxia and ERN1-mediated endoplasmic reticulum stress signaling and correlates with slower proliferation rate of glioma cells without ERN1 function.

  13. Evaluation of an Erns-based enzyme-linked immunosorbent assay to distinguish Classical swine fever virus-infected pigs from pigs vaccinated with CP7_E2alf.

    PubMed

    Pannhorst, Katrin; Fröhlich, Andreas; Staubach, Christoph; Meyer, Denise; Blome, Sandra; Becher, Paul

    2015-07-01

    Infections with Classical swine fever virus (CSFV) are a major economic threat to pig production. To combat CSF outbreaks and to maintain trade, new marker vaccines were developed that allow differentiation of infected from vaccinated animals (DIVA principle). The chimeric pestivirus CP7_E2alf was shown to be safe and efficacious. Its DIVA strategy is based on the detection of CSFV E(rns)-specific antibodies that are only developed on infection. However, for the new marker vaccine to be considered a valuable control tool, a validated discriminatory assay is needed. One promising candidate is the already commercially available enzyme-linked immunosorbent assay, PrioCHECK CSFV E(rns) ELISA (Prionics BV, Lelystad, The Netherlands). Four laboratories of different European Union member states tested 530 serum samples and country-specific field sera from domestic pigs and wild boar. The ELISA displayed a good robustness. However, based on its reproducibility and repeatability, ranges rather than single values for diagnostic sensitivity and specificity were defined. The ELISA displayed a sensitivity of 90-98% with sera from CSFV-infected domestic pigs. A specificity of 89-96% was calculated with sera from domestic pigs vaccinated once with CP7_E2alf. The ELISA detected CSFV infections in vaccinated domestic pigs with a sensitivity of 82-94%. The sensitivity was lower with sera taken ≤21 days post-challenge indicating that the stage of CSFV infection had a considerable influence on testing. Taken together, the PrioCHECK CSFV E(rns) ELISA can be used for detection of CSFV infections in CP7_E2alf-vaccinated and nonvaccinated domestic pig populations, but should only be applied on a herd basis by testing a defined number of animals. © 2015 The Author(s).

  14. The E(2) particle

    SciTech Connect

    Ghosh, Subir; Pal, Probir; Physics Department, Uluberia College, Uluberia, Howrah 711315

    2009-12-15

    Recently it has been advocated [A. G. Cohen and S. L. Glashow, Phys. Rev. Lett. 97, 021601 (2006)] that for describing nature within the minimal symmetry requirement, certain subgroups of the Lorentz group may play a fundamental role. One such group is E(2) which induces a Lie algebraic noncommutative spacetime [M. M. Sheikh-Jabbari and A. Tureanu, Phys. Rev. Lett. 101, 261601 (2008); arXiv:0811.3670] where translation invariance is not fully maintained. We have constructed a consistent structure of noncommutative phase space for this system, and furthermore we have studied an appropriate point particle action on it. Interestingly, the Einstein dispersion relationmore » p{sup 2}=m{sup 2} remains intact. The model is constructed by exploiting a dual canonical phase space following the scheme developed by us earlier [S. Ghosh and P. Pal, Phys. Rev. D 75, 105021 (2007)].« less

  15. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

    PubMed

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-07-22

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation*

    PubMed Central

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-01-01

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

  17. The Papillomavirus E2 proteins

    SciTech Connect

    McBride, Alison A., E-mail: amcbride@nih.gov

    2013-10-15

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

  18. H2A-DUBbing the mammalian epigenome: expanding frontiers for histone H2A deubiquitinating enzymes in cell biology and physiology.

    PubMed

    Belle, Jad I; Nijnik, Anastasia

    2014-05-01

    Posttranslational modifications of histone H2A through the attachment of ubiquitin or poly-ubiquitin conjugates are common in mammalian genomes and play an important role in the regulation of chromatin structure, gene expression, and DNA repair. Histone H2A deubiquitinases (H2A-DUBs) are a group of structurally diverse enzymes that catalyze the removal ubiquitin from histone H2A. In this review we provide a concise summary of the mechanisms that mediate histone H2A ubiquitination in mammalian cells, and review our current knowledge of mammalian H2A-DUBs, their biochemical activities, and recent developments in our understanding of their functions in mammalian physiology. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Differential Gene Expression in Explanted Human Retinal Pigment Epithelial Cells 12-Hours Post-Exposure to 532 nm, 120 ps Pulsed Laser Light

    DTIC Science & Technology

    2004-04-01

    cycling, anaerobic enzymes and kinase enzymes as well as specific cellular channel or receptor components. However, the most striking revelation of the...degradation. Most notably up-regulated were the genes for the enzymes essential in the ubiquitin-proteoasome pathway (UPP) shown to be up-regulated in response...to oxidative stress in eye tissue (1). These were ubiquitin [2.0], 3 ubiquitin-conjugating enzyme genes E2 [2.3], E2D2 [2.3] and E2D3 [2.8]. Also up

  20. A novel UBE2A mutation causes X-linked intellectual disability type Nascimento.

    PubMed

    Tsurusaki, Yoshinori; Ohashi, Ikuko; Enomoto, Yumi; Naruto, Takuya; Mitsui, Jun; Aida, Noriko; Kurosawa, Kenji

    2017-01-01

    X-linked intellectual disability (ID) type Nascimento (MIM #300860), also known as ubiquitin-conjugating enzyme E2 A (UBE2A) deficiency syndrome, is a congenital malformation syndrome characterized by moderate to severe ID, speech impairment, dysmorphic facial features, genital anomalies and skin abnormalities. Here, we report a Japanese patient with severe ID and congenital cataract. We identified a novel hemizygous mutation (c.76G>A, p.Gly26Arg) in UBE2A by whole-exome sequencing.

  1. E-2D Advanced Hawkeye Aircraft (E-2D AHE)

    DTIC Science & Technology

    2015-12-01

    and Homeland Defense. As a part of the E-2D AHE radar modernization effort, the Navy also invested in integrating a full glass cockpit and full...Communication Navigation Surveillance/Air Traffic Management capability. The glass cockpit will also provide the capability for the pilot or co-pilot to...hours at a station distance of 200nm Flat Turn Service Ceiling =>25,000 feet above MSL at mission profile =>25,000 feet above MSL at mission

  2. Atypical E2f functions are critical for pancreas polyploidization

    PubMed Central

    Moreno, Eva; Toussaint, Mathilda J. M.; Tooten, Peter C. J.; van Essen, Saskia C.; van Liere, Elsbeth A.; Youssef, Sameh A.; Bongiovanni, Laura; de Bruin, Alain

    2018-01-01

    The presence of polyploid cells in the endocrine and exocrine pancreas has been reported for four decades. In rodents, pancreatic polyploidization is initiated after weaning and the number of polyploid cells increases with age. Surprisingly the molecular regulators and biological functions of polyploidization in the pancreas are still unknown. We discovered that atypical E2f activity is essential for polyploidization in the pancreas, using an inducible Cre/LoxP approach in new-born mice to delete ubiquitously the atypical E2f transcription factors, E2f7 and E2f8. In contrast to its critical role in embryonic survival, conditional deletion of both of both atypical E2fs in newborn mice had no impact on postnatal survival and mice lived until old age. However, deficiency of E2f7 or E2f8 alone was sufficient to suppress polyploidization in the pancreas and associated with only a minor decrease in blood serum levels of glucose, insulin, amylase and lipase under 4 hours starvation condition compared to wildtype littermates. In mice with fewer pancreatic polyploid cells that were fed ad libitum, no major impact on hormones or enzymes levels was observed. In summary, we identified atypical E2fs to be essential for polyploidization in the pancreas and discovered that postnatal induced loss of both atypical E2fs in many organs is compatible with life until old age. PMID:29329320

  3. Atypical E2f functions are critical for pancreas polyploidization.

    PubMed

    Matondo, Ramadhan B; Moreno, Eva; Toussaint, Mathilda J M; Tooten, Peter C J; van Essen, Saskia C; van Liere, Elsbeth A; Youssef, Sameh A; Bongiovanni, Laura; de Bruin, Alain

    2018-01-01

    The presence of polyploid cells in the endocrine and exocrine pancreas has been reported for four decades. In rodents, pancreatic polyploidization is initiated after weaning and the number of polyploid cells increases with age. Surprisingly the molecular regulators and biological functions of polyploidization in the pancreas are still unknown. We discovered that atypical E2f activity is essential for polyploidization in the pancreas, using an inducible Cre/LoxP approach in new-born mice to delete ubiquitously the atypical E2f transcription factors, E2f7 and E2f8. In contrast to its critical role in embryonic survival, conditional deletion of both of both atypical E2fs in newborn mice had no impact on postnatal survival and mice lived until old age. However, deficiency of E2f7 or E2f8 alone was sufficient to suppress polyploidization in the pancreas and associated with only a minor decrease in blood serum levels of glucose, insulin, amylase and lipase under 4 hours starvation condition compared to wildtype littermates. In mice with fewer pancreatic polyploid cells that were fed ad libitum, no major impact on hormones or enzymes levels was observed. In summary, we identified atypical E2fs to be essential for polyploidization in the pancreas and discovered that postnatal induced loss of both atypical E2fs in many organs is compatible with life until old age.

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  5. pVHL's kryptonite: E2-EPF UCP.

    PubMed

    Ohh, Michael

    2006-08-01

    E2-EPF ubiquitin carrier protein (UCP) is a member of an E2 family of enzymes that catalyzes the ligation of ubiquitin to proteins targeted for destruction by the proteasome. UCP is overexpressed in common human cancers, suggesting its involvement in oncogenesis, but a physiologic target of UCP has not been identified. In a recent report published in Nature Medicine, Jung et al. identified von Hippel-Lindau (VHL) tumor suppressor protein, which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction, as a bona fide substrate of UCP and demonstrated a potential pVHL-HIF pathway-dependent role for UCP in cancer development.

  6. The Astro-E2 Mission

    NASA Technical Reports Server (NTRS)

    Kelley, Richard L.

    2004-01-01

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

  7. How Does (E)-2-(Acetamidomethylene)succinate Bind to Its Hydrolase? From the Binding Process to the Final Result

    PubMed Central

    Zhang, Ji-Long; Zheng, Qing-Chuan; Li, Zheng-Qiang; Zhang, Hong-Xing

    2013-01-01

    The binding of (E)-2-(acetamidomethylene)succinate (E-2AMS) to E-2AMS hydrolase is crucial for biological function of the enzyme and the last step reaction of vitamin B6 biological degradation. In the present study, several molecular simulation methods, including molecular docking, conventional molecular dynamics (MD), steered MD (SMD), and free energy calculation methods, were properly integrated to investigate the detailed binding process of E-2AMS to its hydrolase and to assign the optimal enzyme-substrate complex conformation. It was demonstrated that the substrate binding conformation with trans-form amide bond is energetically preferred conformation, in which E-2AMS's pose not only ensures hydrogen bond formation of its amide oxygen atom with the vicinal oxyanion hole but also provides probability of the hydrophobic interaction between its methyl moiety and the related enzyme's hydrophobic cavity. Several key residues, Arg146, Arg167, Tyr168, Arg179, and Tyr259, orientate the E-2AMS's pose and stabilize its conformation in the active site via the hydrogen bond interaction with E-2AMS. Sequentially, the binding process of E-2AMS to E-2AMS hydrolase was studied by SMD simulation, which shows the surprising conformational reversal of E-2AMS. Several important intermediate structures and some significant residues were identified in the simulation. It is stressed that Arg146 and Arg167 are two pivotal residues responsible for the conformational reversal of E-2AMS in the binding or unbinding. Our research has shed light onto the full binding process of the substrate to E-2AMS hydrolase, which could provide more penetrating insight into the interaction of E-2AMS with the enzyme and would help in the further exploration on the catalysis mechanism. PMID:23308285

  8. E2E: A Summary of the e2e Learning Framework.

    ERIC Educational Resources Information Center

    Learning and Skills Development Agency, London (England).

    This publication is a summary of the E2E (Entry to Employment) Learning Framework that provides guidance on program implementation. (E2E is a new learning program for young people not yet ready or able to enter Modern Apprenticeship programs, a Level 2 program, or employment directly.) Section 2 highlights core values to which all involved should…

  9. A novel UBE2A mutation causes X-linked intellectual disability type Nascimento

    PubMed Central

    Tsurusaki, Yoshinori; Ohashi, Ikuko; Enomoto, Yumi; Naruto, Takuya; Mitsui, Jun; Aida, Noriko; Kurosawa, Kenji

    2017-01-01

    X-linked intellectual disability (ID) type Nascimento (MIM #300860), also known as ubiquitin-conjugating enzyme E2 A (UBE2A) deficiency syndrome, is a congenital malformation syndrome characterized by moderate to severe ID, speech impairment, dysmorphic facial features, genital anomalies and skin abnormalities. Here, we report a Japanese patient with severe ID and congenital cataract. We identified a novel hemizygous mutation (c.76G>A, p.Gly26Arg) in UBE2A by whole-exome sequencing. PMID:28611923

  10. The importance of regulatory ubiquitination in cancer and metastasis

    PubMed Central

    Gallo, L. H.; Ko, J.; Donoghue, D. J.

    2017-01-01

    ABSTRACT Ubiquitination serves as a degradation mechanism of proteins, but is involved in additional cellular processes such as activation of NFκB inflammatory response and DNA damage repair. We highlight the E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases and Deubiquitinases that support the metastasis of a plethora of cancers. E3 ubiquitin ligases also modulate pluripotent cancer stem cells attributed to chemotherapy resistance. We further describe mutations in E3 ubiquitin ligases that support tumor proliferation and adaptation to hypoxia. Thus, this review describes how tumors exploit members of the vast ubiquitin signaling pathways to support aberrant oncogenic signaling for survival and metastasis. PMID:28166483

  11. Prostaglandin E(2) synthase inhibition as a therapeutic target.

    PubMed

    Iyer, Jitesh P; Srivastava, Punit K; Dev, Rishabh; Dastidar, Sunanda G; Ray, Abhijit

    2009-07-01

    Most NSAIDs function by inhibiting biosynthesis of PGE(2) by inhibition of COX-1 and/or COX-2. Since COX-1 has a protective function in the gastro-intestinal tract (GIT), non-selective inhibition of both cycloxy genases leads to moderate to severe gastro-intestinal intolerance. Attempts to identify selective inhibitors of COX-2, led to the identification of celecoxib and rofecoxib. However, long-term use of these drugs has serious adverse effects of sudden myocardial infarction and thrombosis. Drug-mediated imbalance in the levels of prostaglandin I(2) (PGI(2)) and thromboxane A(2) (TXA(2)) with a bias towards TXA(2) may be the primary reason for these events. This resulted in the drugs being withdrawn from the market, leaving a need for an effective and safe anti-inflammatory drug. Recently, the focus of research has shifted to enzymes downstream of COX in the prosta glandin biosynthetic pathway such as prostaglandin E(2) synthases. Microsomal prostaglandin E(2) synthase-1 (mPGES-1) specifically isomerizes PGH(2) to PGE(2), under inflammatory conditions. In this review, we examine the biology of mPGES-1 and its role in disease. Progress in designing molecules that can selectively inhibit mPGES-1 is reviewed. mPGES-1 has the potential to be a target for anti-inflammatory therapy, devoid of adverse GIT and cardiac effects and warrants further investigation.

  12. Enzyme Informatics

    PubMed Central

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  13. The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2

    PubMed Central

    Wickliffe, Katherine E.; Lorenz, Sonja; Wemmer, David E.; Kuriyan, John; Rape, Michael

    2011-01-01

    Ubiquitin chains of different topologies trigger distinct functional consequences, including protein degradation and reorganization of complexes. The assembly of most ubiquitin chains is promoted by E2s, yet how these enzymes achieve linkage specificity is poorly understood. We have discovered that the K11-specific Ube2S orients the donor ubiquitin through an essential non-covalent interaction that occurs in addition to the thioester bond at the E2 active site. The E2-donor ubiquitin complex transiently recognizes the acceptor ubiquitin, primarily through electrostatic interactions. The recognition of the acceptor ubiquitin surface around Lys11, but not around other lysines, generates a catalytically competent active site, which is composed of residues of both Ube2S and ubiquitin. Our studies suggest that monomeric E2s promote linkage-specific ubiquitin chain formation through substrate-assisted catalysis. PMID:21376237

  14. Regulation of the nuclear factor (NF)-kappaB pathway by ISGylation.

    PubMed

    Minakawa, Miki; Sone, Takayuki; Takeuchi, Tomoharu; Yokosawa, Hideyoshi

    2008-12-01

    Post-translational modification with ISG15 (interferon-stimulated gene 15 kDa) (ISGylation) is mediated by a sequential reaction similar to ubiquitination, and various target proteins for ISGylation have been identified. We previously reported that ISGylation of the E2 ubiquitin-conjugating enzyme Ubc13 suppresses its E2 activity. Ubc13 forms a heterodimer with Uev1A, a ubiquitin-conjugating enzyme variant, and the Ubc13-Uev1A complex catalyzes the assembly of a Lys63-linked polyubiquitin chain, which plays a non-proteolytic role in the nuclear factor (NF)-kappaB pathway. In this study, we examined the effect of ISGylation on tumor necrosis factor receptor-associated factor (TRAF)-6/transforming growth factor beta-activated kinase (TAK)-1-dependent NF-kappaB activation. We found that expression of the ISGylation system suppresses NF-kappaB activation via TRAF6 and TAK1 and that the level of polyubiquitinated TRAF6 is reduced by expression of the ISGylation system. Taken together, the results suggest that the NF-kappaB pathway is negatively regulated by ISGylation.

  15. 26 CFR 1.503(e)-2 - Requirements.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 7 2014-04-01 2013-04-01 true Requirements. 1.503(e)-2 Section 1.503(e)-2...) INCOME TAXES (CONTINUED) Exempt Organizations § 1.503(e)-2 Requirements. (a) In general. The requirements... price may not be a valid price for 1,000 bonds and the purchase may therefore not meet the requirements...

  16. 26 CFR 1.503(e)-2 - Requirements.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 7 2011-04-01 2009-04-01 true Requirements. 1.503(e)-2 Section 1.503(e)-2...) INCOME TAXES (CONTINUED) Exempt Organizations § 1.503(e)-2 Requirements. (a) In general. The requirements... price may not be a valid price for 1,000 bonds and the purchase may therefore not meet the requirements...

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 7 2013-04-01 2013-04-01 false Requirements. 1.503(e)-2 Section 1.503(e)-2...) INCOME TAXES (CONTINUED) Exempt Organizations § 1.503(e)-2 Requirements. (a) In general. The requirements... price may not be a valid price for 1,000 bonds and the purchase may therefore not meet the requirements...

  18. The ubiquitin-proteasome system is required for African swine fever replication.

    PubMed

    Barrado-Gil, Lucía; Galindo, Inmaculada; Martínez-Alonso, Diego; Viedma, Sergio; Alonso, Covadonga

    2017-01-01

    Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host's ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.

  19. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  20. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  1. Ectopic High Expression of E2-EPF Ubiquitin Carrier Protein Indicates a More Unfavorable Prognosis in Brain Glioma.

    PubMed

    Zhang, Xiaohui; Zhao, Fangbo; Zhang, Shujun; Song, Yichun

    2017-04-01

    Ubiquitination of proteins meant for elimination is a primary method of eukaryotic cellular protein degradation. The ubiquitin carrier protein E2-EPF is a key degradation enzyme that is highly expressed in many tumors. However, its expression and prognostic significance in brain glioma are still unclear. The aim of this study was to reveal how the level of E2-EPF relates to prognosis in brain glioma. Thirty low-grade and 30 high-grade brain glioma samples were divided into two tissue microarrays each. Levels of E2-EPF protein were examined by immunohistochemistry and immunofluorescence. Quantitative real-time polymerase chain reaction was used to analyze the level of E2-EPF in 60 glioma and 3 normal brain tissue samples. The relationship between E2-EPF levels and prognosis was analyzed by Kaplan-Meier survival curves. E2-EPF levels were low in normal brain tissue samples but high in glioma nuclei. E2-EPF levels gradually increased as glioma grade increased (p < 0.05). Ectopic E2-EPF levels in high-grade glioma were significantly higher than in low-grade glioma (p < 0.01). The 5-year survival rate of glioma patients with high E2-EPF levels was shorter than in patients with low expression (p < 0.05). Furthermore, the 5-year survival rate of patients with ectopic E2-EPF was significantly shorter than patients with only nuclear E2-EPF (p < 0.01). These results suggest that higher E2-EPF levels, especially ectopic, are associated with higher grade glioma and shorter survival. E2-EPF levels may play a key role in predicting the prognosis for patients with brain glioma.

  2. Estradiol-17β, prostaglandin E2 (PGE2) and the prostaglandin E2 receptor are involved in PGE2 positive feedback loop in the porcine endometrium

    PubMed Central

    Waclawik, Agnieszka; Jabbour, Henry N.; Blitek, Agnieszka; Ziecik, Adam J.

    2009-01-01

    Before implantation, the porcine endometrium and trophoblast synthesize elevated amounts of luteoprotective prostaglandin E2 (PGE2). We hypothesized that embryo signal, estradiol-17β (E2) and PGE2 modulate expression of key enzymes in PG synthesis: prostaglandin-endoperoxide synthase-2 (PTGS2), PGE synthase (mPGES-1), PGF synthase (PGFS), and prostaglandin 9-ketoreductase (CBR1); as well as PGE2 receptor (PTGER2 and 4) expression and signaling within the endometrium. We determinated the site of action of PGE2 in endometrium during the estrous cycle and pregnancy. Endometrial tissue explants obtained from gilts (n=6) on days 11-12 of the estrous cycle were treated with vehicle (control), PGE2 (100 nM), E2 (1-100 nM) or phorbol 12-myristate 13-acetate (100 nM, positive control). E2 increased PGE2 secretion through elevating expression of mPGES-1 mRNA and PTGS2 and mPGES-1 protein in endometrial explants. By contrast, E2 decreased PGFS and CBR1 protein expression. E2 also stimulated PTGER2 but not PTGER4 protein content. PGE2 enhanced mPGES-1 and PTGER2 mRNA as well as PTGS2, mPGES-1 and PTGER2 protein expression. PGE2 had no effect on PGFS, CBR1 and PTGER4 expression and PGF2α release. Treatment of endometrial tissue with PGE2 increased cAMP production. Co-treatment with PTGER2 antagonist (AH6809) but not PTGER4 antagonist (GW 627368X) inhibited significantly PGE2-mediated cAMP production. PTGER2 protein was localized in luminal and glandular epithelium and blood vessels of endometrium, and was significantly up-regulated on days 11-12 of pregnancy. Our results suggest that E2, prevents luteolysis through enzymatic modification of PG synthesis and that E2, PGE2 and endometrial PTGER2 are involved in PGE2 positive feedback loop in porcine endometrium. PMID:19359378

  3. Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

    PubMed

    Iglesias, Ainhoa; Murga, Matilde; Laresgoiti, Usua; Skoudy, Anouchka; Bernales, Irantzu; Fullaondo, Asier; Moreno, Bernardino; Lloreta, José; Field, Seth J; Real, Francisco X; Zubiaga, Ana M

    2004-05-01

    E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

  4. The effects of periodontal therapy on intracrevicular prostaglandin E2 concentrations and clinical parameters in pregnancy.

    PubMed

    Yalcin, Funda; Basegmez, Cansu; Isik, Gulden; Berber, Lacin; Eskinazi, Esti; Soydinc, Mahtaban; Issever, Halim; Onan, Utku

    2002-02-01

    The increase in circulating levels of progesterone during pregnancy stimulates production of prostaglandins, especially prostaglandin E2, possibly resulting in pregnancy gingivitis. The purpose of this study is to evaluate the influence of prostaglandin E2 concentrations on gingival tissues in pregnancy and to assess its relationship to clinical parameters. This study evaluates the effects of periodontal treatment on clinical indices including plaque index, gingival index, probing depth, and gingival crevicular fluid prostaglandin E2 levels of 22 pregnant women in their first, second, and third trimesters. Initial periodontal therapy consisting of scaling, root planing, and oral hygiene instruction was performed at the beginning of the first trimester and repeated each trimester. Prostaglandin E2 concentrations in gingival crevicular fluid were determined using a commercially available enzyme immunoassay kit. The statistical tests used were paired sample test and correlation analysis. The results of the study show that periodontal therapy has resulted in an improvement in clinical parameters (P<0.05). There is also a statistically significant decrease in levels of prostaglandin E2 at the second and third trimesters following periodontal therapy (P <0.001). The correlation between prostaglandin E2 concentrations and clinical parameters is found to be non-significant (P >0.05). Our data indicate that levels of prostaglandin E2 in gingival crevicular fluid may be used as a marker of gingival inflammation in order to determine the effects of periodontal therapy in pregnancy. Periodontal therapy that is performed throughout the entire pregnancy period may help prevent the threat of pregnancy gingivitis.

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

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

    2015-01-01

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

  7. [Ubiquitin-proteasome system and sperm DNA repair: An update].

    PubMed

    Zhang, Guo-Wei; Cai, Hong-Cai; Shang, Xue-Jun

    2016-09-01

    The ubiquitin-proteasome system (UPS) is a proteasome system widely present in the human body, which is composed of ubiquitin (Ub), ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), ubiquitin protein ligases (E3), 26S proteasome, and deubiquitinating enzymes (DUBs) and involved in cell cycle regulation, immune response, signal transduction, DNA repair as well as protein degradation. Sperm DNA is vulnerable to interference or damage in the progression of chromosome association and homologous recombination. Recent studies show that UPS participates in DNA repair in spermatogenesis by modulating DNA repair enzymes via ubiquitination, assisting in the identification of DNA damage sites, raising damage repair-related proteins, initiating the DNA repair pathway, maintaining chromosome stability, and ensuring the normal process of spermatogenesis.

  8. Protein quality control at the inner nuclear membrane

    PubMed Central

    Khmelinskii, Anton; Blaszczak, Ewa; Pantazopoulou, Marina; Fischer, Bernd; Omnus, Deike J.; Le Dez, Gaëlle; Brossard, Audrey; Gunnarsson, Alexander; Barry, Joseph D.; Meurer, Matthias; Kirrmaier, Daniel; Boone, Charles; Huber, Wolfgang; Rabut, Gwenaël; Ljungdahl, Per O.; Knop, Michael

    2015-01-01

    The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression1. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER) and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by ER-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc72,3. However, little is known regarding protein quality control at the INM. Here we describe a protein degradation pathway at the INM mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi34. We report that the As complex functions together with the ubiquitin conjugating enzymes Ubc6andUbc7to degrade soluble and integral membrane proteins. Genetic evidence suggest that the Asi ubiquitin ligase defines a pathway distinct from but complementary to ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer (tFT)5, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquity ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalised integral membrane proteins, thus acting to maintain and safeguard the identity of the INM. PMID:25519137

  9. RNF8- and Ube2S-Dependent Ubiquitin Lysine 11-Linkage Modification in Response to DNA Damage.

    PubMed

    Paul, Atanu; Wang, Bin

    2017-05-18

    Ubiquitin modification of proteins plays pivotal roles in the cellular response to DNA damage. Given the complexity of ubiquitin conjugation due to the formation of poly-conjugates of different linkages, functional roles of linkage-specific ubiquitin modification at DNA damage sites are largely unclear. We identify that Lys11-linkage ubiquitin modification occurs at DNA damage sites in an ATM-dependent manner, and ubiquitin-modifying enzymes, including Ube2S E2-conjugating enzyme and RNF8 E3 ligase, are responsible for the assembly of Lys11-linkage conjugates on damaged chromatin, including histone H2A/H2AX. We show that RNF8- and Ube2S-dependent Lys11-linkage ubiquitin conjugation plays an important role in regulating DNA damage-induced transcriptional silencing, distinct from the role of Lys63-linkage ubiquitin in the recruitment of DNA damage repair proteins 53BP1 and BRCA1. Thus, our study highlights the importance of linkage-specific ubiquitination at DNA damage sites, and it reveals that Lys11-linkage ubiquitin modification plays a crucial role in the DNA damage response. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Structure of an E3:E2~Ub Complex Reveals an Allosteric Mechanism Shared among RING/U-box Ligases

    SciTech Connect

    Pruneda, Jonathan N.; Littlefield, Peter J.; Soss, Sarah E.

    2012-09-28

    Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanismby which this class of enzymes facilitates Ub transfer remains enigmatic. Here, we present a structural model for a RING/U-box E3:E2~Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2~Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 side chain and an E2 backbone carbonyl, observed in all structures of active RING/ U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2~Ub. The conformationalmore » biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/ U-box E3:E2~Ub ligase complex and identify the long sought-after source of allostery for RING/UBox activation of E2~Ub conjugates.« less

  11. Primary enzyme quantitation

    DOEpatents

    Saunders, G.C.

    1982-03-04

    The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

  12. Analysis of E2F factors during epidermal differentiation.

    PubMed

    Chang, Wing Y; Dagnino, Lina

    2005-01-01

    The multigene E2F family of transcription factors is central in the control of cell cycle progression. The expression and activity of E2F proteins is tightly regulated transcriptionally and posttranslationally as a function of the proliferation and differentiation status of the cell. In this chapter, we review protocols designed to determine E2F mRNA abundance in tissues by in situ hybridization techniques. The ability to culture primary epidermal keratinocytes and maintain them as either undifferentiated or terminally differentiated cells allows the biochemical and molecular characterization of changes in E2F expression and activity. Thus, we also discuss in detail methods to analyze E2F protein abundance by immunoblot and their ability to bind DNA in cultured cells using electrophoretic mobility shift assays.

  13. The Aryl Hydrocarbon Receptor Binds to E2F1 and Inhibits E2F1-induced Apoptosis

    PubMed Central

    Marlowe, Jennifer L.; Fan, Yunxia; Chang, Xiaoqing; Peng, Li; Knudsen, Erik S.; Xia, Ying

    2008-01-01

    Cellular stress by DNA damage induces checkpoint kinase-2 (CHK2)-mediated phosphorylation and stabilization of the E2F1 transcription factor, leading to induction of apoptosis by activation of a subset of proapoptotic E2F1 target genes, including Apaf1 and p73. This report characterizes an interaction between the aryl hydrocarbon (Ah) receptor (AHR), a ligand-activated transcription factor, and E2F1 that results in the attenuation of E2F1-mediated apoptosis. In Ahr−/− fibroblasts stably transfected with a doxycycline-regulated AHR expression vector, inhibition of AHR expression causes a significant elevation of oxidative stress, γH2A.X histone phosphorylation, and E2F1-dependent apoptosis, which can be blocked by small interfering RNA-mediated knockdown of E2F1 expression. In contrast, ligand-dependent AHR activation protects these cells from etoposide-induced cell death. In cells expressing both proteins, AHR and E2F1 interact independently of the retinoblastoma protein (RB), because AHR and E2F1 coimmunoprecipitate from extracts of RB-negative cells. Additionally, chromatin immunoprecipitation assays indicate that AHR and E2F1 bind to the Apaf1 promoter at a region containing a consensus E2F1 binding site but no AHR binding sites. AHR activation represses Apaf1 and TAp73 mRNA induction by a constitutively active CHK2 expression vector. Furthermore, AHR overexpression blocks the transcriptional induction of Apaf1 and p73 and the accumulation of sub-G0/G1 cells resulting from ectopic overexpression of E2F1. These results point to a proproliferative, antiapoptotic function of the Ah receptor that likely plays a role in tumor progression. PMID:18524851

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

    PubMed

    Braeuning, Albert; Vetter, Silvia

    2012-12-01

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

  15. Sibling rivalry in the E2F family.

    PubMed

    Trimarchi, Jeffrey M; Lees, Jacqueline A

    2002-01-01

    The E2F transcription factor family determines whether or not a cell will divide by controlling the expression of key cell-cycle regulators. The individual E2Fs can be divided into distinct subgroups that act in direct opposition to one another to promote either cellular proliferation or cell-cycle exit and terminal differentiation. What is the underlying molecular basis of this 'push-me-pull-you' regulation, and what are its biological consequences?

  16. Release of colicin E2 from Escherichia coli.

    PubMed

    Pugsley, A P; Rosenbusch, J P

    1981-07-01

    Treatment of Escherichia coli K-12(ColE2.P9) with 500 ng of mitomycin C per ml resulted in rapid and almost synchronous colicin E2 production. Colicin accumulated outside the cytoplasmic membrane, most probably in the periplasmic space. Colicin release occurred during a period in which the turbidity of the culture declined markedly. Periplasmic alkaline phosphatase was released during the same period, but cytoplasmic beta-galactosidase release was delayed.

  17. Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

    NASA Technical Reports Server (NTRS)

    Lecker, Stewart H.; Goldberg, A. L. (Principal Investigator)

    2003-01-01

    PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

  18. Drosophila BRUCE inhibits apoptosis through non-lysine ubiquitination of the IAP-antagonist REAPER

    PubMed Central

    Domingues, C; Ryoo, H D

    2012-01-01

    Active caspases execute apoptosis to eliminate superfluous or harmful cells in animals. In Drosophila, living cells prevent uncontrolled caspase activation through an inhibitor of apoptosis protein (IAP) family member, dIAP1, and apoptosis is preceded by the expression of IAP-antagonists, such as Reaper, Hid and Grim. Strong genetic modifiers of this pathway include another IAP family gene encoding an E2 ubiquitin conjugating enzyme domain, dBruce. Although the genetic effects of dBruce mutants are well documented, molecular targets of its encoded protein have remained elusive. Here, we report that dBruce targets Reaper for ubiquitination through an unconventional mechanism. Specifically, we show that dBruce physically interacts with Reaper, dependent upon Reaper's IAP-binding (IBM) and GH3 motifs. Consistently, Reaper levels were elevated in a dBruce −/− background. Unexpectedly, we found that dBruce also affects the levels of a mutant form of Reaper without any internal lysine residues, which normally serve as conventional ubiquitin acceptor sites. Furthermore, we were able to biochemically detect ubiquitin conjugation on lysine-deficient Reaper proteins, and knockdown of dBruce significantly reduced the extent of this ubiquitination. Our results indicate that dBruce inhibits apoptosis by promoting IAP-antagonist ubiquitination on unconventional acceptor sites. PMID:21886178

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

    PubMed

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

    1997-12-01

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

  20. Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase: DAWIDZIAK et al.

    SciTech Connect

    Dawidziak, Daria M.; Sanchez, Jacint G.; Wagner, Jonathan M.

    Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled-coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher-order oligomerization of the basal coiled-coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.

  1. Evolutionary and biophysical relationships among the papillomavirus E2 proteins.

    PubMed

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

    2009-01-01

    Infection by human papillomavirus (HPV) may result in clinical conditions ranging from benign warts to invasive cancer. The HPV E2 protein represses oncoprotein transcription and is required for viral replication. HPV E2 binds to palindromic DNA sequences of highly conserved four base pair sequences flanking an identical length variable 'spacer'. E2 proteins directly contact the conserved but not the spacer DNA. Variation in naturally occurring spacer sequences results in differential protein affinity that is dependent on their sensitivity to the spacer DNA's unique conformational and/or dynamic properties. This article explores the biophysical character of this core viral protein with the goal of identifying characteristics that associated with risk of virally caused malignancy. The amino acid sequence, 3d structure and electrostatic features of the E2 protein DNA binding domain are highly conserved; specific interactions with DNA binding sites have also been conserved. In contrast, the E2 protein's transactivation domain does not have extensive surfaces of highly conserved residues. Rather, regions of high conservation are localized to small surface patches. Implications to cancer biology are discussed.

  2. E2F4 is required for early eye patterning.

    PubMed

    Ruzhynsky, Vladimir A; Furimsky, Marosh; Park, David S; Wallace, Valerie A; Slack, Ruth S

    2009-01-01

    Increasingly, studies reveal novel functions for cell cycle proteins during development. Here, we investigated the role of E2F4 in eye development. E2F4-deficient mouse embryos exhibit severe early eye patterning defects, which are evident from embryonic day 11.5 and characterized by aberrant shape of the optic cup, coloboma as well as abnormal eye pigmentation. Loss of E2F4 is associated with proximal-distal patterning defects in the optic vesicle. These defects are characterized by the expansion of optic stalk marker gene expression to the optic cup and reduced expression of ventral optic cup markers. These defects are associated with a split of Shh expression domain at the ventral midline of the forebrain and expansion of the Shh activity into the ventral optic cup. Despite these patterning defects, early neuronal differentiation and Shh expression in the retina are not affected by E2F4 deletion. Overall, the results of our studies show a novel role of E2F4 in the early eye development. 2009 S. Karger AG, Basel.

  3. Revisiting Grodzins systematics of B(E2) values

    DOE PAGES

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

    2017-04-03

    Using Grodzins formalism, we analyze systematics of our latest evaluated B(E2) data for all the even–even nuclei in Z=2–104. The analysis indicates a low predictive power of systematics for a large number of cases, and a strong correlation between B(E2) fit values and nuclear structure effects. These findings provide a strong rationale for introduction of individual or elemental (grouped by Z) fit parameters. The current estimates of quadrupole collectivities for systematics of even–even nuclei yield complementary values for comparison with experimental results and theoretical calculations. Furthermore, the lists of fit parameters and predicted B(E2) values are given and possible implicationsmore » are discussed.« less

  4. B (e 2 ;21+→01+) value in 90Kr

    NASA Astrophysics Data System (ADS)

    Régis, J.-M.; Jolie, J.; Saed-Samii, N.; Warr, N.; Pfeiffer, M.; Blanc, A.; Jentschel, M.; Köster, U.; Mutti, P.; Soldner, T.; Simpson, G. S.; Drouet, F.; Vancraeyenest, A.; de France, G.; Clément, E.; Stezowski, O.; Ur, C. A.; Urban, W.; Regan, P. H.; Podolyák, Zs.; Larijani, C.; Townsley, C.; Carroll, R.; Wilson, E.; Fraile, L. M.; Mach, H.; Paziy, V.; Olaizola, B.; Vedia, V.; Bruce, A. M.; Roberts, O. J.; Smith, J. F.; Kröll, T.; Hartig, A.-L.; Ignatov, A.; Ilieva, S.; Thürauf, M.; Lalkovski, S.; Ivanova, D.; Kisyov, S.; Korten, W.; Salsac, M.-D.; Zielińska, M.; Mǎrginean, N.; Ghitǎ, D. G.; Licǎ, R.; Petrache, C. M.; Astier, A.; Leguillon, R.

    2014-12-01

    A smooth onset of collectivity in 88 ,92 ,94 ,96Kr has been determined from reported B (E 2 ;21+→01+) and E (21+) values. This is in contrast to the sudden onset in even-even Zr, Mo, and Sr isotopes. Our objective was to complete the systematics by determining the B (E 2 ;21+→01+) value in 90Kr, which was produced by cold-neutron-induced fission of 235U . The lifetime of the 21+ state in 90Kr was measured via the electronic γ -γ timing technique using the EXILL and FATIMA spectrometers. Based on the measured mean lifetime of τ = 15(10) ps, the B (E 2 ;21+→01+) value of 13 -5+26 W.u. in 90Kr is determined for the first time and the smooth onset of deformation in the even-even Kr isotopes beyond neutron number N =50 is confirmed.

  5. Catalytic domain of PDC-E2 contains epitopes recognized by antimitochondrial antibodies in primary biliary cirrhosis.

    PubMed

    Braun, Sandra; Berg, Christoph; Buck, Sandra; Gregor, Michael; Klein, Reinhild

    2010-02-28

    To search for further immunodominant peptides of the pyruvate dehydrogenase complex E2-component (PDC-E2) recognized by antimitochondrial antibodies (AMA) in primary biliary cirrhosis (PBC). Sera from 95 patients with PBC were tested by enzyme-linked immunosorbent assay against 33 synthetic overlapping peptides (25 amino acids; aa) covering the entire length of the E2-subunit of PDC-E2. Furthermore, the inner lipoyl peptide 167-184 was used in an unlipoylated and a lipoylated form as well as coupled to ovalbumin. Sera from 11 AMA negative/ANA positive PBC patients, 63 patients with other liver disorders and 22 healthy blood donors served as controls. Of the 95 PBC-sera, 74% reacted with the peptide 475-499 and 58% with the peptide 407-431 located within the catalytic domain of PDC-E2. Patients with other disorders or healthy controls were positive in only up to 18%. Antibodies to the unlipoylated and lipoylated peptide 167-184 within the inner lipoyl domain were found in only 5% and 11% of the PBC sera, respectively; using ovalbumin-coupled peptides, the incidence increased up to 57% (unlipoylated form). Peptides within the catalytic site of PDC-E2 rather than the previously reported lipoyl binding peptide 167-184 may represent major immunodominant epitopes recognized by AMA in PBC.

  6. Preparation of sumoylated substrates for biochemical analysis.

    PubMed

    Knipscheer, Puck; Klug, Helene; Sixma, Titia K; Pichler, Andrea

    2009-01-01

    Covalent modification of proteins with SUMO (small ubiquitin related modifier) affects many cellular processes like transcription, nuclear transport, DNA repair and cell cycle progression. Although hundreds of SUMO targets have been identified, for several of them the function remains obscure. In the majority of cases sumoylation is investigated via "loss of modification" analysis by mutating the relevant target lysine. However, in other cases this approach is not successful since mapping of the modification site is problematic or mutation does not cause an obvious phenotype. These latter cases ask for different approaches to investigate the target modification. One possibility is to choose the opposite approach, a "gain in modification" analysis by producing both SUMO modified and unmodified protein in vitro and comparing them in functional assays. Here, we describe the purification of the ubiquitin conjugating enzyme E2-25K, its in vitro sumoylation with recombinant enzymes and the subsequent separation and purification of the modified and the unmodified forms.

  7. Ubiquitin Ligases: Structure, Function, and Regulation.

    PubMed

    Zheng, Ning; Shabek, Nitzan

    2017-06-20

    Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.

  8. Targeted Delivery of Ubiquitin-Conjugated BH3 Peptide-Based Mcl-1 Inhibitors into Cancer Cells

    PubMed Central

    2015-01-01

    BH3 peptides are key mediators of apoptosis and have served as the lead structures for the development of anticancer therapeutics. Previously, we reported the application of a simple cysteine-based side chain cross-linking chemistry to NoxaBH3 peptides that led to the generation of the cross-linked NoxaBH3 peptides with increased cell permeability and higher inhibitory activity against Mcl-1 (Muppidi, A., Doi, K., Edwardraja, S., Drake, E. J., Gulick, A. M., Wang, H.-G., Lin, Q. (2012) J. Am. Chem. Soc.134, 1473422920569). To deliver cross-linked NoxaBH3 peptides selectively into cancer cells for enhanced efficacy and reduced systemic toxicity, here we report the conjugation of the NoxaBH3 peptides with the extracellular ubiquitin, a recently identified endogenous ligand for CXCR4, a chemokine receptor overexpressed in cancer cells. The resulting ubiquitin-NoxaBH3 peptide conjugates showed increased inhibitory activity against Mcl-1 and selective killing of the CXCR4-expressing cancer cells. The successful delivery of the NoxaBH3 peptides by ubiquitin into cancer cells suggests that the ubiquitin/CXCR4 axis may serve as a general route for the targeted delivery of anticancer agents. PMID:24410055

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

    PubMed Central

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

    1996-01-01

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

  10. Elevated Liver Enzymes

    MedlinePlus

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  11. Production and actions of the anandamide metabolite prostamide E2 in the renal medulla.

    PubMed

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

    2012-09-01

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

  12. 26 CFR 31.3231(e)-2 - Contribution base.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

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

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  15. ENVIRONMENTAL EFFECTS OF DREDGING AND DISPOSAL (E2-D2)

    EPA Science Inventory

    US Army Corps of Engineers public web site for the "Environmental Effects of Dredging and Disposal" ("E2-D2") searchable database of published reports and studies about environmental impacts associated with dredging and disposal operations. Many of the reports and studies are ava...

  16. Prostaglandin E2 Regulation of Chondrocyte Proliferation and Differentiation

    DTIC Science & Technology

    1994-05-01

    lipopolysaccharide- and TNF-induced cartilage breakdown in bovine nasal cartilage(121’. The use of medications that modulate PGE2 production may have an adverse...Levine, P. Goldhaber. 1972. Evidence that the bone resorption stimulating factor produced by mouse fibrosarcoma cells is prostaglandin E2. J. Exp. Med

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

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Williams, Zachary D.; Oriti, Salvatore M.

    2012-01-01

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

  20. Formulation of Ames 24E2 IR-black coating

    NASA Technical Reports Server (NTRS)

    Smith, Sheldon M.

    1991-01-01

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

  1. Up-regulation of cyclooxygenase-2 by product-prostaglandin E2

    NASA Technical Reports Server (NTRS)

    Tjandrawinata, R. R.; Hughes-Fulford, M.

    1997-01-01

    The development of prostate cancer has been linked to high level of dietary fat intake. Our laboratory investigates the connection between cancer cell growth and fatty acid products. Studying human prostatic carcinoma PC-3 cells, we found that prostaglandin E2 (PGE2) increased cell growth and up-regulated the gene expression of its own synthesizing enzyme, cyclooxygenase-2 (COX-2). PGE2 increased COX-2 mRNA expression dose-dependently with the highest levels of stimulation seen at the 3-hour period following PGE2 addition. The NSAID flurbiprofen (5 microM), in the presence of exogenous PGE2, inhibited the up-regulation of COX-2 mRNA and cell growth. These data suggest that the levels of local intracellular PGE2 play a major role in the growth of prostate cancer cells through an activation of COX-2 gene expression.

  2. Hyperforin, an Anti-Inflammatory Constituent from St. John's Wort, Inhibits Microsomal Prostaglandin E2 Synthase-1 and Suppresses Prostaglandin E2 Formation in vivo

    PubMed Central

    Koeberle, Andreas; Rossi, Antonietta; Bauer, Julia; Dehm, Friederike; Verotta, Luisella; Northoff, Hinnak; Sautebin, Lidia; Werz, Oliver

    2010-01-01

    The acylphloroglucinol hyperforin (Hyp) from St. John's wort possesses anti-inflammatory and anti-carcinogenic properties which were ascribed among others to the inhibition of 5-lipoxygenase. Here, we investigated whether Hyp also interferes with prostanoid generation in biological systems, particularly with key enzymes participating in prostaglandin (PG)E2 biosynthesis, i.e., cyclooxygenases (COX)-1/2 and microsomal PGE2 synthase (mPGES)-1 which play key roles in inflammation and tumorigenesis. Similar to the mPGES-1 inhibitors MK-886 and MD-52, Hyp significantly suppressed PGE2 formation in whole blood assays starting at 0.03–1 μM, whereas the concomitant generation of COX-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, thromboxane B2, and 6-keto PGF1α was not significantly suppressed up to 30 μM. In cell-free assays, Hyp efficiently blocked the conversion of PGH2 to PGE2 mediated by mPGES-1 (IC50 = 1 μM), and isolated COX enzymes were not (COX-2) or hardly (COX-1) suppressed. Intraperitoneal (i.p.) administration of Hyp (4 mg kg−1) to rats impaired exudate volume and leukocyte numbers in carrageenan-induced pleurisy associated with reduced PGE2 levels, and Hyp (given i.p.) inhibited carrageenan-induced mouse paw edema formation (ED50 = 1 mg kg−1) being superior over indomethacin (ED50 = 5 mg kg−1). We conclude that the suppression of PGE2 biosynthesis in vitro and in vivo by acting on mPGES-1 critically contributes to the anti-inflammatory efficiency of Hyp. PMID:21687502

  3. Design and Development of Microsomal Prostaglandin E2 Synthase-1 Inhibitors: Challenges and Future Directions.

    PubMed

    Koeberle, Andreas; Laufer, Stefan A; Werz, Oliver

    2016-07-14

    Microsomal prostaglandin E2 synthase (mPGES)-1 is responsible for the massive prostaglandin E2 (PGE2) formation during inflammation. Increasing evidence reveals mPGES-1 inhibitors as a safe alternative to nonsteroidal anti-inflammatory drugs. The first selective mPGES-1 inhibitors recently entered clinical trials. Major challenges for drug development have been the high plasma protein binding of lead structures, interspecies discrepancies, nuisance inhibition, sophisticated enzyme assays, and limited structural information about the mPGES-1 inhibitor binding site. Since most of these drawbacks could be solved during the past few years, we are standing at the threshold of a new era of mPGES-1-targeting anti-inflammatory drugs. This perspective introduces mPGES-1 as a key player within the network of eicosanoid biosynthesis and summarizes our current understanding of its structure and mechanism. Moreover, we present high-throughput and in silico screening techniques and discuss the structure-activity relationship and pharmacological potential of major mPGES-1 inhibitor classes in light of recent insights from pharmacophore models and cocrystallization studies.

  4. Insolubilization process increases enzyme stability

    NASA Technical Reports Server (NTRS)

    Billingham, J.; Lyn, J.

    1971-01-01

    Enzymes complexed with polymeric matrices contain properties suggesting application to enzyme-controlled reactions. Stability of insolubilized enzyme derivatives is markedly greater than that of soluble enzymes and physical form of insolubilized enzymes is useful in column and batch processes.

  5. Astro-E2 Magnesium Diboride High Current Leads

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  6. Mutations in STAMBP, encoding a deubiquitinating enzyme, cause Microcephaly-Capillary Malformation syndrome

    PubMed Central

    McDonell, Laura M.; Mirzaa, Ghayda M.; Alcantara, Diana; Schwartzentruber, Jeremy; Carter, Melissa T.; Lee, Leo J.; Clericuzio, Carol L.; Graham, John M.; Morris-Rosendahl, Deborah J.; Polster, Tilman; Acsadi, Gyula; Townshend, Sharron; Williams, Simon; Halbert, Anne; Isidor, Bertrand; Smyser, Christopher D.; Paciorkowski, Alex R.; Willing, Marcia; Woulfe, John; Das, Soma; Beaulieu, Chandree L.; Marcadier, Janet; Geraghty, Michael T.; Frey, Brendan J.; Majewski, Jacek; Bulman, Dennis E.; Dobyns, William B.; O’Driscoll, Mark; Boycott, Kym M.

    2014-01-01

    Microcephaly-capillary malformation (MIC-CAP) syndrome exhibits severe microcephaly with progressive cortical atrophy, intractable epilepsy, profound developmental delay and multiple small capillary malformations on the skin. We employed whole-exome sequencing of five patients with MIC-CAP syndrome and identified novel recessive mutations in STAMBP, a gene encoding the deubiquitinating (DUB) isopeptidase STAMBP (STAM-binding protein)/AMSH (Associated Molecule with the SH3 domain of STAM), that plays a key role in cell surface receptor-mediated endocytosis and sorting. Patient cell lines showed reduced STAMBP expression associated with accumulation of ubiquitin-conjugated protein aggregates, elevated apoptosis and insensitive activation of the RAS-MAPK and PI3K-AKT-mTOR pathways. The latter cellular phenotype is significant considering the established connection between these pathways and their association with vascular and capillary malformations. Furthermore, our findings of a congenital human disorder caused by a defective DUB protein that functions in endocytosis, implicates ubiquitin-conjugate aggregation and elevated apoptosis as factors potentially influencing the progressive neuronal loss underlying MIC-CAP. PMID:23542699

  7. Novel contraceptive targets to inhibit ovulation: the prostaglandin E2 pathway

    PubMed Central

    Duffy, Diane M.

    2015-01-01

    BACKGROUND Prostaglandin E2 (PGE2) is an essential intrafollicular regulator of ovulation. In contrast with the one-gene, one-protein concept for synthesis of peptide signaling molecules, production and metabolism of bioactive PGE2 requires controlled expression of many proteins, correct subcellular localization of enzymes, coordinated PGE2 synthesis and metabolism, and prostaglandin transport in and out of cells to facilitate PGE2 action and degradation. Elevated intrafollicular PGE2 is required for successful ovulation, so disruption of PGE2 synthesis, metabolism or transport may yield effective contraceptive strategies. METHODS This review summarizes case reports and studies on ovulation inhibition in women and macaques treated with cyclooxygenase inhibitors published from 1987 to 2014. These findings are discussed in the context of studies describing levels of mRNA, protein, and activity of prostaglandin synthesis and metabolic enzymes as well as prostaglandin transporters in ovarian cells. RESULTS The ovulatory surge of LH regulates the expression of each component of the PGE2 synthesis-metabolism-transport pathway within the ovulatory follicle. Data from primary ovarian cells and cancer cell lines suggest that enzymes and transporters can cooperate to optimize bioactive PGE2 levels. Elevated intrafollicular PGE2 mediates key ovulatory events including cumulus expansion, follicle rupture and oocyte release. Inhibitors of the prostaglandin-endoperoxide synthase 2 (PTGS2) enzyme (also known as cyclooxygenase-2 or COX2) reduce ovulation rates in women. Studies in macaques show that PTGS2 inhibitors can reduce the rates of cumulus expansion, oocyte release, follicle rupture, oocyte nuclear maturation and fertilization. A PTGS2 inhibitor reduced pregnancy rates in breeding macaques when administered to simulate emergency contraception. However, PTGS2 inhibition did not prevent pregnancy in monkeys when administered to simulate monthly contraceptive use. CONCLUSION

  8. Metal Dependence of the Xylose Isomerase from Piromyces sp. E2 Explored by Activity Profiling and Protein Crystallography

    PubMed Central

    2017-01-01

    Xylose isomerase from Piromyces sp. E2 (PirXI) can be used to equip Saccharomyces cerevisiae with the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters, and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in Escherichia coli, analyzed the metal dependence of its catalytic properties, and determined 12 crystal structures in the presence of different metals, substrates, and substrate analogues. The activity assays revealed that various bivalent metals can activate PirXI for xylose isomerization. Among these metals, Mn2+ is the most favorable for catalytic activity. Furthermore, the enzyme shows the highest affinity for Mn2+, which was established by measuring the activation constants (Kact) for different metals. Metal analysis of the purified enzyme showed that in vivo the enzyme binds a mixture of metals that is determined by metal availability as well as affinity, indicating that the native metal composition can influence activity. The crystal structures show the presence of an active site similar to that of other xylose isomerases, with a d-xylose binding site containing two tryptophans and a catalytic histidine, as well as two metal binding sites that are formed by carboxylate groups of conserved aspartates and glutamates. The binding positions and conformations of the metal-coordinating residues varied slightly for different metals, which is hypothesized to contribute to the observed metal dependence of the isomerase activity. PMID:29045784

  9. E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells.

    PubMed

    Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

    2015-01-01

    E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.

  10. E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells

    PubMed Central

    Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

    2015-01-01

    E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells. PMID:25892555

  11. UUCD: a family-based database of ubiquitin and ubiquitin-like conjugation.

    PubMed

    Gao, Tianshun; Liu, Zexian; Wang, Yongbo; Cheng, Han; Yang, Qing; Guo, Anyuan; Ren, Jian; Xue, Yu

    2013-01-01

    In this work, we developed a family-based database of UUCD (http://uucd.biocuckoo.org) for ubiquitin and ubiquitin-like conjugation, which is one of the most important post-translational modifications responsible for regulating a variety of cellular processes, through a similar E1 (ubiquitin-activating enzyme)-E2 (ubiquitin-conjugating enzyme)-E3 (ubiquitin-protein ligase) enzyme thioester cascade. Although extensive experimental efforts have been taken, an integrative data resource is still not available. From the scientific literature, 26 E1s, 105 E2s, 1003 E3s and 148 deubiquitination enzymes (DUBs) were collected and classified into 1, 3, 19 and 7 families, respectively. To computationally characterize potential enzymes in eukaryotes, we constructed 1, 1, 15 and 6 hidden Markov model (HMM) profiles for E1s, E2s, E3s and DUBs at the family level, separately. Moreover, the ortholog searches were conducted for E3 and DUB families without HMM profiles. Then the UUCD database was developed with 738 E1s, 2937 E2s, 46 631 E3s and 6647 DUBs of 70 eukaryotic species. The detailed annotations and classifications were also provided. The online service of UUCD was implemented in PHP + MySQL + JavaScript + Perl.

  12. [Human drug metabolizing enzymes. II. Conjugation enzymes].

    PubMed

    Vereczkey, L; Jemnitz, K; Gregus, Z

    1998-09-01

    In this review we focus on human conjugation enzymes (UDP-glucuronyltransferases, methyl-trasferases, N-acetyl-transferases, O-acetyl-transferases, Amidases/carboxyesterases, sulfotransferases, Glutation-S-transferases and the enzymes involved in the conjugation with amino acids) that participate in the metabolism of xenobiotics. Although conjugation reactions in most of the cases result in detoxication, more and more publications prove that the reactions catalysed by these enzymes very often lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity (liver, neuro-, embryotoxicity, allergy, carcinogenecity). We have summarised the data available on these enzymes concerning their catalytic profile and specificity, inhibition, induction properties, their possible role in the generation of toxic compounds, their importance in clinical practice and drug development.

  13. Rh2E2, a novel metabolic suppressor, specifically inhibits energy-based metabolism of tumor cells

    PubMed Central

    Bai, Li-Ping; Jiang, Zhi-Hong; Guo, Yue; Kong, Ah-Ng Tony; Wang, Rui; Kam, Richard Kin Ting; Law, Betty Yuen Kwan; Hsiao, Wendy Wen Luen; Chan, Ka Man; Wang, Jingrong; Chan, Rick Wai Kit; Guo, Jianru; Zhang, Wei; Yen, Feng Gen; Zhou, Hua; Leung, Elaine Lai Han; Yu, Zhiling; Liu, Liang

    2016-01-01

    Energy metabolism in cancer cells is often increased to meet their higher proliferative rate and biosynthesis demands. Suppressing cancer cell metabolism using agents like metformin has become an attractive strategy for treating cancer patients. We showed that a novel ginsenoside derivative, Rh2E2, is as effective as aspirin in preventing the development of AOM/DSS-induced colorectal cancer and suppresses tumor growth and metastasis in a LLC-1 xenograft. A sub-chronic and acute toxicity LD50 test of Rh2E2 showed no harmful reactions at the maximum oral dosage of 5000 mg/kg body weight in mice. Proteomic profiling revealed that Rh2E2 specifically inhibited ATP production in cancer cells via down-regulation of metabolic enzymes involving glycolysis, fatty acid β-oxidation and the tricarboxylic acid cycle, leading to specific cytotoxicity and S-phase cell cycle arrest in cancer cells. Those findings suggest that Rh2E2 possesses a novel and safe anti-metabolic agent for cancer patients by specific reduction of energy-based metabolism in cancer cells. PMID:26799418

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

    PubMed Central

    Braeuning, Albert; Vetter, Silvia

    2012-01-01

    Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays. PMID:22789175

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

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

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

    PubMed

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

    2014-09-28

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

  17. E2F mediates enhanced alternative polyadenylation in proliferation.

    PubMed

    Elkon, Ran; Drost, Jarno; van Haaften, Gijs; Jenal, Mathias; Schrier, Mariette; Oude Vrielink, Joachim A F; Agami, Reuven

    2012-07-02

    The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation.

  18. E2F mediates enhanced alternative polyadenylation in proliferation

    PubMed Central

    2012-01-01

    Background The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. Results Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. Conclusions Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation. PMID:22747694

  19. Detection of classical swine fever virus E2 gene in cattle serum samples from cattle herds of Meghalaya.

    PubMed

    Chakraborty, A K; Karam, A; Mukherjee, P; Barkalita, L; Borah, P; Das, S; Sanjukta, R; Puro, K; Ghatak, S; Shakuntala, I; Sharma, I; Laha, R G; Sen, A

    2018-03-01

    The present study focused on the detection and genetic characterisation of 5' untranslated region (5'UTR) and E2 gene of classical swine fever virus (CSFV, family Flaviviridae , genus Pestivirus ) from bovine population of the northeastern region of India. A total of 134 cattle serum samples were collected from organised cattle farms and were screened for CSFV antigen with a commercial antigen capture enzyme linked immunosorbent assay (Ag-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). A total of 10 samples were positive for CSFV antigen by ELISA, while all of them were positive in PCR for 5'UTR region. Full length E2 region of CSFV were successfully amplified from two positive samples and used for subsequent phylogenetic analysis and determination of protein 3D structure which showed similarity with reported CSFV isolate from Assam of sub-genogroup 2.1, with minor variations in protein structure.

  20. Profiling the orphan enzymes.

    PubMed

    Sorokina, Maria; Stam, Mark; Médigue, Claudine; Lespinet, Olivier; Vallenet, David

    2014-06-06

    The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called "orphan enzymes". The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to "local orphan enzymes" that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new activities.

  1. Magnetic imaging of antiferromagnetic and superconducting phases in R bxF e2 -yS e2 crystals

    NASA Astrophysics Data System (ADS)

    Hazi, J.; Mousavi, T.; Dudin, P.; van der Laan, G.; Maccherozzi, F.; Krzton-Maziopa, A.; Pomjakushina, E.; Conder, K.; Speller, S. C.

    2018-02-01

    High-temperature superconducting (HTS) cuprate materials, with the ability to carry large electrical currents with no resistance at easily reachable temperatures, have stimulated enormous scientific and industrial interest since their discovery in the 1980's. However, technological applications of these promising compounds have been limited by their chemical and microstructural complexity and the challenging processing strategies required for the exploitation of their extraordinary properties. The lack of theoretical understanding of the mechanism for superconductivity in these HTS materials has also hindered the search for new superconducting systems with enhanced performance. The unexpected discovery in 2008 of HTS iron-based compounds has provided an entirely new family of materials for studying the crucial interplay between superconductivity and magnetism in unconventional superconductors. Alkali-metal-doped iron selenide (AxF e2 -yS e2 , A =alkali metal ) compounds are of particular interest owing to the coexistence of superconductivity at relatively high temperatures with antiferromagnetism. Intrinsic phase separation on the mesoscopic scale is also known to occur in what were intended to be single crystals of these compounds, making it difficult to interpret bulk property measurements. Here, we use a combination of two advanced microscopy techniques to provide direct evidence of the magnetic properties of the individual phases. First, x-ray linear dichroism studies in a photoelectron emission microscope, and supporting multiplet calculations, indicate that the matrix (majority) phase is antiferromagnetic whereas the minority phase is nonmagnetic at room temperature. Second, cryogenic magnetic force microscopy demonstrates unambiguously that superconductivity occurs only in the minority phase. The correlation of these findings with previous microstructural studies and bulk measurements paves the way for understanding the intriguing electronic and magnetic

  2. Evolutionary dynamics of enzymes.

    PubMed

    Demetrius, L

    1995-08-01

    This paper codifies and rationalizes the large diversity in reaction rates and substrate specificity of enzymes in terms of a model which postulates that the kinetic properties of present-day enzymes are the consequence of the evolutionary force of mutation and selection acting on a class of primordial enzymes with poor catalytic activity and broad substrate specificity. Enzymes are classified in terms of their thermodynamic parameters, activation enthalpy delta H* and activation entropy delta S*, in their kinetically significant transition states as follows: type 1, delta H* > 0, delta S* < 0; type 2, delta H* < or = 0, delta S* < or = 0; type 3, delta H* > 0, delta S* > 0. We study the evolutionary dynamics of these three classes of enzymes subject to mutation, which acts at the level of the gene which codes for the enzyme and selection, which acts on the organism that contains the enzyme. Our model predicts the following evolutionary trends in the reaction rate and binding specificity for the three classes of molecules. In type 1 enzymes, evolution results in random, non-directional changes in the reaction rate and binding specificity. In type 2 and 3 enzymes, evolution results in a unidirectional increase in both the reaction rate and binding specificity. We exploit these results in order to codify the diversity in functional properties of present-day enzymes. Type 1 molecules will be described by intermediate reaction rates and broad substrate specificity. Type 2 enzymes will be characterized by diffusion-controlled rates and absolute substrate specificity. The type 3 catalysts can be further subdivided in terms of their activation enthalpy into two classes: type 3a (delta H* small) and type 3b (delta H* large). We show that type 3a will be represented by the same functional properties that identify type 2, namely, diffusion-controlled rates and absolute substrate specificity, whereas type 3b will be characterized by non-diffusion-controlled rates and absolute

  3. Roles of prostaglandin E2 in the cochlea.

    PubMed

    Nakagawa, Takayuki

    2011-06-01

    Prostaglandins are one of the major groups of chemical mediators in the mammalian body. Among prostaglandins, prostaglandin E2 (PGE2) is the most abundant prostanoid in humans and involved in regulating many different fundamental biological functions. PGE2 signaling is mediated by four distinct E-prostanoid receptors (EPs) namely EP1-4. Recently, accumulating evidence indicates critical, but complex roles of EP signaling in the pathogenesis of neuronal diseases depending on the context of neuronal injury. Four distinct EPs are expressed in the stria vascularis, spiral ligament, spiral ganglion and organ of Corti, indicating an involvement of EP signaling in the cochlear function. Activation of EP4 in cochleae significantly attenuates noise-induced damage in cochleae, and activation of EP2 or EP4 induces the formation of vascular endothelial growth factor in cochleae. These findings strongly suggest that individual EP signaling may be involved in the maintenance of the cochlear sensory system similarly to the central nervous system. This review highlights recent findings on EP signaling in the central nervous system, and presents its possible roles in regulation of blood flow, protection of sensory cells and immune responses in cochleae. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Influence of prostaglandin E2 on parturition in cattle.

    PubMed

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

    2007-09-22

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

  5. Prevotella intermedia induces prostaglandin E2 via multiple signaling pathways.

    PubMed

    Guan, S-M; Fu, S-M; He, J-J; Zhang, M

    2011-01-01

    Prostaglandin E(2) (PGE(2)) plays important roles in the bone resorption of inflammatory diseases such as rheumatoid arthritis and periodontitis via specific prostaglandin receptors (i.e., EP1-EP4). In this study, the authors examined whether Prevotella intermedia regulates PGE(2) production and EP expression in human periodontal ligament fibroblasts (hPDLs); they also explored the potential signaling pathways involved in PGE(2) production. P. intermedia induced PGE(2) production and cyclooxygenase-2 (COX-2) expression in a dose- and time-dependent manner. Indomethacin and NS-398 completely abrogated the P. intermedia-induced PGE(2) production without modulating COX-2 expression. Specific inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38, phosphatidylinositol 3-kinase, and protein kinase C--but not c-AMP and protein kinase A--significantly attenuated the P. intermedia-induced COX-2 and PGE(2) expression. P. intermedia reduced EP1 expression in a concentration- and time-dependent manner. The results indicate that the COX-2-dependent induction of PGE(2) by P. intermedia in hPDLs is mediated by multiple signaling pathways.

  6. Enzymes for improved biomass conversion

    DOEpatents

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  7. Magnetically responsive enzyme powders

    NASA Astrophysics Data System (ADS)

    Pospiskova, Kristyna; Safarik, Ivo

    2015-04-01

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (-20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties.

  8. Catalyzed enzyme electrodes

    DOEpatents

    Zawodzinski, Thomas A.; Wilson, Mahlon S.; Rishpon, Judith; Gottesfeld, Shimshon

    1993-01-01

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  9. Profiling the orphan enzymes

    PubMed Central

    2014-01-01

    The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new

  10. Dissipative Dynamics of Enzymes

    NASA Astrophysics Data System (ADS)

    Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni

    2014-11-01

    We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C ; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

  11. Dissipative Dynamics of Enzymes

    NASA Astrophysics Data System (ADS)

    Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni; Zocchi LabMolecular Biophysics Team

    2015-03-01

    We explore enzyme conformational dynamics at sub - Å resolution, specifically temperature effects. The ensemble averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor 2 as the temperature is raised from 10 C to 45 C; the elastic parameter K shows a similar decrease. Thus when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

  12. Dissipative dynamics of enzymes.

    PubMed

    Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni

    2014-11-07

    We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

  13. Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome

    PubMed Central

    McGinty, Robert K.; Henrici, Ryan C.; Tan, Song

    2014-01-01

    The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in higher eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the molecular mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear. Here we present the crystal structure of the Ring1B/Bmi1/UbcH5c E3-E2 complex (the PRC1 ubiquitylation module) bound to its nucleosome core particle substrate. The structure shows how a chromatin enzyme achieves substrate specificity by interacting with multiple nucleosome surfaces spatially distinct from the site of catalysis. Our structure further reveals an unexpected role for the ubiquitin E2 enzyme in substrate recognition, and provides insight into how the related histone H2A E3 ligase, BRCA1, interacts with and ubiquitylates the nucleosome. PMID:25355358

  14. Prostaglandin E2 signals white-to-brown adipogenic differentiation

    PubMed Central

    García-Alonso, Verónica; Clària, Joan

    2014-01-01

    The formation of new adipocytes from precursor cells is a crucial aspect of normal adipose tissue function. During the adipogenic process, adipocytes differentiated from mesenchymal stem cells give rise to two main types of fat: white adipose tissue (WAT) characterized by the presence of adipocytes containing large unilocular lipid droplets, and brown adipose tissue (BAT) composed by multilocular brown adipocytes packed with mitochondria. WAT is not only important for energy storage but also as an endocrine organ regulating whole body homeostasis by secreting adipokines and other mediators, which directly impact metabolic functions in obesity. By contrast, BAT is specialized in dissipating energy in form of heat and has salutary effects in combating obesity and associated disorders. Unfortunately, WAT is the predominant fat type, whereas BAT is scarce and located in discrete pockets in adult humans. Luckily, another type of brown adipocytes, called beige or brite (brown-in-white) adipocytes, with similar functions to those of “classical” brown adipocytes has recently been identified in WAT. In this review, a close look is given into the role of bioactive lipid mediators in the regulation of adipogenesis, with a special emphasis on the role of the microsomal prostaglandin E (PGE) synthase-1, a terminal enzyme in PGE2 biosynthesis, as a key regulator of white-to-brown adipogenesis in WAT. PMID:26317053

  15. Targeted enzyme prodrug therapies.

    PubMed

    Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

    2010-09-01

    The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

  16. Enzymes in Fermented Fish.

    PubMed

    Giyatmi; Irianto, H E

    Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

  17. Mycophenolate mofetil decreases endothelial prostaglandin E2 in response to allogeneic T cells or cytokines.

    PubMed

    Blaheta, R A; Nelson, K; Oppermann, E; Leckel, K; Harder, S; Cinatl, J; Weber, S; Shipkova, M; Encke, A; Markus, B H

    2000-05-15

    Prostaglandin E2 (PGE2) is a powerful endogenous immune suppressant and interferes with various T-cell functions. However, it is not known in detail whether immunosuppressive drugs influence the PGE2-driven immune response in transplant patients. Therefore, we investigated the effect of several immunosuppressive compounds, in particular the novel drug mycophenolate mofetil (MMF), on endothelial PGE2 release. Endothelial cells (HUVEC) were activated by either allogeneic CD4+ or CD8+ T cells, or by the cytokines interleukin-1 or gamma-interferon. Using an enzyme-linked immunosorbent assay, we analyzed PGE2 release of the activated HWEC in the presence of MMF, cyclosporine, or tacrolimus. As verapamil and mibefradil also possess immunosuppressive properties, they were included in the study as well. Activation of HUVEC with interleukin-1 or T cells resulted in a drastic accumulation of PGE2 in the supernatant. Cyclosporine or tacrolimus had no effect on PGE2 release. However, Ca2+ channel blockers, when applied at higher dosages, caused a significant increase in PGE2. Interestingly, MMF strongly diminished the PGE2 level in the cell culture supernatant in a concentration-dependent manner. The results demonstrate an inhibitory effect of MMF on PGE2 production, which may lower the benefits of the PGE2-triggered immune response after organ transplantation.

  18. Prostaglandin E2 Regulates Liver versus Pancreas Cell Fate Decisions and Endodermal Outgrowth

    PubMed Central

    Nissim, Sahar; Sherwood, Richard I.; Wucherpfennig, Julia; Saunders, Diane; Harris, James M.; Esain, Virginie; Carroll, Kelli J.; Frechette, Gregory M.; Kim, Andrew J.; Hwang, Katie L.; Cutting, Claire C.; Elledge, Susanna; North, Trista E.; Goessling, Wolfram

    2014-01-01

    SUMMARY The liver and pancreas arise from common endodermal progenitors. How these distinct cell fates are specified is poorly understood. Here, we describe prostaglandin E2 (PGE2) as a regulator of endodermal fate specification during development. Modulating PGE2 activity has opposing effects on liver-versus-pancreas specification in zebrafish embryos as well as mouse endodermal progenitors. The PGE2 synthetic enzyme cox2a and receptor ep2a are patterned such that cells closest to PGE2 synthesis acquire a liver fate whereas more distant cells acquire a pancreas fate. PGE2 interacts with the bmp2b pathway to regulate fate specification. At later stages of development, PGE2 acting via the ep4a receptor promotes outgrowth of both the liver and pancreas. PGE2 remains important for adult organ growth, as it modulates liver regeneration. This work provides in vivo evidence that PGE2 may act as a morphogen to regulate cell fate decisions and outgrowth of the embryonic endodermal anlagen. PMID:24530296

  19. E2 protein cage as a multifunctional nanoplatform

    NASA Astrophysics Data System (ADS)

    Dalmau Mallorqui, Merce

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

  20. Structure, Dynamics, and Interaction of Mycobacterium tuberculosis (Mtb) DprE1 and DprE2 Examined by Molecular Modeling, Simulation, and Electrostatic Studies

    PubMed Central

    Bhutani, Isha; Loharch, Saurabh; Gupta, Pawan; Madathil, Rethi; Parkesh, Raman

    2015-01-01

    The enzymes decaprenylphosphoryl-β-D-ribose oxidase (DprE1) and decaprenylphosphoryl-β-D-ribose-2-epimerase (DprE2) catalyze epimerization of decaprenylphosporyl ribose (DPR) todecaprenylphosporyl arabinose (DPA) and are critical for the survival of Mtb. Crystal structures of DprE1 so far reported display significant disordered regions and no structural information is known for DprE2. We used homology modeling, protein threading, molecular docking and dynamics studies to investigate the structural and dynamic features of Mtb DprE1 and DprE2 and DprE1-DprE2 complex. A three-dimensional model for DprE2 was generated using the threading approach coupled with ab initio modeling. A 50 ns simulation of DprE1 and DprE2 revealed the overall stability of the structures. Principal Component Analysis (PCA) demonstrated the convergence of sampling in both DprE1 and DprE2. In DprE1, residues in the 269–330 area showed considerable fluctuation in agreement with the regions of disorder observed in the reported crystal structures. In DprE2, large fluctuations were detected in residues 95–113, 146–157, and 197–226. The study combined docking and MD simulation studies to map and characterize the key residues involved in DprE1-DprE2 interaction. A 60 ns MD simulation for DprE1-DprE2 complex was also performed. Analysis of data revealed that the docked complex is stabilized by H-bonding, hydrophobic and ionic interactions. The key residues of DprE1 involved in DprE1-DprE2 interactions belong to the disordered region. We also examined the docked complex of DprE1-BTZ043 to investigate the binding pocket of DprE1 and its interactions with the inhibitor BTZ043. In summary, we hypothesize that DprE1-DprE2 interaction is crucial for the synthesis of DPA and DprE1-DprE2 complex may be a new therapeutic target amenable to pharmacological validation. The findings have important implications in tuberculosis (TB) drug discovery and will facilitate drug development efforts against TB

  1. Structure, dynamics, and interaction of Mycobacterium tuberculosis (Mtb) DprE1 and DprE2 examined by molecular modeling, simulation, and electrostatic studies.

    PubMed

    Bhutani, Isha; Loharch, Saurabh; Gupta, Pawan; Madathil, Rethi; Parkesh, Raman

    2015-01-01

    The enzymes decaprenylphosphoryl-β-D-ribose oxidase (DprE1) and decaprenylphosphoryl-β-D-ribose-2-epimerase (DprE2) catalyze epimerization of decaprenylphosporyl ribose (DPR) todecaprenylphosporyl arabinose (DPA) and are critical for the survival of Mtb. Crystal structures of DprE1 so far reported display significant disordered regions and no structural information is known for DprE2. We used homology modeling, protein threading, molecular docking and dynamics studies to investigate the structural and dynamic features of Mtb DprE1 and DprE2 and DprE1-DprE2 complex. A three-dimensional model for DprE2 was generated using the threading approach coupled with ab initio modeling. A 50 ns simulation of DprE1 and DprE2 revealed the overall stability of the structures. Principal Component Analysis (PCA) demonstrated the convergence of sampling in both DprE1 and DprE2. In DprE1, residues in the 269-330 area showed considerable fluctuation in agreement with the regions of disorder observed in the reported crystal structures. In DprE2, large fluctuations were detected in residues 95-113, 146-157, and 197-226. The study combined docking and MD simulation studies to map and characterize the key residues involved in DprE1-DprE2 interaction. A 60 ns MD simulation for DprE1-DprE2 complex was also performed. Analysis of data revealed that the docked complex is stabilized by H-bonding, hydrophobic and ionic interactions. The key residues of DprE1 involved in DprE1-DprE2 interactions belong to the disordered region. We also examined the docked complex of DprE1-BTZ043 to investigate the binding pocket of DprE1 and its interactions with the inhibitor BTZ043. In summary, we hypothesize that DprE1-DprE2 interaction is crucial for the synthesis of DPA and DprE1-DprE2 complex may be a new therapeutic target amenable to pharmacological validation. The findings have important implications in tuberculosis (TB) drug discovery and will facilitate drug development efforts against TB.

  2. Functional synergy between DP-1 and E2F-1 in the cell cycle-regulating transcription factor DRTF1/E2F.

    PubMed Central

    Bandara, L R; Buck, V M; Zamanian, M; Johnston, L H; La Thangue, N B

    1993-01-01

    It is widely believed that the cellular transcription factor DRTF1/E2F integrates cell cycle events with the transcription apparatus because during cell cycle progression in mammalian cells it interacts with molecules that are important regulators of cellular proliferation, such as the retinoblastoma tumour suppressor gene product (pRb), p107, cyclins and cyclin-dependent kinases. Thus, pRb, which negatively regulates early cell cycle progression and is frequently mutated in tumour cells, and the Rb-related protein p107, bind to and repress the transcriptional activity of DRTF1/E2F. Viral oncoproteins, such as adenovirus E1a and SV40 large T antigen, overcome such repression by sequestering pRb and p107 and in so doing are likely to activate genes regulated by DRTF1/E2F, such as cdc2, c-myc and DHFR. Two sequence-specific DNA binding proteins, E2F-1 and DP-1, which bind to the E2F site, contain a small region of similarity. The functional relationship between them has, however, been unclear. We report here that DP-1 and E2F-1 exist in a DNA binding complex in vivo and that they bind efficiently and preferentially as a heterodimer to the E2F site. Moreover, studies in yeast and Drosophila cells indicate that DP-1 and E2F-1 interact synergistically in E2F site-dependent transcriptional activation. Images PMID:8223441

  3. HPV16 E2 gene disruption and polymorphisms of E2 and LCR: some significant associations with cervical cancer in Indian women.

    PubMed

    Bhattacharjee, Bornali; Sengupta, Sharmila

    2006-02-01

    We evaluated the status of the HPV16 E2 gene (disrupted or intact), nucleotide sequence alterations within intact E2 genes and LCR of HPV16 isolates in a group of CaCx cases (invasive squamous cell carcinomas, n = 81) and population controls (normal cervical scrapes, n = 27) from Indian women. E2 disruption was detected by amplifying the entire E2 gene with single set of primers, while overlapping primers were used to determine if any particular region got selectively disrupted. Nucleotide variations in E2 and LCR were analyzed by PCR amplification followed by bi-directional sequencing. The associations between the viral factors and CaCx were analyzed using Fisher's Exact or Chi-squared test and interpreted as OR (95% CI) and P values. E2 disruption was significantly higher among the cases [3.38 (1.07-10.72); P = 0.02], which was maximum in the region between nucleotides 3650 and 3872 (DNA-binding region). The European (E) variant was found to be the prevalent subgroup (87.76% among cases and 96.30% among the controls), and the remaining samples were Asian-American variants. Among the E subgroup, variation at position 7450 (T > C) within the E2-binding site-IV was found to be significantly higher among the E2 undisrupted cases (21/37; 56.76%), compared to controls (5/18; 27.78%) [3.41 (1.01-11.55); P = 0.03]. Besides HPV16 E2 disruption, LCR 7450T > C variation within undisrupted E2 of E subgroup appears to be a major factor contributing to the risk of CaCx development in Indian women. Furthermore, polymorphisms in the E2 gene of HPV16 may not be significant for disease risk.

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

    USDA-ARS?s Scientific Manuscript database

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

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

    USDA-ARS?s Scientific Manuscript database

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

  6. Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

    PubMed

    Ali, Amjad; Nisar, Muhammad; Idrees, Muhammad; Rafique, Shazia; Iqbal, Muhammad

    2015-05-01

    Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the

  7. E2F8 as a Novel Therapeutic Target for Lung Cancer

    PubMed Central

    Park, Sin-Aye; Platt, James; Lee, Jong Woo; López-Giráldez, Francesc; Herbst, Roy S.

    2015-01-01

    Background: The E2F members have been divided into transcription activators (E2F1-E2F3) and repressors (E2F4-E2F8). E2F8 with E2F7 has been known to play an important physiologic role in embryonic development and cell cycle regulation by repressing E2F1. However, the function of E2F8 in cancer cells is unknown. Methods: E2F8 expression was assessed by immunoblotting or immunofluorescence staining in human lung cancer (LC) cells and tissues from LC patients (n = 45). Cell proliferation, colony formation, and invasion analysis were performed to evaluate the role of E2F8 in LC. Microarray analysis was used to determine the target genes of E2F8. The regulation of E2F8 on the expression of ubiquitin-like PHD and RING domain-containing 1 (UHRF1), one of E2F8 target genes, was determined using chromatin immunoprecipitation and promoter activity assays. Human LC xenograft models were used to determine the effects of inhibiting E2F8 by siRNAs (n = 7 per group) or antisense morpholino (n = 8 per group) on tumor growth. Survival was analyzed using the Kaplan-Meier method and group differences by the Student’s t test. All statistical tests were two-sided. Results: LC tumors overexpressed E2F8 compared with normal lung tissues. Depletion of E2F8 inhibited cell proliferation and tumor growth. E2F8 knockdown statistically significantly reduced the expression of UHRF1 (~60%-70%, P < .001), and the direct binding of E2F8 on the promoter of UHRF1 was identified. Kaplan-Meier analysis with a public database showed prognostic significance of aberrant E2F8 expression in LC (HR = 1.91 95% CI = 1.21 to 3.01 in chemo-naïve patients, P = .0047). Conclusions: We demonstrated that E2F8 is overexpressed in LC and is required for the growth of LC cells. These findings implicate E2F8 as a novel therapeutic target for LC treatment. PMID:26089541

  8. E2F8 as a Novel Therapeutic Target for Lung Cancer.

    PubMed

    Park, Sin-Aye; Platt, James; Lee, Jong Woo; López-Giráldez, Francesc; Herbst, Roy S; Koo, Ja Seok

    2015-09-01

    The E2F members have been divided into transcription activators (E2F1-E2F3) and repressors (E2F4-E2F8). E2F8 with E2F7 has been known to play an important physiologic role in embryonic development and cell cycle regulation by repressing E2F1. However, the function of E2F8 in cancer cells is unknown. E2F8 expression was assessed by immunoblotting or immunofluorescence staining in human lung cancer (LC) cells and tissues from LC patients (n = 45). Cell proliferation, colony formation, and invasion analysis were performed to evaluate the role of E2F8 in LC. Microarray analysis was used to determine the target genes of E2F8. The regulation of E2F8 on the expression of ubiquitin-like PHD and RING domain-containing 1 (UHRF1), one of E2F8 target genes, was determined using chromatin immunoprecipitation and promoter activity assays. Human LC xenograft models were used to determine the effects of inhibiting E2F8 by siRNAs (n = 7 per group) or antisense morpholino (n = 8 per group) on tumor growth. Survival was analyzed using the Kaplan-Meier method and group differences by the Student's t test. All statistical tests were two-sided. LC tumors overexpressed E2F8 compared with normal lung tissues. Depletion of E2F8 inhibited cell proliferation and tumor growth. E2F8 knockdown statistically significantly reduced the expression of UHRF1 (~60%-70%, P < .001), and the direct binding of E2F8 on the promoter of UHRF1 was identified. Kaplan-Meier analysis with a public database showed prognostic significance of aberrant E2F8 expression in LC (HR = 1.91 95% CI = 1.21 to 3.01 in chemo-naïve patients, P = .0047). We demonstrated that E2F8 is overexpressed in LC and is required for the growth of LC cells. These findings implicate E2F8 as a novel therapeutic target for LC treatment. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  9. Different enzyme kinetic models.

    PubMed

    Seibert, Eleanore; Tracy, Timothy S

    2014-01-01

    As described in Chapter 2 , a large number of enzymatic reactions can be adequately described by Michaelis-Menten kinetics. The Michaelis-Menten equation represents a rectangular hyperbola, with a y-asymptote at the V max value. In many cases, more complex kinetic models are required to explain the observed data. Atypical kinetic profiles are believed to arise from the simultaneous binding of multiple molecules within the active site of the enzyme (Tracy and Hummel, Drug Metab Rev 36:231-242, 2004). Several cytochromes P450 have large active sites that enable binding of multiple molecules (Wester et al. J Biol Chem 279:35630-35637, 2004; Yano et al. J Biol Chem 279:38091-38094, 2004). Thus, atypical kinetics are not uncommon in in vitro drug metabolism studies. This chapter covers enzyme kinetic reactions in which a single enzyme has multiple binding sites for substrates and/or inhibitors as well as reactions catalyzed by multiple enzymes.

  10. Enzyme Kinetics in Microgravity

    NASA Astrophysics Data System (ADS)

    Liu, C. C.; Licata, V. J.

    2010-04-01

    The kinetics of some enzymes have been found to be enhanced by the microgravity environment. This is a relatively small effect, but is sufficient to have physiological effects and to impact pharmaceutical therapy in microgravity.

  11. Starch Biorefinery Enzymes.

    PubMed

    Läufer, Albrecht

    2017-03-07

    Nature uses enzymes to build and convert biomass; mankind uses the same enzymes and produces them on a large scale to make optimum use of biomass in biorefineries. Bacterial α-amylases and fungal glucoamylases have been the workhorses of starch biorefineries for many decades. Pullulanases were introduced in the 1980s. Proteases, cellulases, hemicellulases, and phytases have been on the market for a few years as process aids, improving yields, performance, and costs. Detailed studies of the complex chemical structures of biomass and of the physicochemical limitations of industrial biorefineries have led enzyme developers to produce novel tailor-made solutions for improving yield and profitability in the industry. This chapter reviews the development of enzyme applications in the major starch biorefining processes.

  12. Indicators: Sediment Enzymes

    EPA Pesticide Factsheets

    Sediment enzymes are proteins that are produced by microorganisms living in the sediment or soil. They are indicators of key ecosystem processes and can help determine which nutrients are affecting the biological community of a waterbody.

  13. RNA as an Enzyme.

    ERIC Educational Resources Information Center

    Cech, Thomas R.

    1986-01-01

    Reviews current findings that explain RNA's function as an enzyme in addition to being an informational molecule. Highlights recent research efforts and notes changes in the information base on RNA activity. Includes models and diagrams of RNA activity. (ML)

  14. 40 CFR Figure E-2 to Subpart E of... - Product Manufacturing Checklist

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 5 2010-07-01 2010-07-01 false Product Manufacturing Checklist E Figure E-2 to Subpart E of Part 53 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Equivalent Methods for PM2.5 or PM10â2.5 Pt. 53, Subpt. E, Fig. E-2 Figure E-2 to Subpart E of Part 53...

  15. E2fl1 is a meiosis-specific transcription factor in the protist Tetrahymena thermophila

    PubMed Central

    Zhang, Jing; Tian, Miao; Miao, Wei

    2017-01-01

    ABSTRACT Members of the E2F family of transcription factors have been reported to regulate the expression of genes involved in cell cycle control, DNA replication, and DNA repair in multicellular eukaryotes. Here, E2FL1, a meiosis-specific E2F transcription factor gene, was identified in the model ciliate Tetrahymena thermophila. Loss of this gene resulted in meiotic arrest prior to anaphase I. The cytological experiments revealed that the meiotic homologous pairing was not affected in the absence of E2FL1, but the paired homologous chromosomes did not separate and assumed a peculiar tandem arrangement. This is the first time that an E2F family member has been shown to regulate meiotic events. Moreover, BrdU incorporation showed that DSB processing during meiosis was abnormal upon the deletion of E2FL1. Transcriptome sequencing analysis revealed that E2FL1 knockout decreased the expression of genes involved in DNA replication and DNA repair in T. thermophila, suggesting that the function of E2F is highly conserved in eukaryotes. In addition, E2FL1 deletion inhibited the expression of related homologous chromosome segregation genes in T. thermophila. The result may explain the meiotic arrest phenotype at anaphase I. Finally, by searching for E2F DNA-binding motifs in the entire T. thermophila genome, we identified 714 genes containing at least one E2F DNA-binding motif; of these, 235 downregulated represent putative E2FL1 target genes. PMID:27892792

  16. 40 CFR Figure E-2 to Subpart E of... - Product Manufacturing Checklist

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 5 2011-07-01 2011-07-01 false Product Manufacturing Checklist E Figure E-2 to Subpart E of Part 53 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Equivalent Methods for PM2.5 or PM10â2.5 Pt. 53, Subpt. E, Fig. E-2 Figure E-2 to Subpart E of Part 53...

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

    PubMed Central

    Lee, Mi-Young; Moreno, Carlos S.

    2014-01-01

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

  18. E2F activators signal and maintain centrosome amplification in breast cancer cells.

    PubMed

    Lee, Mi-Young; Moreno, Carlos S; Saavedra, Harold I

    2014-07-01

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

  19. Overproduction of ligninolytic enzymes

    SciTech Connect

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  20. CpG methylation of HPV 16 LCR at E2 binding site proximal to P97 is associated with cervical cancer in presence of intact E2.

    PubMed

    Bhattacharjee, Bornali; Sengupta, Sharmila

    2006-10-25

    Human papillomavirus type 16 (HPV-16) E2 protein negatively regulates transcription of the E6 and E7 genes. This study was done to test the hypothesis that methylation of the HPV 16 long control region (LCR) is overrepresented among cervical cancer (CaCx) cases compared to cytologically normal controls harboring intact E2 gene. Methylation of the E2 binding site (E2BS-I), proximal to the P97 promoter, was assessed by HpaII/ MspI restriction digestion while McrBC digestion was used to assess LCR-E6 (7289-540) for 57 CaCx samples and 15 normal controls. E2BS-I methylation was found to be significantly higher (56.14%) in cases compared to (20%) controls [OR(age-adjusted) (95% CI): 4.53 (1.05-19.43) p=0.042]. The difference between cases (54.39%) and controls (40%) with respect to LCR-E6 methylation status [OR(age-adjusted) (95% CI): 1.77(0.5-6.3); p=0.38] was not significant. Sequencing of a randomly selected set of 13 methylated malignant samples revealed absence or rare presence, of methylation at CpGs 7579, 7535, 7683 and 7862 respectively. Methylation was found to be more at CpGs within E2 binding sites proximal to the P97 promoter. These results indicate the involvement of E2 binding site methylation in presence of intact E2, leading to loss of E2 repressor activity in CaCx.

  1. Intravenous anesthetic propofol suppresses prostaglandin E2 production in murine dendritic cells.

    PubMed

    Inada, Takefumi; Kubo, Kozue; Ueshima, Hironobu; Shingu, Koh

    2011-01-01

    Propofol is an intravenous anesthetic that is widely used for anesthesia and sedation. Dendritic cells (DC) are one of the crucial immune cells that bridge innate and adaptive immunity, in which DC process antigens during innate immune responses to present them to naïve T-cells, leading to an establishment of adaptive immunity. Prostaglandin (PG)-E(2) may be secreted by DC into the microenvironment, considerably influencing DC phenotype and function, and thus determining the fate of adaptive immunity. Since propofol suppresses PGE(2) production in murine macrophages, the primary purpose of the present study was to determine whether propofol also suppresses PGE(2) production in DC. Assuming a positive finding of such suppression, we tested whether this also leads to alterations of interleukin (IL)-12 and IL-10 production and DC surface marker expression, both of which can be modulated by PGE(2). In bone marrow-derived DC, propofol significantly suppressed the PGE(2) production after lipopolysaccharide stimulation. Cyclo-oxygenase (COX) protein expression and arachidonic acid release were unaffected, while COX enzyme activity was significantly inhibited by propofol. The propofol-induced COX inhibition did not lead to the increased production of cysteinyl leukotrienes and leukotriene-B(4). Endogenous COX inhibition with propofol, as well as with the selective COX-2 inhibitor, NS-398, did not affect IL-12 and IL-10 production from DC. The surface expression of I-A(b) and CD40 on DC was not changed, while that of CD86 slightly increased, with both propofol and NS-398; expression of CD80 was not affected with propofol, but increased slightly with NS-398. Finally, endogenous COX inhibition with either propofol or NS-398 did not significantly affect the ability of DC to induce allogeneic T-cell proliferation. It is concluded that the intravenous anesthetic propofol suppresses COX enzyme activity in DC, with no consequences with respect to IL-12/IL-10 production and

  2. Stretch independent regulation of prostaglandin E(2) production within the isolated guinea-pig lamina propria.

    PubMed

    Nile, Christopher J; de Vente, Jan; Gillespie, James I

    2010-02-01

    To use an isolated preparation of the guinea-pig bladder lamina propria (LP) to investigate the effects of adenosine tri-phosphate (ATP) and nitric oxide (NO) on the release of prostaglandin E(2) (PGE(2)). The bladders of female guinea-pigs (200-400 g) were isolated and opened to expose the urothelial surface. The LP was dissected free of the underlying detrusor muscle and cut into strips from the dome to base. Strips were then incubated in Krebs buffer at 37 degrees C. Each tissue piece was then exposed to the stable ATP analogue, BzATP, and a NO donor, diethylamine-NONOate (DEANO), and the effect on PGE(2) output into the supernatant determined using the Parameter(TM) PGE(2) enzyme immunoassay kit (R & D Systems, Abingdon, UK). Experiments were repeated in the presence of purinergic receptor and cyclooxygenase (COX) enzymes, COX I and COX II, antagonists. The cellular location of COX I, COX II and neuronal NO synthase (nNOS) within the bladder LP was also determined by immunohistochemistry. PGE(2) production was significantly increased by BzATP. Antagonist studies showed the purinergic stimulation involved both P(2)X and P(2)Y receptors. The BzATP response was inhibited by the COX inhibitor indomethacin (COX I >COX II) but not by DUP 697 (COX II >COX I). Thus, BzATP stimulation occurs because of COX I stimulation. NO had no effect on PGE(2) production over the initial 10 min of an exposure. However, PGE(2) output was increased 100 min after exposure to the NO donor. In the presence of NO, the BzATP stimulation was abolished. Immunohistochemistry was used to confirm the location of COX I to the basal and inner intermediate urothelial layers and to cells within the diffuse layer of LP interstitial cells. In addition, nNOS was also located in the basal urothelial layers whilst COX II was found in the interstitial cell layers. There is complex interaction between ATP and NO to modulate PGE(2) release from the bladder LP in the un-stretched preparation. Such

  3. HP-41CV Flight Performance Advisory System (FPAS) for the E-2C, E-2B, and C-2A Aircraft

    DTIC Science & Technology

    1982-06-01

    NPS67-82- 003 NAVAL POSTGRADUATE SCHOOL Monterey, California DTIC HP-41CV FLIGHT PERFORMANCE ADVISORY SYSTEM (FPAS) FOR THE E-2C, E-2B, AND C-2A...A’P-𔃻"’f .00 ____________ 4. TITLE9 (and Subtil) SL TYPE OF REPORT & PERIOD COVERED H1P-41CV FLIGHT PERFORMANCE ADVISORY SYSTEM (FPAS) TECHNICAL REPORT...complement the original design of a Flight Performance Advisory System (FPAS) for the E-2C aircraft. The original design fulfilled the requirements of AE 3001

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

    PubMed

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

    2014-11-01

    Human papillomavirus 16 (HPV16) is causative in human cancer. The E2 protein regulates transcription from and replication of the viral genome; the role of E2 in regulating the host genome has been less well studied. We have expressed HPV16 E2 (E2) stably in U2OS cells; these cells tolerate E2 expression well and gene expression analysis identified 74 genes showing differential expression specific to E2. Analysis of published gene expression data sets during cervical cancer progression identified 20 of the genes as being altered in a similar direction as the E2 specific genes. In addition, E2 altered the splicing of many genes implicated in cancer and cell motility. The E2 expressing cells showed no alteration in cell growth but were altered in cell motility, consistent with the E2 induced altered splicing predicted to affect this cellular function. The results present a model system for investigating E2 regulation of the host genome. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. E2f1–3 Are Critical for Myeloid Development*

    PubMed Central

    Trikha, Prashant; Sharma, Nidhi; Opavsky, Rene; Reyes, Andres; Pena, Clarissa; Ostrowski, Michael C.; Roussel, Martine F.; Leone, Gustavo

    2011-01-01

    Hematopoietic development involves the coordinated activity of differentiation and cell cycle regulators. In current models of mammalian cell cycle control, E2f activators (E2f1, E2f2, and E2f3) are portrayed as the ultimate transcriptional effectors that commit cells to enter and progress through S phase. Using conditional gene knock-out strategies, we show that E2f1–3 are not required for the proliferation of early myeloid progenitors. Rather, these E2fs are critical for cell survival and proliferation at two distinct steps of myeloid development. First, E2f1–3 are required as transcriptional repressors for the survival of CD11b+ myeloid progenitors, and then they are required as activators for the proliferation of CD11b+ macrophages. In bone marrow macrophages, we show that E2f1–3 respond to CSF1-Myc mitogenic signals and serve to activate E2f target genes and promote their proliferation. Together, these findings expose dual functions for E2f1–3 at distinct stages of myeloid development in vivo, first as repressors in cell survival and then as activators in cell proliferation. In summary, this work places E2f1–3 in a specific signaling cascade that is critical for myeloid development in vivo. PMID:21115501

  6. E2F1 transcription is induced by genotoxic stress through ATM/ATR activation.

    PubMed

    Carcagno, Abel L; Ogara, María F; Sonzogni, Silvina V; Marazita, Mariela C; Sirkin, Pablo F; Ceruti, Julieta M; Cánepa, Eduardo T

    2009-05-01

    E2F1, a member of the E2F family of transcription factors, plays a critical role in controlling both cell cycle progression and apoptotic cell death in response to DNA damage and oncogene activation. Following genotoxic stresses, E2F1 protein is stabilized by phosphorylation and acetylation driven to its accumulation. The aim of the present work was to examine whether the increase in E2F1 protein levels observed after DNA damage is only a reflection of an increase in E2F1 protein stability or is also the consequence of enhanced transcription of the E2F1 gene. The data presented here demonstrates that UV light and other genotoxics induce the transcription of E2F1 gene in an ATM/ATR dependent manner, which results in increasing E2F1 mRNA and protein levels. After genotoxic stress, transcription of cyclin E, an E2F1 target gene, was significantly induced. This induction was the result of two well-differentiated effects, one of them dependent on de novo protein synthesis and the other on the protein stabilization. Our results strongly support a transcriptional effect of DNA damaging agents on E2F1 expression. The results presented herein uncover a new mechanism involving E2F1 in response to genotoxic stress.

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

    PubMed

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

    2016-04-01

    The E2 is at the center of a cascade of Ub1 transfers, and it links activation of the Ub1 by E1 to its eventual E3-catalyzed attachment to substrate. Although the genome-wide analysis of this family has been performed in some species, little is known about analysis of E2 genes in banana. In this study, 74 E2 genes of banana were identified and phylogenetically clustered into thirteen subgroups. The predicted banana E2 genes were distributed across all 11 chromosomes at different densities. Additionally, the E2 domain, gene structure and motif compositions were analyzed. The expression of all of the banana E2 genes was analyzed in the root, stem, leaf, flower organs, five stages of fruit development and under abiotic stresses. All of the banana E2 genes, with the exception of few genes in each group, were expressed in at least one of the organs and fruit developments, which indicated that the E2 genes might involve in various aspects of the physiological and developmental processes of the banana. Quantitative RT-PCR (qRT-PCR) analysis identified that 45 E2s under drought and 33 E2s under salt were induced. To the best of our knowledge, this report describes the first genome-wide analysis of the banana E2 gene family, and the results should provide valuable information for understanding the classification, cloning and putative functions of this family. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. Increased urinary prostaglandin E2 metabolite: A potential therapeutic target of Gitelman syndrome.

    PubMed

    Peng, Xiaoyan; Jiang, Lanping; Chen, Chen; Qin, Yan; Yuan, Tao; Wang, Ou; Xing, Xiaoping; Li, Xuemei; Nie, Min; Chen, Limeng

    2017-01-01

    Gitelman syndrome (GS), an inherited autosomal recessive salt-losing renal tubulopathy caused by mutations in SLC12A3 gene, has been associated with normal prostaglandin E2 (PGE2) levels since 1995 by a study involving 11 clinically diagnosed patients. However, it is difficult to explain why cyclooxygenase-2 (COX2) inhibitors, which pharmacologically reduce PGE2 synthesis, are helpful to patients with GS, and few studies performed in the last 20 years have measured PGE2 levels. The relationships between the clinical manifestations and PGE2 levels were never thoroughly analyzed. This study involved 39 GS patients diagnosed by SLC12A3 gene sequencing. Plasma and 24-h urine samples as well as the clinical data were collected at admission. PGE2 and PGEM levels were detected in plasma and urine samples by enzyme immunoassays. The in vivo function of the sodium-chloride co-transporter (NCC) in GS patients was evaluated using a modified thiazide test. The association among PGE2 levels, clinical manifestations and the function of NCC in GS patients were analyzed. Significantly higher levels of urinary and plasma PGEM were observed in GS patients than in the healthy volunteers. Higher urinary PGEM levels indicated more severe clinical manifestations and NCC dysfunction estimated by the increase of Cl- clearance. A higher PGEM level was found in male GS patients, who showed earlier onset age and more severe hypokalemia, hypochloremia and metabolic alkalosis than female GS patients. No relationship between renin angiotensin aldosterone system activation and PGEM level was observed. Higher urinary PGEM levels indicated more severe clinical manifestations and NCC dysfunction in GS patients. COX2 inhibition might be a potential therapeutic target in GS patients with elevated PGEM levels.

  9. Random-walk enzymes.

    PubMed

    Mak, Chi H; Pham, Phuong; Afif, Samir A; Goodman, Myron F

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C→U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  10. Random-walk enzymes

    PubMed Central

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-01-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C → U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics. PMID:26465508

  11. Random-walk enzymes

    NASA Astrophysics Data System (ADS)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C →U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  12. RNF168 forms a functional complex with RAD6 during the DNA damage response

    PubMed Central

    Liu, Chao; Wang, Degui; Wu, Jiaxue; Keller, Jennifer; Ma, Teng; Yu, Xiaochun

    2013-01-01

    Summary Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, E2 ubiquitin conjugating enzymes are crucial for catalyzing substrate ubiquitination that recruits downstream DNA repair factors to DNA lesions. To identify novel E2 conjugating enzymes important for initiating the DNA-damage-induced ubiquitination cascade, we screened most of the known E2 enzymes and found that RAD6A and RAD6B function together with RNF168 in the ionizing radiation (IR)-induced DNA damage response. Similarly to RNF168-deficient cells, RAD6A- or RAD6B-deficient cells exhibit a reduction in DNA-damage-induced protein ubiquitination. Correspondingly, DNA-damage-induced foci formation of DNA damage repair proteins, such as BRCA1 and 53BP1, is impaired in the absence of RAD6A or RAD6B. Moreover, the RNF168–RAD6 complex targeted histone H1.2 for ubiquitination in vitro and regulated DNA-damage-induced histone H1.2 ubiquitination in vivo. Collectively, these data demonstrate that RNF168, in complex with RAD6A or RAD6B, is activated in the DNA-damage-induced protein ubiquitination cascade. PMID:23525009

  13. Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity*

    PubMed Central

    Ranaweera, Ruchira S.; Yang, Xiaolu

    2013-01-01

    The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes. PMID:23671280

  14. The NAMPT/E2F2/SIRT1 axis promotes proliferation and inhibits p53-dependent apoptosis in human melanoma cells.

    PubMed

    Zhao, Hailong; Tang, Weiwei; Chen, Xiaowen; Wang, Siyu; Wang, Xianyan; Xu, Haiyan; Li, Lijuan

    2017-11-04

    Melanoma is the most common primary malignant neoplasm in adults, causing more deaths than any other skin cancer, necessitating the development of new target-based approaches. Current evidence suggests SIRT1, the mammalian nicotinamide adenine dinucleotide (NAD + )-dependent protein deacetylase, and nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting NAD + biosynthetic enzyme, together comprise a novel systemic regulatory network to play a pivotal role in cell proliferation and apoptosis. Nevertheless, how the regulation of this cofactor interfaces with signal transduction network remains poorly understood in melanoma. Here, we report NAMPT is highly expressed in melanomaassociated with poor overall survival in patients. Pharmacological and genetic inhibition of NAMPT decreased NAD + levels and melanoma cell proliferation capacity, and NAMPT knockdown induced apoptosis through the activity of the tumor suppressor p53. Next, we demonstrate NAMPT regulates the transcription factor E2F family member 2 (E2F2) in the apoptosis process. Downstream, E2F2 control the mRNA and protein levels of SIRT1. Finally, we find NAMPT mediates the apoptosis resistance of melanoma cells through NAMPT-E2F2-SIRT1 axis, more than NAD + -driven transcriptional program. Accordingly, our results demonstrated that NAMPT is a prognostic marker in melanoma, and the identificationofNAMPT-E2F2-SIRT1 pathway establishes another link between NAMPT and apoptosis events in melanoma, with therapeutic implications for this deadly cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Crystal structures of two bacterial HECT-like E3 ligases in complex with a human E2 reveal atomic details of pathogen-host interactions

    SciTech Connect

    Lin, David Yin-wei; Diao, Jianbo; Chen, Jue

    2012-12-10

    In eukaryotes, ubiquitination is an important posttranslational process achieved through a cascade of ubiquitin-activating (E1), conjugating (E2), and ligase (E3) enzymes. Many pathogenic bacteria deliver virulence factors into the host cell that function as E3 ligases. How these bacterial 'Trojan horses' integrate into the eukaryotic ubiquitin system has remained a mystery. Here we report crystal structures of two bacterial E3s, Salmonella SopA and Escherichia coli NleL, both in complex with human E2 UbcH7. These structures represent two distinct conformational states of the bacterial E3s, supporting the necessary structural rearrangements associated with ubiquitin transfer. The E2-interacting surface of SopA and NleLmore » has little similarity to those of eukaryotic E3s. However, both bacterial E3s bind to the canonical surface of E2 that normally interacts with eukaryotic E3s. Furthermore, we show that a glutamate residue on E3 is involved in catalyzing ubiquitin transfer from E3 to the substrate, but not from E2 to E3. Together, these results provide mechanistic insights into the ubiquitin pathway and a framework for understanding molecular mimicry in bacterial pathogenesis.« less

  16. TFDP3 was expressed in coordination with E2F1 to inhibit E2F1-mediated apoptosis in prostate cancer.

    PubMed

    Ma, Yueyun; Xin, Yijuan; Li, Rui; Wang, Zhe; Yue, Qiaohong; Xiao, Fengjing; Hao, Xiaoke

    2014-03-10

    TFDP3 has been previously identified as an inhibitor of E2F molecules. It has been shown to suppress E2F1-induced apoptosis dependent P53 and to play a potential role in carcinogenesis. However, whether it indeed helps cancer cells tolerate apoptosis stress in cancer tissues remains unknown. TFDP3 expression was assessed by RT-PCR, in situ hybridization and immunohistochemistry in normal human tissues, cancer tissues and prostate cancer tissues. The association between TFDP3 and E2F1 in prostate cancer development was analyzed in various stages. Apoptosis was evaluated with annexin-V and propidium iodide staining and flow-cytometry. The results show that, in 96 samples of normal human tissues, TFDP3 could be detected in the cerebrum, esophagus, stomach, small intestine, bronchus, breast, ovary, uterus, and skin, but seldom in the lung, muscles, prostate, and liver. In addition, TFDP3 was highly expressed in numerous cancer tissues, such as brain-keratinous, lung squamous cell carcinoma, testicular seminoma, cervical carcinoma, skin squamous cell carcinoma, gastric adenocarcinoma, liver cancer, and prostate cancer. Moreover, TFDP3 was positive in 23 (62.2%) of 37 prostate cancer samples regardless of stage. Furthermore, immunohistochemistry results show that TFDP3 was always expressed in coordination with E2F1 at equivalent expression levels in prostate cancer tissues, and was highly expressed particularly in samples of high stage. When E2F1 was extrogenously expressed in LNCap cells, TFDP3 could be induced, and the apoptosis induced by E2F1 was significantly decreased. It was demonstrated that TFDP3 was a broadly expressed protein corresponding to E2F1 in human tissues, and suggested that TFDP3 is involved in prostate cancer cell survival by suppressing apoptosis induced by E2F1. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Carboxylesterases: General detoxifying enzymes

    PubMed Central

    Hatfield, M. Jason; Umans, Robyn A.; Hyatt, Janice L.; Edwards, Carol C; Wierdl, Monika; Tsurkan, Lyudmila; Taylor, Michael R.; Potter, Philip M.

    2016-01-01

    Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents). PMID:26892220

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

    PubMed Central

    Zappia, Maria Paula; Frolov, Maxim V.

    2016-01-01

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

  19. E2F8 is essential for polyploidization in mammalian cells.

    PubMed

    Pandit, Shusil K; Westendorp, Bart; Nantasanti, Sathidpak; van Liere, Elsbeth; Tooten, Peter C J; Cornelissen, Peter W A; Toussaint, Mathilda J M; Lamers, Wouter H; de Bruin, Alain

    2012-11-01

    Polyploidization is observed in all mammalian species and is a characteristic feature of hepatocytes, but its molecular mechanism and biological significance are unknown. Hepatocyte polyploidization in rodents occurs through incomplete cytokinesis, starts after weaning and increases with age. Here, we show in mice that atypical E2F8 is induced after weaning and required for hepatocyte binucleation and polyploidization. A deficiency in E2f8 led to an increase in the expression level of E2F target genes promoting cytokinesis and thereby preventing polyploidization. In contrast, loss of E2f1 enhanced polyploidization and suppressed the polyploidization defect of hepatocytes deficient for atypical E2Fs. In addition, E2F8 and E2F1 were found on the same subset of target promoters. Contrary to the long-standing hypothesis that polyploidization indicates terminal differentiation and senescence, we show that prevention of polyploidization through inactivation of atypical E2Fs has, surprisingly, no impact on liver differentiation, zonation, metabolism and regeneration. Together, these results identify E2F8 as a repressor and E2F1 as an activator of a transcriptional network controlling polyploidization in mammalian cells.

  20. [Eukaryotic expression and application of HCV Hebei strain E2 extracellular core region].

    PubMed

    Ye, Chuantao; Bian, Peiyu; Weng, Daihui; Zhang, Hui; Yang, Jing; Zhang, Ying; Lei, Yingfeng; Jia, Zhansheng

    2016-06-01

    Objective To express core region of HCV1b (Hebei strain) E2 protein (E2c) by eukaryotic system, and establish the detection method of specific anti-HCV E2 antibody in the sera from hepatitis C patients. Methods Based on the literature, the E2c gene was modified from the HCV1b gene and synthesized via overlapping PCR. Thereafter, the E2c gene including tissue-type plasminogen activator (tPA) signal peptide was cloned into the pCI-neo eukaryotic expression vector, and the product was named pCI-tpa-1bE2c. After HEK293T cells were transfected with pCI-tpa-1bE2c, the supernatant was collected, condensed and purified. Its specificity was identified by Western blotting. Galanthus nivalis agglutinin (GNA)-based ELISA was used to detect the antibody against HCVE2 in the sera from hepatitis C patients. Results Modified HCV E2c protein was successfully expressed in HEK293T cells and the GNA-based ELISA was developed for detecting the antibody against HCV E2 in the sera from hepatitis C patients. Conclusion HCV-1bE2c protein can be effectively expressed in HEK293T cells and applied clinically.

  1. CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes

    PubMed Central

    Singh, Randeep K.; Dagnino, Lina

    2017-01-01

    The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation. Analysis of the structural determinants that govern these processes revealed a central role for S403 and T433. In particular, substitution of these two amino acid residues with non-phosphorylatable alanine (E2F1 ST/A) interferes with E2F1 nuclear export, K11- and K48-linked polyubiquitylation and degradation in differentiated keratinocytes. In contrast, replacement of S403 and T433 with phosphomimetic aspartic acid to generate a pseudophosphorylated E2F1 mutant protein (E2F1 ST/D) generates a protein that is regulated in a manner indistinguishable from that of wild type E2F1. Cdh1 is an activating cofactor that interacts with the anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, promoting proteasomal degradation of various substrates. We found that Cdh1 associates with E2F1 in keratinocytes. Inhibition or RNAi-mediated silencing of Cdh1 prevents E2F1 degradation in response to differentiation signals. Our results reveal novel regulatory mechanisms that jointly modulate post-translational modifications and downregulation of E2F1, which are necessary for proper epidermal keratinocyte differentiation. PMID:27903963

  2. CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes.

    PubMed

    Singh, Randeep K; Dagnino, Lina

    2017-01-17

    The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation. Analysis of the structural determinants that govern these processes revealed a central role for S403 and T433. In particular, substitution of these two amino acid residues with non-phosphorylatable alanine (E2F1 ST/A) interferes with E2F1 nuclear export, K11- and K48-linked polyubiquitylation and degradation in differentiated keratinocytes. In contrast, replacement of S403 and T433 with phosphomimetic aspartic acid to generate a pseudophosphorylated E2F1 mutant protein (E2F1 ST/D) generates a protein that is regulated in a manner indistinguishable from that of wild type E2F1. Cdh1 is an activating cofactor that interacts with the anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, promoting proteasomal degradation of various substrates. We found that Cdh1 associates with E2F1 in keratinocytes. Inhibition or RNAi-mediated silencing of Cdh1 prevents E2F1 degradation in response to differentiation signals. Our results reveal novel regulatory mechanisms that jointly modulate post-translational modifications and downregulation of E2F1, which are necessary for proper epidermal keratinocyte differentiation.

  3. [Micro fabricated enzyme battery].

    PubMed

    Sasaki, S; Karube, I

    1996-10-01

    Although various work has been done in the field of implantable micro actuators such as artificial organs and micro surgery robots, a suitable electric power supply for these is yet to be developed. For this purpose a micro fabricated enzyme fuel cell was developed which uses glucose contained in the human body as a fuel. In order to obtain enough voltage each cell was formed as part of a serial array on a silicon wafer. Glucose solution enters the cells by a capillary effect. In this article fuel cells already developed using biocatalysts are described, and the future possibility of a micro fabricated enzyme battery is discussed.

  4. Oxysterol biosynthetic enzymes.

    PubMed

    Russell, D W

    2000-12-15

    Oxysterols, herein defined as derivatives of cholesterol with a hydroxyl group on the side chain, play several roles in lipid metabolism. Members of this class regulate the expression of genes that participate in both sterol and fat metabolism, serve as substrates for the synthesis of bile acids, and are intermediates in the transfer of sterols from the periphery to the liver. Three abundant naturally occurring oxysterols are 24-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. The cholesterol hydroxylase enzymes that synthesize each of these have been isolated over the last several years and their study has produced insight into the biology of oxysterols. This article focuses on the properties of these enzymes.

  5. Natural products inhibiting the ubiquitin-proteasome proteolytic pathway, a target for drug development.

    PubMed

    Tsukamoto, Sachiko; Yokosawa, Hideyoshi

    2006-01-01

    The ubiquitin-proteasome proteolytic pathway plays a major role in selective protein degradation and regulates various cellular events including cell cycle progression, transcription, DNA repair, signal transduction, and immune response. Ubiquitin, a highly conserved small protein in eukaryotes, attaches to a target protein prior to degradation. The polyubiquitin chain tagged to the target protein is recognized by the 26S proteasome, a high-molecular-mass protease subunit complex, and the protein portion is degraded by the 26S proteasome. The potential of specific proteasome inhibitors, which act as anti-cancer agents, is now under intensive investigation, and bortezomib (PS-341), a proteasome inhibitor, has been recently approved by FDA for multiple myeloma treatment. Since ubiquitination of proteins requires the sequential action of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3), and polyubiquitination is a prerequisite for proteasome-mediated protein degradation, inhibitors of E1, E2, and E3 are reasonably thought to be drug candidates for treatment of diseases related to ubiquitination. Recently, various compounds inhibiting the ubiquitin-proteasome pathway have been isolated from natural resources. We also succeeded in isolating inhibitors against the proteasome and E1 enzyme from marine natural resources. In this review, we summarize the structures and biological activities of natural products that inhibit the ubiquitin-proteasome proteolytic pathway.

  6. Toying with Enzyme Catalysis.

    ERIC Educational Resources Information Center

    Richards, Debbie

    1998-01-01

    Describes a set of manipulatives that are used to establish a secure understanding of the concepts related to the environmental factors that affect the activities of enzymes. Includes a description of the model components and procedures for construction of the model. (DDR)

  7. Enzyme leaps fuel antichemotaxis

    PubMed Central

    Jee, Ah-Young; Dutta, Sandipan; Cho, Yoon-Kyoung

    2018-01-01

    There is mounting evidence that enzyme diffusivity is enhanced when the enzyme is catalytically active. Here, using superresolution microscopy [stimulated emission-depletion fluorescence correlation spectroscopy (STED-FCS)], we show that active enzymes migrate spontaneously in the direction of lower substrate concentration (“antichemotaxis”) by a process analogous to the run-and-tumble foraging strategy of swimming microorganisms and our theory quantifies the mechanism. The two enzymes studied, urease and acetylcholinesterase, display two families of transit times through subdiffraction-sized focus spots, a diffusive mode and a ballistic mode, and the latter transit time is close to the inverse rate of catalytic turnover. This biochemical information-processing algorithm may be useful to design synthetic self-propelled swimmers and nanoparticles relevant to active materials. Executed by molecules lacking the decision-making circuitry of microorganisms, antichemotaxis by this run-and-tumble process offers the biological function to homogenize product concentration, which could be significant in situations when the reactant concentration varies from spot to spot. PMID:29255047

  8. Embedded enzymes catalyse capture

    NASA Astrophysics Data System (ADS)

    Kentish, Sandra

    2018-05-01

    Membrane technologies for carbon capture can offer economic and environmental advantages over conventional amine-based absorption, but can suffer from limited gas flux and selectivity to CO2. Now, a membrane based on enzymes embedded in hydrophilic pores is shown to exhibit combined flux and selectivity that challenges the state of the art.

  9. Cold-Adapted Enzymes

    NASA Astrophysics Data System (ADS)

    Georlette, D.; Bentahir, M.; Claverie, P.; Collins, T.; D'amico, S.; Delille, D.; Feller, G.; Gratia, E.; Hoyoux, A.; Lonhienne, T.; Meuwis, M.-a.; Zecchinon, L.; Gerday, Ch.

    In the last few years, increased attention has been focused on enzymes produced by cold-adapted micro-organisms. It has emerged that psychrophilic enzymes represent an extremely powerful tool in both protein folding investigations and for biotechnological purposes. Such enzymes are characterised by an increased thermosensitivity and, most of them, by a higher catalytic efficiency at low and moderate temperatures, when compared to their mesophilic counterparts. The high thermosensitivity probably originates from an increased flexibility of either a selected area of the molecular edifice or the overall protein structure, providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Structure modelling and recent crystallographic data have allowed to elucidate the structural parameters that could be involved in this higher resilience. It was demonstrated that each psychrophilic enzyme adopts its own adaptive strategy. It appears, moreover, that there is a continuum in the strategy of protein adaptation to temperature, as the previously mentioned structural parameters are implicated in the stability of thermophilic proteins. Additional 3D crystal structures, site-directed and random mutagenesis experiments should now be undertaken to further investigate the stability-flexibility-activity relationship.

  10. Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen.

    PubMed

    Kong, Leopold; Lee, David E; Kadam, Rameshwar U; Liu, Tong; Giang, Erick; Nieusma, Travis; Garces, Fernando; Tzarum, Netanel; Woods, Virgil L; Ward, Andrew B; Li, Sheng; Wilson, Ian A; Law, Mansun

    2016-10-24

    Hepatitis C virus (HCV) is a major cause of liver disease, affecting over 2% of the world's population. The HCV envelope glycoproteins E1 and E2 mediate viral entry, with E2 being the main target of neutralizing antibody responses. Structural investigations of E2 have produced templates for vaccine design, including the conserved CD81 receptor-binding site (CD81bs) that is a key target of broadly neutralizing antibodies (bNAbs). Unfortunately, immunization with recombinant E2 and E1E2 rarely elicits sufficient levels of bNAbs for protection. To understand the challenges for eliciting bNAb responses against the CD81bs, we investigated the E2 CD81bs by electron microscopy (EM), hydrogen-deuterium exchange (HDX), molecular dynamics (MD), and calorimetry. By EM, we observed that HCV1, a bNAb recognizing the N-terminal region of the CD81bs, bound a soluble E2 core construct from multiple angles of approach, suggesting components of the CD81bs are flexible. HDX of multiple E2 constructs consistently indicated the entire CD81bs was flexible relative to the rest of the E2 protein, which was further confirmed by MD simulations. However, E2 has a high melting temperature of 84.8 °C, which is more akin to proteins from thermophilic organisms. Thus, recombinant E2 is a highly stable protein overall, but with an exceptionally flexible CD81bs. Such flexibility may promote induction of nonneutralizing antibodies over bNAbs to E2 CD81bs, underscoring the necessity of rigidifying this antigenic region as a target for rational vaccine design.

  11. Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1

    PubMed Central

    Zheng, Shunsheng; Moehlenbrink, Jutta; Lu, Yi-Chien; Zalmas, Lykourgos-Panagiotis; Sagum, Cari A.; Carr, Simon; McGouran, Joanna F.; Alexander, Leila; Fedorov, Oleg; Munro, Shonagh; Kessler, Benedikt; Bedford, Mark T.; Yu, Qiang; La Thangue, Nicholas B.

    2014-01-01

    Summary The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase (PRMT) 1 and symmetric dimethylating PRMT5, and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favours proliferation by antagonising methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN down-regulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity. PMID:24076217

  12. E2F1 transcription factor and its impact on growth factor and cytokine signaling.

    PubMed

    Ertosun, Mustafa Gokhan; Hapil, Fatma Zehra; Osman Nidai, Ozes

    2016-10-01

    E2F1 is a transcription factor involved in cell cycle regulation and apoptosis. The transactivation capacity of E2F1 is regulated by pRb. In its hypophosphorylated form, pRb binds and inactivates DNA binding and transactivating functions of E2F1. The growth factor stimulation of cells leads to activation of CDKs (cyclin dependent kinases), which in turn phosphorylate Rb and hyperphosphorylated Rb is released from E2F1 or E2F1/DP complex, and free E2F1 can induce transcription of several genes involved in cell cycle entry, induction or inhibition of apoptosis. Thus, growth factors and cytokines generally utilize E2F1 to direct cells to either fate. Furthermore, E2F1 regulates expressions of various cytokines and growth factor receptors, establishing positive or negative feedback mechanisms. This review focuses on the relationship between E2F1 transcription factor and cytokines (IL-1, IL-2, IL-3, IL-6, TGF-beta, G-CSF, LIF), growth factors (EGF, KGF, VEGF, IGF, FGF, PDGF, HGF, NGF), and interferons (IFN-α, IFN-β and IFN-γ). Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. NF-E2 Overexpression Delays Erythroid Maturation and Increases Erythrocyte Production

    PubMed Central

    Mutschler, Manuel; Magin, Angela S.; Buerge, Martina; Roelz, Roland; Schanne, Daniel H.; Will, Britta; Pilz, Ingo H.; Migliaccio, Anna Rita; Pahl, Heike L.

    2009-01-01

    Summary The transcription factor Nuclear Factor-Erythroid 2 (NF-E2) is overexpressed in the vast majority of patients with polycythaemia vera (PV). In murine models, NF-E2 overexpression increases proliferation and promotes cellular viability in the absence of erythropoietin (EPO). EPO-independent growth is a hallmark of PV. We therefore hypothesized that NF-E2 overexpression contributes to erythrocytosis, the pathognomonic feature of PV. Consequently, we investigated the effect of NF-E2 overexpression in healthy CD34+ cells. NF-E2 overexpression led to a delay in erythroid maturation, manifested by a belated appearance of glycophorin A-positive erythroid precursors. Maturation delay was similarly observed in primary PV patient erythroid cultures compared to healthy controls. Protracted maturation led to a significant increase in the accumulated number of erythroid cells both in PV cultures and in CD34+ cells overexpressing NF-E2. Similarly, NF-E2 overexpression altered erythroid colony formation, leading to an increase in BFU-E formation. These data indicate that NF-E2 overexpression delays the early phase of erythroid maturation, resulting in an expansion of erythroid progenitors, thereby increasing the number of erythrocytes derived from one CD34+ cell. These data propose a role for NF-E2 in mediating the erythrocytosis of PV. PMID:19466964

  14. The Enzyme Function Initiative†

    PubMed Central

    Gerlt, John A.; Allen, Karen N.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.; Cronan, John E.; Dunaway-Mariano, Debra; Imker, Heidi J.; Jacobson, Matthew P.; Minor, Wladek; Poulter, C. Dale; Raushel, Frank M.; Sali, Andrej; Shoichet, Brian K.; Sweedler, Jonathan V.

    2011-01-01

    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts. PMID

  15. The Enzyme Function Initiative.

    PubMed

    Gerlt, John A; Allen, Karen N; Almo, Steven C; Armstrong, Richard N; Babbitt, Patricia C; Cronan, John E; Dunaway-Mariano, Debra; Imker, Heidi J; Jacobson, Matthew P; Minor, Wladek; Poulter, C Dale; Raushel, Frank M; Sali, Andrej; Shoichet, Brian K; Sweedler, Jonathan V

    2011-11-22

    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include (1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation), (2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia, (3) computational and bioinformatic tools for using the strategy, (4) provision of experimental protocols and/or reagents for enzyme production and characterization, and (5) dissemination of data via the EFI's Website, http://enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal, and pharmaceutical efforts.

  16. Dual Functions of α-Ketoglutarate Dehydrogenase E2 in the Krebs Cycle and Mitochondrial DNA Inheritance in Trypanosoma brucei

    PubMed Central

    Sykes, Steven E.

    2013-01-01

    The dihydrolipoyl succinyltransferase (E2) of the multisubunit α-ketoglutarate dehydrogenase complex (α-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional α-KD, bloodstream form (BF) trypanosomes express α-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, α-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for α-KDE2 in kDNA maintenance was revealed in α-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that α-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis. PMID:23125353

  17. Dual functions of α-ketoglutarate dehydrogenase E2 in the Krebs cycle and mitochondrial DNA inheritance in Trypanosoma brucei.

    PubMed

    Sykes, Steven E; Hajduk, Stephen L

    2013-01-01

    The dihydrolipoyl succinyltransferase (E2) of the multisubunit α-ketoglutarate dehydrogenase complex (α-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional α-KD, bloodstream form (BF) trypanosomes express α-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, α-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for α-KDE2 in kDNA maintenance was revealed in α-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that α-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis.

  18. Efficient activation of transcription in yeast by the BPV1 E2 protein.

    PubMed Central

    Stanway, C A; Sowden, M P; Wilson, L E; Kingsman, A J; Kingsman, S M

    1989-01-01

    The full-length gene product encoded by the E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV1) is a transcriptional transactivator. It is believed to mediate its effect on the BPV1 long control region (LCR) by binding to motifs with the consensus sequence ACCN6GGT. The minimal functional cis active site, called the E2 response element (E2RE), in mammalian cells comprises two copies of this motif. Here we have shown that E2 can function in Saccharomyces cerevisiae by placing an E2RE upstream of a synthetic yeast assay promoter which consists of a TATA motif and an mRNA initiation site, spaced correctly. This E2RE-minimal promoter is only transcriptionally active in the presence of E2 protein and the resulting mRNA is initiated at the authentic start site. This is the first report of a mammalian viral transactivator functioning in yeast. The level of activation by E2 via the E2RE was the same as observed with the highly efficient authentic PGK promoter where the upstream activation sequence is composed of three distinct elements. Furthermore a single E2 motif which is insufficient in mammalian cells as an activation site was as efficiently utilized in yeast as the E2RE (2 motifs). Previous studies have shown that mammalian cellular activators can function in yeast and our data now extend this to viral-specific activators. Our data indicate however that while the mechanism of transactivation is broadly conserved there may be significant differences at the detailed level. Images PMID:2539584

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

    PubMed

    Xanthoulis, Athanasios; Tiniakos, Dina G

    2013-06-07

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

  20. Enzyme nanoparticle fabrication: magnetic nanoparticle synthesis and enzyme immobilization.

    PubMed

    Johnson, Patrick A; Park, Hee Joon; Driscoll, Ashley J

    2011-01-01

    Immobilized enzymes are drawing significant attention for potential commercial applications as biocatalysts by reducing operational expenses and by increasing process utilization of the enzymes. Typically, immobilized enzymes have greater thermal and operational stability at various pH values, ionic strengths and are more resistant to denaturation that the soluble native form of the enzyme. Also, immobilized enzymes can be recycled by utilizing the physical or chemical properties of the supporting material. Magnetic nanoparticles provide advantages as the supporting material for immobilized enzymes over competing materials such as: higher surface area that allows for greater enzyme loading, lower mass transfer resistance, less fouling effect, and selective, nonchemical separation from the reaction mixture by an applied a magnetic field. Various surface modifications of magnetic nanoparticles, such as silanization, carbodiimide activation, and PEG or PVA spacing, aid in the binding of single or multienzyme systems to the particles, while cross-linking using glutaraldehyde can also stabilize the attached enzymes.

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

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... variable life insurance separate accounts. 270.6e-2 Section 270.6e-2 Commodity and Securities Exchanges...-2 Exemptions for certain variable life insurance separate accounts. (a) A separate account, and the... a life insurance company pursuant to the insurance laws or code of (i) any state or territory of the...

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

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... variable life insurance separate accounts. 270.6e-2 Section 270.6e-2 Commodity and Securities Exchanges...-2 Exemptions for certain variable life insurance separate accounts. (a) A separate account, and the... a life insurance company pursuant to the insurance laws or code of (i) any state or territory of the...

  3. 17 CFR 270.6e-2 - Exemptions for certain variable life insurance separate accounts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... variable life insurance separate accounts. 270.6e-2 Section 270.6e-2 Commodity and Securities Exchanges...-2 Exemptions for certain variable life insurance separate accounts. (a) A separate account, and the... a life insurance company pursuant to the insurance laws or code of (i) any state or territory of the...

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

    SciTech Connect

    Chang, Szu-Wei; Lu, Pei-Yu; Guo, Jih-Huong

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

  5. New perspective of Grodzins E × B(E2) ↑ product rule

    NASA Astrophysics Data System (ADS)

    Gupta, J. B.; Katoch, Vikas

    In the collective spectra of atomic nuclei, the level energy E(21+) varies with atomic number Z and neutron number N. Also the E2 decay-reduced transition probability B(E2, 01+ → 2 1+) is related to the energy E(21+). The product E(21+) × B(E2) ↑ is constant according to Grodzins product rule, independent of the vibration or rotational status of the nucleus. The product rule is often used for determining B(E2) from the known E(21+). However, the variation of the product with various parameters is also suggested in the literature. Hence, a detailed global study of this rule for (Z = 54‑‑78, 66 < N < 126) region is warranted. We use a novel method of displaying the linear relation of B(E2) ↑ with 1/E(21+) for the isotopes of each element (Xe-Pt), instead of their variation with N,Z or A. Through our work, we firmly establish the global validity of the Grodzins relation of B(E2), being proportional to the moment of inertia, except for the deviation in specific cases. Our B(E2) ↑ versus 1/E plots provide a transparent view of the variation of the low-energy nuclear structure. This gives a new perspective of their nuclear structure. Also the various theoretical interpretations of B(E2)s and the energy E(21+) are reviewed.

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

    NASA Astrophysics Data System (ADS)

    Singh, Nitu; Senapati, Sanjib; Bose, Kakoli

    2016-02-01

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

  7. Synthetic Helizyme Enzymes.

    DTIC Science & Technology

    1987-08-18

    NOTATION 17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number) FIELD GROUP SUB-GROUP I Synthetic enzymes...chymotrypsin; molecular modeling; 03 peptide synthesis 19. ABSTRACT (Continue on reverse if necessary and identify by block number) The object of this...for AChE. Additionally, synthetic models ofcL- chymotrypsin built using cyclo- dextrins show catalytic activity over a limited pH range.2 Using L

  8. Amperometric Enzyme Electrodes

    DTIC Science & Technology

    1989-12-01

    form of carbon (glascy carbon , graphite, reticulated vitreous carbon , carbon paste, fiber or foil). Carbon is favored for enzyme immoblization...the surface for covalent bonding. The most frequently used electrode material, glassy carbon , often displays complex behavior. Although attempts have...Mixed Carbon Paste Electrode with an Immobilized Layer of D-Gluconate Dehydrogenase from Bacteral Membranes," Agric. Biol. Chelm., 51 (1987), 747-754

  9. Characterization of a new chimeric marker vaccine candidate with a mutated antigenic E2-epitope.

    PubMed

    Reimann, Ilona; Depner, Klaus; Utke, Katrin; Leifer, Immanuel; Lange, Elke; Beer, Martin

    2010-04-21

    A new chimeric pestivirus "CP7_E1E2alf_TLA", based on the infectious cDNA of bovine viral diarrhea virus (BVDV) strain CP7, was constructed. The substitution of BVDV E1 and E2 with the respective proteins of classical swine fever (CSF) strain Alfort 187 allows an optimal heterodimerization of E1 and E2 in the chimeric virus, which is beneficial for efficient and authentic virus assembly and growth. In addition, for implementation of E2-based marker diagnostics, the previously described antigenic CSFV-specific TAVSPTTLR epitope was exchanged with the corresponding E2-epitope of BVDV strain CP7. Recombinant virus CP7_E1E2alf_TLA displayed a growth defect, and was not reacting with monoclonal antibodies used in commercial E2 antibody blocking ELISAs. Therefore, efficacy as well as marker properties of CP7_E1E2alf_TLA were investigated in an animal experiment with both a high dose and a low dose vaccine preparation. All CP7_E1E2alf_TLA-vaccinated animals seroconverted until day 28 post-vaccination with neutralizing antibodies. Furthermore, at the day of challenge infection CP7_E1E2alf_TLA-immunized animals showed distinct lower ELISA values in a commercial CSFV E2 antibody test in comparison to the C-strain vaccinated controls. However, E2-ELISA reactivity as well as neutralizing titers were directly connected to the dosage used for vaccination, and only the low dose group had E2-ELISA values below threshold until challenge infection. Following challenge infection with highly virulent CSFV strain Koslov, all vaccinees were protected, however, short-term fever episodes and very limited CSFV genome detection with very low copy numbers could be observed. In conclusion, manipulation of the TAVSPTTLR-epitope within the tested chimeric virus resulted in an slightly reduced efficacy, but the E2 marker properties unexpectedly did not allow a clear differentiation of infected from vaccinated animals in some cases. Copyright 2009 Elsevier B.V. All rights reserved.

  10. Uronic polysaccharide degrading enzymes.

    PubMed

    Garron, Marie-Line; Cygler, Miroslaw

    2014-10-01

    In the past several years progress has been made in the field of structure and function of polysaccharide lyases (PLs). The number of classified polysaccharide lyase families has increased to 23 and more detailed analysis has allowed the identification of more closely related subfamilies, leading to stronger correlation between each subfamily and a unique substrate. The number of as yet unclassified polysaccharide lyases has also increased and we expect that sequencing projects will allow many of these unclassified sequences to emerge as new families. The progress in structural analysis of PLs has led to having at least one representative structure for each of the families and for two unclassified enzymes. The newly determined structures have folds observed previously in other PL families and their catalytic mechanisms follow either metal-assisted or Tyr/His mechanisms characteristic for other PL enzymes. Comparison of PLs with glycoside hydrolases (GHs) shows several folds common to both classes but only for the β-helix fold is there strong indication of divergent evolution from a common ancestor. Analysis of bacterial genomes identified gene clusters containing multiple polysaccharide cleaving enzymes, the Polysaccharides Utilization Loci (PULs), and their gene complement suggests that they are organized to process completely a specific polysaccharide. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Micellar Polymer Encapsulation of Enzymes.

    PubMed

    Besic, Sabina; Minteer, Shelley D

    2017-01-01

    Although enzymes are highly efficient and selective catalysts, there have been problems incorporating them into fuel cells. Early enzyme-based fuel cells contained enzymes in solution rather than immobilized on the electrode surface. One problem utilizing an enzyme in solution is an issue of transport associated with long diffusion lengths between the site of bioelectrocatalysis and the electrode. This issue drastically decreases the theoretical overall power output due to the poor electron conductivity. On the other hand, enzymes immobilized at the electrode surface have eliminated the issue of poor electron conduction due to close proximity of electron transfer between electrode and the biocatalyst. Another problem is inefficient and short term stability of catalytic activity within the enzyme that is suspended in free flowing solution. Enzymes in solutions are only stable for hours to days, whereas immobilized enzymes can be stable for weeks to months and now even years. Over the last decade, there has been substantial research on immobilizing enzymes at electrode surfaces for biofuel cell and sensor applications. The most commonly used techniques are sandwich or wired. Sandwich techniques are powerful and successful for enzyme immobilization; however, the enzymes optimal activity is not retained due to the physical distress applied by the polymer limiting its applications as well as the non-uniform distribution of the enzyme and the diffusion of analyte through the polymer is slowed significantly. Wired techniques have shown to extend the lifetime of an enzyme at the electrode surface; however, this technique is very hard to master due to specific covalent bonding of enzyme and polymer which changes the three-dimensional configuration of enzyme and with that decreases the optimal catalytic activity. This chapter details encapsulation techniques where an enzyme will be immobilized within the pores/pockets of the hydrophobically modified micellar polymers such as

  12. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-11-25

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups. 19 figs.

  13. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, Matthew R.; Bednarski, Mark D.; Gruber, Patrick R.

    1997-01-01

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups.

  14. Competing E2 and SN2 Mechanisms for the F- + CH3CH2I Reaction.

    PubMed

    Yang, Li; Zhang, Jiaxu; Xie, Jing; Ma, Xinyou; Zhang, Linyao; Zhao, Chenyang; Hase, William L

    2017-02-09

    Anti-E2, syn-E2, inv-, and ret-S N 2 reaction channels for the gas-phase reaction of F - + CH 3 CH 2 I were characterized with a variety of electronic structure calculations. Geometrical analysis confirmed synchronous E2-type transition states for the elimination of the current reaction, instead of nonconcerted processes through E1cb-like and E1-like mechanisms. Importantly, the controversy concerning the reactant complex for anti-E2 and inv-S N 2 paths has been clarified in the present work. A positive barrier of +19.2 kcal/mol for ret-S N 2 shows the least feasibility to occur at room temperature. Negative activation energies (-16.9, -16.0, and -4.9 kcal/mol, respectively) for inv-S N 2, anti-E2, and syn-E2 indicate that inv-S N 2 and anti-E2 mechanisms significantly prevail over the eclipsed elimination. Varying the leaving group for a series of reactions F - + CH 3 CH 2 Y (Y = F, Cl, Br, and I) leads to monotonically decreasing barriers, which relates to the gradually looser TS structures following the order F > Cl > Br > I. The reactivity of each channel nearly holds unchanged except for the perturbation between anti-E2 and inv-S N 2. RRKM calculation reveals that the reaction of the fluorine ion with ethyl iodide occurs predominately via anti-E2 elimination, and the inv-S N 2 pathway is suppressed, although it is energetically favored. This phenomenon indicates that, in evaluating the competition between E2 and S N 2 processes, the kinetic or dynamical factors may play a significant role. By comparison with benchmark CCSD(T) energies, MP2, CAM-B3LYP, and M06 methods are recommended to perform dynamics simulations of the title reaction.

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

    SciTech Connect

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

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

  16. How alkyl halide structure affects E2 and SN2 reaction barriers: E2 reactions are as sensitive as SN2 reactions.

    PubMed

    Rablen, Paul R; McLarney, Brett D; Karlow, Brandon J; Schneider, Jean E

    2014-02-07

    High-level electronic structure calculations, including a continuum treatment of solvent, are employed to elucidate and quantify the effects of alkyl halide structure on the barriers of SN2 and E2 reactions. In cases where such comparisons are available, the results of these calculations show close agreement with solution experimental data. Structural factors investigated include α- and β-methylation, adjacency to unsaturated functionality (allyl, benzyl, propargyl, α to carbonyl), ring size, and α-halogenation and cyanation. While the influence of these factors on SN2 reactivity is mostly well-known, the present study attempts to provide a broad comparison of both SN2 and E2 reactivity across many cases using a single methodology, so as to quantify relative reactivity trends. Despite the fact that most organic chemistry textbooks say far more about how structure affects SN2 reactions than about how it affects E2 reactions, the latter are just as sensitive to structural variation as are the former. This sensitivity of E2 reactions to structure is often underappreciated.

  17. Treating Wastewater With Immobilized Enzymes

    NASA Technical Reports Server (NTRS)

    Jolly, Clifford D.

    1991-01-01

    Experiments show enzymes are immobilized on supporting materials to make biocatalyst beds for treatment of wastewater. With suitable combination of enzymes, concentrations of various inorganic and organic contaminants, including ammonia and urea, reduced significantly.

  18. SciTech Connect

    Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.

    DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

  19. Aerobic batch degradation of 17-beta estradiol (E2) by activated sludge: effects of spiking E2 concentrations, MLVSS and temperatures.

    PubMed

    Li, Fusheng; Yuasa, Akira; Obara, Aya; Mathews, Alexander P

    2005-05-01

    Aerobic batch degradation of 17beta estradiol (E2) spiked into the activated sludge liquor from a sewage treatment plant was studied; and the likely impacts of E2's initial concentrations (C0), microbial population densities (MLVSS) and temperatures (TEMPT) were examined for a variety of combinations of these three factors: C0 = 10, 30 and 50 microgl(-1); MLVSS = 1750, 875 and 435 mgl(-1); and TEMPT = 5, 20 and 35 degrees C. The results, together with those obtained through two control runs performed using a killed sludge sample, demonstrated clearly that E2 was eliminated from the aqueous phase readily under appropriate MLVSS and temperature levels, with the role of sorption by biomass being less significant. By fitting observed concentration data with a first-order rate expression, the degradation rate constants (k) under all experimental conditions were estimated. The magnitude of k changed markedly in the range of 0.23-4.79 h(-1), following a general order that the higher the MLVSS was, the higher the rate constant, and that the higher the temperature, the higher the rate constant. An obvious increasing trend of the biomass-modified average rate constant (k') with increases in the temperature was also presented: the k' values at 5, 20 and 35 degrees C were 0.79, 1.77 and 3.29l MLVSS g(-1)h(-1), respectively. Furthermore, based upon the estimated k values, the temperature coefficients (theta) over the ranges of 5-20 and 20-35 degrees C were determined. In similarity with the magnitude of theta reported for ordinary BOD-based organic matrices in domestic wastewater, the theta values of E2 varied in the range of 1.026-1.09, suggesting that the temperature impacts on the degradation rates of E2 and BOD constituents are probably similar.

  20. Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats

    PubMed Central

    Lecker, Stewart H.; Solomon, Vered; Price, S. Russ; Kwon, Yong Tae; Mitch, William E.; Goldberg, Alfred L.

    1999-01-01

    Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40–50% greater rates of conjugation of 125I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E214k and E3α. A specific substrate of this pathway, α-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E214k inhibited this increase in ubiquitination rates. Both E214k and E3α were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E214k and E3α (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E214k and E3α content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E214k and E3α in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism. J. Clin. Invest. 104:1411–1420 (1999). PMID:10562303

  1. The Catalytic Function of Enzymes.

    ERIC Educational Resources Information Center

    Splittgerber, Allan G.

    1985-01-01

    Discusses: structure of the enzyme molecule; active site; reaction mechanism; transition state; factors affecting enzyme reaction rates, concentration of enzyme; concentration of substrate; product concentration; temperature effects and pH effects; factors causing a lowering of activation energy; proximity and orientation effects; substrate strain…

  2. Repression of transcriptional activity of C/EBPalpha by E2F-dimerization partner complexes.

    PubMed

    Zaragoza, Katrin; Bégay, Valérie; Schuetz, Anja; Heinemann, Udo; Leutz, Achim

    2010-05-01

    The transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) coordinates proliferation arrest and the differentiation of myeloid progenitors, adipocytes, hepatocytes, keratinocytes, and cells of the lung and placenta. C/EBPalpha transactivates lineage-specific differentiation genes and inhibits proliferation by repressing E2F-regulated genes. The myeloproliferative C/EBPalpha BRM2 mutant serves as a paradigm for recurrent human C-terminal bZIP C/EBPalpha mutations that are involved in acute myeloid leukemogenesis. BRM2 fails to repress E2F and to induce adipogenesis and granulopoiesis. The data presented here show that, independently of pocket proteins, C/EBPalpha interacts with the dimerization partner (DP) of E2F and that C/EBPalpha-E2F/DP interaction prevents both binding of C/EBPalpha to its cognate sites on DNA and transactivation of C/EBP target genes. The BRM2 mutant, in addition, exhibits enhanced interaction with E2F-DP and reduced affinity toward DNA and yet retains transactivation potential and differentiation competence that becomes exposed when E2F/DP levels are low. Our data suggest a tripartite balance between C/EBPalpha, E2F/DP, and pocket proteins in the control of proliferation, differentiation, and tumorigenesis.

  3. SciTech Connect

    Jeon, En Hee; Pak, Jung Hun; Kim, Mi Jin

    Highlights: Black-Right-Pointing-Pointer We isolated a novel E2 ubiquitin-conjugating enzyme from leaves of wild rice plants. Black-Right-Pointing-Pointer The OgUBC1 was highly expressed in leaves treated with SA and UV-B radiation. Black-Right-Pointing-Pointer The recombinant OgUBC1 has an enzymatic activity of E2 in vitro. Black-Right-Pointing-Pointer The OgUBC1 could protect disruption of plant cells by UV-B radiation. Black-Right-Pointing-Pointer OgUBC1 confers disease resistance and UV-B tolerance in transgenic Arabidopsis plants. -- Abstract: A previously unidentified gene encoding ubiquitin-conjugating enzyme was isolated from leaves of wild rice plant treated with wounding and microbe-associated molecular patterns. The OgUBC1 gene was composed of 148 amino acids and containedmore » a typical active site and 21 ubiquitin thioester intermediate interaction residues and 4 E3 interaction residues. Both exogenous application of salicylic acid and UV-B irradiation triggered expression of OgUBC1 in leaves of wild rice. Recombinant OgUBC1 proteins bound to ubiquitins in vitro, proposing that the protein might act as E2 enzyme in planta. Heterologous expression of the OgUBC1 in Arabidopsis thaliana protected plants from cellular damage caused by an excess of UV-B radiation. A stable expression of chalcone synthase gene was detected in leaves of OgUBC1-expressing Arabidopsis, resulting in producing higher amounts of anthocyanin than those in wild-type Col-0 plants. Additionally, both pathogenesis-related gene1 and 5 were transcribed in the transgenic Arabidopsis in the absence of pathogen infection. The OgUBC1-expressing plants were resistant to the infection of Botrytis cinerea. Taken together, we suggested that the OgUBC1 is involved in ubiquitination process important for cellular response against biotic and abiotic stresses in plants.« less

  4. Forward genetic screens identify a role for the mitochondrial HER2 in E-2-hexenal responsiveness.

    PubMed

    Scala, Alessandra; Mirabella, Rossana; Goedhart, Joachim; de Vries, Michel; Haring, Michel A; Schuurink, Robert C

    2017-11-01

    This work adds a new player, HER2, downstream of the perception of E-2-hexenal, a green leaf volatile, and shows that E-2-hexenal specifically changes the redox status of the mitochondria. It is widely accepted that plants produce and respond to green leaf volatiles (GLVs), but the molecular components involved in transducing their perception are largely unknown. The GLV E-2-hexenal inhibits root elongation in seedlings and, using this phenotype, we isolated E-2-hexenal response (her) Arabidopsis thaliana mutants. Using map-based cloning we positioned the her2 mutation to the At5g63620 locus, resulting in a phenylalanine instead of serine on position 223. Knockdown and overexpression lines of HER2 confirmed the role of HER2, which encodes an oxidoreductase, in the responsiveness to E-2-hexenal. Since E-2-hexenal is a reactive electrophile species, which are known to influence the redox status of cells, we utilized redox sensitive GFP2 (roGFP2) to determine the redox status of E-2-hexenal-treated root cells. Since the signal peptide of HER2 directed mCherry to the mitochondria, we targeted the expression of roGFP2 to this organelle besides the cytosol. E-2-hexenal specifically induced a change in the redox status in the mitochondria. We did not see a difference in the redox status in her2 compared to wild-type Arabidopsis. Still, the mitochondrial redox status did not change with Z-3-hexenol, another abundant GLV. These results indicate that HER2 is involved in transducing the perception of E-2-hexenal, which changes the redox status of the mitochondria.

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

    PubMed

    Evangelou, Konstantinos; Havaki, Sophia; Kotsinas, Athanassios

    2014-08-07

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-07-01

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

  7. Copy number variations of E2F1: a new genetic risk factor for testicular cancer.

    PubMed

    Rocca, Maria Santa; Di Nisio, Andrea; Marchiori, Arianna; Ghezzi, Marco; Opocher, Giuseppe; Foresta, Carlo; Ferlin, Alberto

    2017-03-01

    Testicular germ cell tumor (TGCT) is one of the most heritable forms of cancer. In last years, many evidence suggested that constitutional genetic factors, mainly single nucleotide polymorphisms, can increase its risk. However, the possible contribution of copy number variations (CNVs) in TGCT susceptibility has not been substantially addressed. Indeed, an increasing number of studies have focused on the effect of CNVs on gene expression and on the role of these structural genetic variations as risk factors for different forms of cancer. E2F1 is a transcription factor that plays an important role in regulating cell growth, differentiation, apoptosis and response to DNA damage. Therefore, deficiency or overexpression of this protein might significantly influence fundamental biological processes involved in cancer development and progression, including TGCT. We analyzed E2F1 CNVs in 261 cases with TGCT and 165 controls. We found no CNVs in controls, but 17/261 (6.5%) cases showed duplications in E2F1 Blot analysis demonstrated higher E2F1 expression in testicular samples of TGCT cases with three copies of the gene. Furthermore, we observed higher phosphorylation of Akt and mTOR in samples with E2F1 duplication. Interestingly, normal, non-tumoral testicular tissue in patient with E2F1 duplication showed lower expression of E2F1 and lower AKT/mTOR phosphorylation with respect to adjacent tumor tissue. Furthermore, increased expression of E2F1 obtained in vitro in NTERA-2 testicular cell line induced increased AKT/mTOR phosphorylation. This study suggests for the first time an involvement of E2F1 CNVs in TGCT susceptibility and supports previous preliminary data on the importance of AKT/mTOR signaling pathway in this cancer. © 2017 Society for Endocrinology.

  8. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  9. Estradiol-17beta, prostaglandin E2 (PGE2), and the PGE2 receptor are involved in PGE2 positive feedback loop in the porcine endometrium.

    PubMed

    Waclawik, Agnieszka; Jabbour, Henry N; Blitek, Agnieszka; Ziecik, Adam J

    2009-08-01

    Before implantation, the porcine endometrium and trophoblast synthesize elevated amounts of luteoprotective prostaglandin estradiol-17beta (E(2)) (PGE(2)). We hypothesized that embryo signal, E(2), and PGE(2) modulate expression of key enzymes in PG synthesis: PG-endoperoxide synthase-2 (PTGS2), microsomal PGE synthase (mPGES-1), PGF synthase (PGFS), and PG 9-ketoreductase (CBR1) as well as PGE(2) receptor (PTGER2 and -4) expression and signaling within the endometrium. We determined the site of action of PGE(2) in endometrium during the estrous cycle and pregnancy. Endometrial tissue explants obtained from gilts (n = 6) on d 11-12 of the estrous cycle were treated with vehicle (control), PGE(2) (100 nM), E(2) (1-100 nm), or phorbol 12-myristate 13-acetate (100 nm, positive control). E(2) increased PGE(2) secretion through elevating expression of mPGES-1 mRNA and PTGS2 and mPGES-1 protein in endometrial explants. By contrast, E(2) decreased PGFS and CBR1 protein expression. E(2) also stimulated PTGER2 but not PTGER4 protein content. PGE(2) enhanced mPGES-1 and PTGER2 mRNA as well as PTGS2, mPGES-1, and PTGER2 protein expression. PGE(2) had no effect on PGFS, CBR1, and PTGER4 expression and PGF(2alpha) release. Treatment of endometrial tissue with PGE(2) increased cAMP production. Cotreatment with PTGER2 antagonist (AH6809) but not PTGER4 antagonist (GW 627368X) inhibited significantly PGE(2)-mediated cAMP production. PTGER2 protein was localized in luminal and glandular epithelium and blood vessels of endometrium and was significantly up-regulated on d 11-12 of pregnancy. Our results suggest that E(2) prevents luteolysis through enzymatic modification of PG synthesis and that E(2), PGE(2), and endometrial PTGER2 are involved in a PGE(2) positive feedback loop in porcine endometrium.

  10. The enzymic hydrolysis of amygdalin

    PubMed Central

    Haisman, D. R.; Knight, D. J.

    1967-01-01

    Chromatographic examination has shown that the enzymic hydrolysis of amygdalin by an almond β-glucosidase preparation proceeds consecutively: amygdalin was hydrolysed to prunasin and glucose; prunasin to mandelonitrile and glucose; mandelonitrile to benzaldehyde and hydrocyanic acid. Gentiobiose was not formed during the enzymic hydrolysis. The kinetics of the production of mandelonitrile and hydrocyanic acid from amygdalin by the action of the β-glucosidase preparation favour the probability that three different enzymes are involved, each specific for one hydrolytic stage, namely, amygdalin lyase, prunasin lyase and hydroxynitrile lyase. Cellulose acetate electrophoresis of the enzyme preparation showed that it contained a number of enzymically active components. PMID:4291788

  11. Synthetic, switchable enzymes

    PubMed Central

    Norris, Vic; Krylov, Sergey N.; Agarwal, Pratul K.; White, Glenn J.

    2017-01-01

    The construction of switchable, radiation-controlled, aptameric enzymes alias swenzymes is, in principle, feasible. We propose a strategy to make such catalysts from two (or more) aptamers each selected to bind specifically to one of the substrates in, for example, a two-substrate reaction. Construction of a combinatorial library of candidate swenzymes entails selecting a set of a million aptamers that bind one substrate and a second set of a million aptamers that bind the second substrate; the aptamers in these sets are then linked pairwise by a linker so bringing together the substrates. In the presence of the substrates, some linked aptamer pairs catalyze the reaction when exposed to external energy in the form of a specific frequency of low intensity, non-ionizing electromagnetic or acoustic radiation. Such swenzymes are detected via a separate, product-capturing, aptamer that changes conformation on capturing the product; this altered conformation allows it (1) to bind to every potential swenzyme in its vicinity (thereby giving a higher probability of capture to the swenzymes that generate the product) and (2) to bind to a sequence on a magnetic bead (thereby permitting purification of the swenzyme plus product-capturing aptamer by precipitation). Attempts to implement the swenzyme strategy may help elucidate fundamental problems in enzyme catalysis. PMID:28448969

  12. Synthetic, Switchable Enzymes.

    PubMed

    Norris, Vic; Krylov, Sergey N; Agarwal, Pratul K; White, Glenn J

    2017-01-01

    The construction of switchable, radiation-controlled, aptameric enzymes - "swenzymes" - is, in principle, feasible. We propose a strategy to make such catalysts from 2 (or more) aptamers each selected to bind specifically to one of the substrates in, for example, a 2-substrate reaction. Construction of a combinatorial library of candidate swenzymes entails selecting a set of a million aptamers that bind one substrate and a second set of a million aptamers that bind the second substrate; the aptamers in these sets are then linked pairwise by a linker, thus bringing together the substrates. In the presence of the substrates, some linked aptamer pairs catalyze the reaction when exposed to external energy in the form of a specific frequency of low-intensity, nonionizing electromagnetic or acoustic radiation. Such swenzymes are detected via a separate product-capturing aptamer that changes conformation on capturing the product; this altered conformation allows it (1) to bind to every potential swenzyme in its vicinity (thereby giving a higher probability of capture to the swenzymes that generate the product) and (2) to bind to a sequence on a magnetic bead (thereby permitting purification of the swenzyme plus product-capturing aptamer by precipitation). Attempts to implement the swenzyme strategy may help elucidate fundamental problems in enzyme catalysis. © 2017 S. Karger AG, Basel.

  13. Characterising Complex Enzyme Reaction Data

    PubMed Central

    Rahman, Syed Asad; Thornton, Janet M.

    2016-01-01

    The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution. PMID:26840640

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

    PubMed

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

    2015-11-10

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

  15. Enhancement of B(E2) Collectivity in the sdg-Interacting Boson Model

    NASA Astrophysics Data System (ADS)

    Long, GuiLu; Ji, HuaYing; Zhu, ShengJiang

    1999-12-01

    It is pointed out that the difficulty in boson models in explaining the large B(E2) experimental data is not due to the lack of collectivity in boson model wavefunction. This long standing problem of reduction in collectivity of wavefunction in boson models can be solved by choosing an appropriate E2 transition operator in the SU(3) limit of the sdg-interacting boson model. The ratio B(E2, L + 2 → L) / B(E2, 2 → 0) can be almost any large number. The project supported by The Science Fund of China Nuclear Industry under grant No. J95AY5021, National Natural Science Foundation of China under grant No. 19775026, Excellent Young University Teachers Fund of National Education Committee of China

  16. Novel functions for the transcription factor E2F4 in development and disease

    PubMed Central

    Sage, Julien

    2016-01-01

    ABSTRACT The E2F family of transcription factors is a key determinant of cell proliferation in response to extra- and intra-cellular signals. Within this family, E2F4 is a transcriptional repressor whose activity is critical to engage and maintain cell cycle arrest in G0/G1 in conjunction with members of the retinoblastoma (RB) family. However, recent observations challenge this paradigm and indicate that E2F4 has a multitude of functions in cells besides this cell cycle regulatory role, including in embryonic and adult stem cells, during regenerative processes, and in cancer. Some of these new functions are independent of the RB family and involve direct activation of target genes. Here we review the canonical functions of E2F4 and discuss recent evidence expanding the role of this transcription factor, with a focus on cell fate decisions in tissue homeostasis and regeneration. PMID:27753528

  17. Characterization of biosynthesis and modes of action of prostaglandin E2 and prostacyclin in guinea pig mesenteric lymphatic vessels.

    PubMed

    Rehal, Sonia; Blanckaert, Pauline; Roizes, Simon; von der Weid, Pierre-Yves

    2009-12-01

    Rhythmical transient constrictions of the lymphatic vessels provide the means for efficient lymph drainage and interstitial tissue fluid balance. This activity is critical during inflammation, to avoid or limit oedema resulting from increased vascular permeability, mediated by the release of various inflammatory mediators. In this study, we investigated the mechanisms by which prostaglandin E(2) (PGE(2)) and prostacyclin modulate lymphatic contractility in isolated guinea pig mesenteric lymphatic vessels. Quantitative RT-PCR was used to assess the expression of mRNA for enzymes and receptors involved in the production and action of PGE(2) and prostacyclin in mesenteric collecting lymphatic vessels. Frequency and amplitude of lymphatic vessel constriction were measured in the presence of these prostaglandins and the role of their respective EP and IP receptors assessed. Prostaglandin E(2) and prostacyclin decreased concentration-dependently the frequency, without affecting the amplitude, of lymphatic constriction. Data obtained in the presence of the EP(4) receptor antagonists, GW627368x (1 microM) and AH23848B (30 microM) and the IP receptor antagonist CAY10441 (0.1 microM) suggest that PGE(2) predominantly activates EP(4), whereas prostacyclin mainly stimulates IP receptors. Inhibition of responses to either prostaglandin with H89 (10 microM) or glibenclamide (1 microM) suggested a role for the activation of protein kinase A and ATP-sensitive K(+) channels. Our findings characterized the inhibition of lymphatic pumping induced by PGE(2) or prostacyclin in guinea pig mesenteric lymphatics. This action is likely to impair oedema resolution and to contribute to the pro-inflammatory actions of these prostaglandins.

  18. Put a RING on it: regulation and inhibition of RNF8 and RNF168 RING finger E3 ligases at DNA damage sites

    PubMed Central

    Bartocci, Cristina; Denchi, Eros Lazzerini

    2013-01-01

    RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligases comprise a large family of enzymes that in combination with an E2 ubiquitin-conjugating enzyme, modify target proteins by attaching ubiquitin moieties. A number of RING E3s play an essential role in the cellular response to DNA damage highlighting a crucial contribution for ubiquitin-mediated signaling to the genome surveillance pathway. Among the RING E3s, RNF8 and RNF168 play a critical role in the response to double stranded breaks, one of the most deleterious types of DNA damage. These proteins act as positive regulators of the signaling cascade that initiates at DNA lesions. Inactivation of these enzymes is sufficient to severely impair the ability of cells to respond to DNA damage. Given their central role in the pathway, several layers of regulation act at this nodal signaling point. Here we will summarize current knowledge on the roles of RNF8 and RNF168 in maintaining genome integrity with particular emphasis on recent insights into the multiple layers of regulation that act on these enzymes to fine-tune the cellular response to DNA lesions. PMID:23847653

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

    PubMed Central

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

    2014-01-01

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

  20. The B(E2;4^+1->2^+1) / B(E2;2^+1->0^+1) Ratio in Even-Even Nuclei

    NASA Astrophysics Data System (ADS)

    Loelius, C.; Sharon, Y. Y.; Zamick, L.; G"Urdal, G.

    2009-10-01

    We considered 207 even-even nuclei throughout the chart of nuclides for which the NNDC Tables had data on the energies and lifetimes of the 2^+1 and 4^+1 states. Using these data we calculated for each nucleus the electric quadrupole transition strengths B(E2;4^+1->2^+1) and B(E2;2^+1->0^+1), as well as their ratio. The internal conversion coefficients were obtained by using the NNDC HSICC calculator. For each nucleus we plotted the B(E2) ratio against A, N, and Z. We found that for close to 90% of the nuclei considered the ratio had values between 0.5 and 2.5. Most of the outliers had magic numbers of protons or neutrons. Our ratio results were compared with the theoretical predictions for this ratio by different models--10/7 in the rotational model and 2 in the simplest vibrational model. In the rotational regions (for 150 < A < 180 and A > 220) the ratios were indeed close to 10/7. For the few nuclei thought to be vibrational the ratios were usually less than 2. Otherwise, we got a wide scatter of ratio values. Hence other models, including the NpNn scheme, must be considered in interpreting these results.

  1. E2F1-mediated human POMC expression in ectopic Cushing's syndrome.

    PubMed

    Araki, Takako; Liu, Ning-Ai; Tone, Yukiko; Cuevas-Ramos, Daniel; Heltsley, Roy; Tone, Masahide; Melmed, Shlomo

    2016-11-01

    Cushing's syndrome is caused by excessive adrenocorticotropic hormone (ACTH) secretion derived from pituitary corticotroph tumors (Cushing disease) or from non-pituitary tumors (ectopic Cushing's syndrome). Hypercortisolemic features of ectopic Cushing's syndrome are severe, and no definitive treatment for paraneoplastic ACTH excess is available. We aimed to identify subcellular therapeutic targets by elucidating transcriptional regulation of the human ACTH precursor POMC (proopiomelanocortin) and ACTH production in non-pituitary tumor cells and in cell lines derived from patients with ectopic Cushing's syndrome. We show that ectopic hPOMC transcription proceeds independently of pituitary-specific Tpit/Pitx1 and demonstrate a novel E2F1-mediated transcriptional mechanism regulating hPOMC We identify an E2F1 cluster binding to the proximal hPOMC promoter region (-42 to +68), with DNA-binding activity determined by the phosphorylation at Ser-337. hPOMC mRNA expression in cancer cells was upregulated (up to 40-fold) by the co-expression of E2F1 and its heterodimer partner DP1. Direct and indirect inhibitors of E2F1 activity suppressed hPOMC gene expression and ACTH by modifying E2F1 DNA-binding activity in ectopic Cushing's cell lines and primary tumor cells, and also suppressed paraneoplastic ACTH and cortisol levels in xenografted mice. E2F1-mediated hPOMC transcription is a potential target for suppressing ACTH production in ectopic Cushing's syndrome. © 2016 Society for Endocrinology.

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

    PubMed Central

    Julian, Lisa M.; Blais, Alexandre

    2015-01-01

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

  3. Geraniol suppresses prostate cancer growth through down-regulation of E2F8.

    PubMed

    Lee, Sanghoon; Park, Yu Rang; Kim, Su-Hwa; Park, Eun-Jung; Kang, Min Ji; So, Insuk; Chun, Jung Nyeo; Jeon, Ju-Hong

    2016-10-01

    Geraniol, an acyclic dietary monoterpene, has been found to suppress cancer survival and growth. However, the molecular mechanism underlying the antitumor action of geraniol has not been investigated at the genome-wide level. In this study, we analyzed the microarray data obtained from geraniol-treated prostate cancer cells. Geraniol potently altered a gene expression profile and primarily down-regulated cell cycle-related gene signatures, compared to linalool, another structurally similar monoterpene that induces no apparent phenotypic changes. Master regulator analysis using the prostate cancer-specific regulatory interactome identified that the transcription factor E2F8 as a specific target molecule regulates geraniol-specific cell cycle signatures. Subsequent experiments confirmed that geraniol down-regulated E2F8 expression and the knockdown of E2F8 was sufficient to suppress cell growth by inducing G 2 /M arrest. Epidemiological analysis showed that E2F8 is up-regulated in metastatic prostate cancer and associated with poor prognosis. These results indicate that E2F8 is a crucial transcription regulator controlling cell cycle and survival in prostate cancer cells. Therefore, our study provides insight into the role of E2F8 in prostate cancer biology and therapeutics. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  4. Positive and negative regulation of V(D)J recombination by the E2A proteins.

    PubMed

    Bain, G; Romanow, W J; Albers, K; Havran, W L; Murre, C

    1999-01-18

    A key feature of B and T lymphocyte development is the generation of antigen receptors through the rearrangement and assembly of the germline variable (V), diversity (D), and joining (J) gene segments. However, the mechanisms responsible for regulating developmentally ordered gene rearrangements are largely unknown. Here we show that the E2A gene products are essential for the proper coordinated temporal regulation of V(D)J rearrangements within the T cell receptor (TCR) gamma and delta loci. Specifically, we show that E2A is required during adult thymocyte development to inhibit rearrangements to the gamma and delta V regions that normally recombine almost exclusively during fetal thymocyte development. The continued rearrangement of the fetal Vgamma3 gene segment in E2A-deficient adult thymocytes correlates with increased levels of Vgamma3 germline transcripts and increased levels of double-stranded DNA breaks at the recombination signal sequence bordering Vgamma3. Additionally, rearrangements to a number of Vgamma and Vdelta gene segments used predominantly during adult development are significantly reduced in E2A-deficient thymocytes. Interestingly, at distinct stages of T lineage development, both the increased and decreased rearrangement of particular Vdelta gene segments is highly sensitive to the dosage of the E2A gene products, suggesting that the concentration of the E2A proteins is rate limiting for the recombination reaction involving these Vdelta regions.

  5. Cell cycle regulator E2F4 is essential for the development of the ventral telencephalon.

    PubMed

    Ruzhynsky, Vladimir A; McClellan, Kelly A; Vanderluit, Jacqueline L; Jeong, Yongsu; Furimsky, Marosh; Park, David S; Epstein, Douglas J; Wallace, Valerie A; Slack, Ruth S

    2007-05-30

    Early forebrain development is characterized by extensive proliferation of neural precursors coupled with complex structural transformations; however, little is known regarding the mechanisms by which these processes are integrated. Here, we show that deficiency of the cell cycle regulatory protein, E2F4, results in the loss of ventral telencephalic structures and impaired self-renewal of neural precursor cells. The mechanism underlying aberrant ventral patterning lies in a dramatic loss of Sonic hedgehog (Shh) expression specifically in this region. The E2F4-deficient phenotype can be recapitulated by interbreeding mice heterozygous for E2F4 with those lacking one allele of Shh, suggesting a genetic interaction between these pathways. Treatment of E2F4-deficient cells with a Hh agonist rescues stem cell self-renewal and cells expressing the homeodomain proteins that specify the ventral telencephalic structures. Finally, we show that E2F4 deficiency results in impaired activity of Shh forebrain-specific enhancers. In conclusion, these studies establish a novel requirement for the cell cycle regulatory protein, E2F4, in the development of the ventral telencephalon.

  6. Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells

    NASA Technical Reports Server (NTRS)

    Tjandrawinata, R. R.; Dahiya, R.; Hughes-Fulford, M.

    1997-01-01

    Prostaglandins are synthesized from arachidonic acid by the enzyme cyclo-oxygenase. There are two isoforms of cyclooxygenases: COX-1 (a constitutive form) and COX-2 (an inducible form). COX-2 has recently been categorized as an immediate-early gene and is associated with cellular growth and differentiation. The purpose of this study was to investigate the effects of exogenous dimethylprostaglandin E2 (dmPGE2) on prostate cancer cell growth. Results of these experiments demonstrate that administration of dmPGE2 to growing PC-3 cells significantly increased cellular proliferation (as measured by the cell number), total DNA content and endogenous PGE2 concentration. DmPGE2 also increased the steady-state mRNA levels of its own inducible synthesizing enzyme, COX-2, as well as cellular growth to levels similar to those seen with fetal calf serum and phorbol ester. The same results were observed in other human cancer cell types, such as the androgen-dependent LNCaP cells, breast cancer MDA-MB-134 cells and human colorectal carcinoma DiFi cells. In PC-3 cells, the dmPGE2 regulation of the COX-2 mRNA levels was both time dependent, with maximum stimulation seen 2 h after addition, and dose dependent on dmPGE2 concentration, with maximum stimulation seen at 5 microg ml(-1). The non-steroidal anti-inflammatory drug flurbiprofen (5 microM), in the presence of exogenous dmPGE2, inhibited the up-regulation of COX-2 mRNA and PC-3 cell growth. Taken together, these data suggest that PGE2 has a specific role in the maintenance of human cancer cell growth and that the activation of COX-2 expression depends primarily upon newly synthesized PGE2, perhaps resulting from changes in local cellular PGE2 concentrations.

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

    PubMed

    Weger-Lucarelli, James; Aliota, Matthew T; Wlodarchak, Nathan; Kamlangdee, Attapon; Swanson, Ryan; Osorio, Jorge E

    2015-12-16

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

  8. E2F3a gene expression has prognostic significance in childhood acute lymphoblastic leukemia.

    PubMed

    Wang, Kai-Ling; Mei, Yan-Yan; Cui, Lei; Zhao, Xiao-Xi; Li, Wei-Jing; Gao, Chao; Liu, Shu-Guang; Jiao, Ying; Liu, Fei-Fei; Wu, Min-Yuan; Ding, Wei; Li, Zhi-Gang

    2014-10-01

    To study E2F3a expression and its clinical significance in children with acute lymphoblastic leukemia (ALL). We quantified E2F3a expression at diagnosis in 148 children with ALL by real-time PCR. In the test cohort (n = 48), receiver operating characteristic (ROC) curve was used to find the best cut-off point to divide the patients into E2F3a low- and high-expression groups. The prognostic significance of E2F3a expression was investigated in the test cohort and confirmed in the validation cohort (n = 100). The correlations of E2F3a expression with the clinical features and treatment outcome of these patients were analyzed. ROC curve analysis indicated that the best cut-off point of E2F3a expression was 0.3780. In the test cohort, leukemia-free survival (LFS) and event-free survival (EFS) of the low-expression group were lower than those of the high-expression group (log rank: P = 0.026 for both). This finding was verified in the validation cohort. LFS, EFS, and overall survival were also lower in the low-expression group than in the high-expression group (log rank, P = 0.015, 0.008, and 0.002 respectively). E2F3a low expression was correlated with the existence of BCR-ABL fusion. An algorithm composed of E2F3a expression and minimal residual disease (MRD) could predict relapse or induction failure more precisely than current risk stratification. These results were still significant in the ALL patients without BCR-ABL fusion. Low expression of E2F3a was associated with inferior prognosis in childhood ALL. An algorithm composed of E2F3a expression and MRD could predict relapse or induction failure more precisely than that of the current risk stratification. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

    PubMed

    Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

    2016-01-01

    Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

  10. Tuning BRCA1 and BARD1 activity to investigate RING ubiquitin ligase mechanisms.

    PubMed

    Stewart, Mikaela D; Duncan, Emily D; Coronado, Ernesto; DaRosa, Paul A; Pruneda, Jonathan N; Brzovic, Peter S; Klevit, Rachel E

    2017-03-01

    The tumor-suppressor protein BRCA1 works with BARD1 to catalyze the transfer of ubiquitin onto protein substrates. The N-terminal regions of BRCA1 and BARD1 that contain their RING domains are responsible for dimerization and ubiquitin ligase activity. This activity is a common feature among hundreds of human RING domain-containing proteins. RING domains bind and activate E2 ubiquitin-conjugating enzymes to promote ubiquitin transfer to substrates. We show that the identity of residues at specific positions in the RING domain can tune activity levels up or down. We report substitutions that create a structurally intact BRCA1/BARD1 heterodimer that is inactive in vitro with all E2 enzymes. Other substitutions in BRCA1 or BARD1 RING domains result in hyperactivity, revealing that both proteins have evolved attenuated activity. Loss of attenuation results in decreased product specificity, providing a rationale for why nature has tuned BRCA1 activity. The ability to tune BRCA1 provides powerful tools for understanding its biological functions and provides a basis to assess mechanisms for rescuing the activity of cancer-associated variations. Beyond the applicability to BRCA1, we show the identity of residues at tuning positions that can be used to predict and modulate the activity of an unrelated RING E3 ligase. These findings provide valuable insights into understanding the mechanism and function of RING E3 ligases like BRCA1. © 2017 The Protein Society.

  11. Inhibition of E2F1 activity and cell cycle progression by arsenic via retinoblastoma protein.

    PubMed

    Sheldon, Lynn A

    2017-01-01

    The regulation of cell cycle progression by steroid hormones and growth factors is important for maintaining normal cellular processes including development and cell proliferation. Deregulated progression through the G1/S and G2/M cell cycle transitions can lead to uncontrolled cell proliferation and cancer. The transcription factor E2F1, a key cell cycle regulator, targets genes encoding proteins that regulate cell cycle progression through the G1/S transition as well as proteins important in DNA repair and apoptosis. E2F1 expression and activity is inhibited by inorganic arsenic (iAs) that has a dual role as a cancer therapeutic and as a toxin that leads to diseases including cancer. An understanding of what underlies this dichotomy will contribute to understanding how to use iAs as a more effective therapeutic and also how to treat cancers that iAs promotes. Here, we show that quiescent breast adenocarcinoma MCF-7 cells treated with 17-β estradiol (E2) progress through the cell cycle, but few cells treated with E2 + iAs progress from G1 into S-phase due to a block in cell cycle progression. Our data support a model in which iAs inhibits the dissociation of E2F1 from the tumor suppressor, retinoblastoma protein (pRB) due to changes in pRB phosphorylation which leads to decreased E2F1 transcriptional activity. These findings present an explanation for how iAs can disrupt cell cycle progression through E2F1-pRB and has implications for how iAs acts as a cancer therapeutic as well as how it may promote tumorigenesis through decreased DNA repair.

  12. DGAT enzymes and triacylglycerol biosynthesis

    PubMed Central

    Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.

    2008-01-01

    Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, and the use of molecular tools, including mice deficient in either enzyme, has shed light on their functions. Although DGAT enzymes are involved in TG synthesis, they have distinct protein sequences and differ in their biochemical, cellular, and physiological functions. Both enzymes may be useful as therapeutic targets for diseases. Here we review the current knowledge of DGAT enzymes, focusing on new advances since the cloning of their genes, including possible roles in human health and diseases. PMID:18757836

  13. Detergent-resistant membrane association of NS2 and E2 during hepatitis C virus replication.

    PubMed

    Shanmugam, Saravanabalaji; Saravanabalaji, Dhanaranjani; Yi, MinKyung

    2015-04-01

    Previously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-β-cyclodextrin (MβCD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by MβCD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production. The biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these

  14. Phage lytic enzymes: a history.

    PubMed

    Trudil, David

    2015-02-01

    There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of 'bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

  15. Enzyme Mimics: Advances and Applications.

    PubMed

    Kuah, Evelyn; Toh, Seraphina; Yee, Jessica; Ma, Qian; Gao, Zhiqiang

    2016-06-13

    Enzyme mimics or artificial enzymes are a class of catalysts that have been actively pursued for decades and have heralded much interest as potentially viable alternatives to natural enzymes. Aside from having catalytic activities similar to their natural counterparts, enzyme mimics have the desired advantages of tunable structures and catalytic efficiencies, excellent tolerance to experimental conditions, lower cost, and purely synthetic routes to their preparation. Although still in the midst of development, impressive advances have already been made. Enzyme mimics have shown immense potential in the catalysis of a wide range of chemical and biological reactions, the development of chemical and biological sensing and anti-biofouling systems, and the production of pharmaceuticals and clean fuels. This Review concerns the development of various types of enzyme mimics, namely polymeric and dendrimeric, supramolecular, nanoparticulate and proteinic enzyme mimics, with an emphasis on their synthesis, catalytic properties and technical applications. It provides an introduction to enzyme mimics and a comprehensive summary of the advances and current standings of their applications, and seeks to inspire researchers to perfect the design and synthesis of enzyme mimics and to tailor their functionality for a much wider range of applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Enzyme actuated bioresponsive hydrogels

    NASA Astrophysics Data System (ADS)

    Wilson, Andrew Nolan

    Bioresponsive hydrogels are emerging with technological significance in targeted drug delivery, biosensors and regenerative medicine. Conferred with the ability to respond to specific biologically derived stimuli, the design challenge is in effectively linking the conferred biospecificity with an engineered response tailored to the needs of a particular application. Moreover, the fundamental phenomena governing the response must support an appropriate dynamic range and limit of detection. The design of these systems is inherently complicated due to the high interdependency of the governing phenomena that guide the sensing, transduction, and the actuation response of hydrogels. To investigate the dynamics of these materials, model systems may be used which seek to interrogate the system dynamics by uni-variable experimentation and limit confounding phenomena such as: polymer-solute interactions, polymer swelling dynamics and biomolecular reaction-diffusion concerns. To this end, a model system, alpha-chymotrypsin (Cht) (a protease) and a cleavable peptide-chromogen (pro-drug) covalently incorporated into a hydrogel, was investigated to understand the mechanisms of covalent loading and release by enzymatic cleavage in bio-responsive delivery systems. Using EDC and Sulfo-NHS, terminal carboxyl groups of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, a cleavable chromogen, were conjugated to primary amines of a hydrated poly(HEMA)-based hydrogel. Hydrogel discs were incubated in buffered Cht causing enzyme-mediated cleavage of the peptide and concomitant release of the chromophore for monitoring. To investigate substrate loading and the effects of hydrogel morphology on the system, the concentration of the amino groups (5, 10, 20, and 30 mol%) and the cross-linked density (1, 5, 7, 9 and 12 mol%) were independently varied. Loading-Release Efficiency of the chromogen was shown to exhibit a positive relation to increasing amino groups (AEMA). The release rates demonstrated a

  17. Mutation of zebrafish dihydrolipoamide branched-chain transacylase E2 results in motor dysfunction and models maple syrup urine disease

    PubMed Central

    Friedrich, Timo; Lambert, Aaron M.; Masino, Mark A.; Downes, Gerald B.

    2012-01-01

    SUMMARY Analysis of zebrafish mutants that demonstrate abnormal locomotive behavior can elucidate the molecular requirements for neural network function and provide new models of human disease. Here, we show that zebrafish quetschkommode (que) mutant larvae exhibit a progressive locomotor defect that culminates in unusual nose-to-tail compressions and an inability to swim. Correspondingly, extracellular peripheral nerve recordings show that que mutants demonstrate abnormal locomotor output to the axial muscles used for swimming. Using positional cloning and candidate gene analysis, we reveal that a point mutation disrupts the gene encoding dihydrolipoamide branched-chain transacylase E2 (Dbt), a component of a mitochondrial enzyme complex, to generate the que phenotype. In humans, mutation of the DBT gene causes maple syrup urine disease (MSUD), a disorder of branched-chain amino acid metabolism that can result in mental retardation, severe dystonia, profound neurological damage and death. que mutants harbor abnormal amino acid levels, similar to MSUD patients and consistent with an error in branched-chain amino acid metabolism. que mutants also contain markedly reduced levels of the neurotransmitter glutamate within the brain and spinal cord, which probably contributes to their abnormal spinal cord locomotor output and aberrant motility behavior, a trait that probably represents severe dystonia in larval zebrafish. Taken together, these data illustrate how defects in branched-chain amino acid metabolism can disrupt nervous system development and/or function, and establish zebrafish que mutants as a model to better understand MSUD. PMID:22046030

  18. Stochastic E2F activation and reconciliation of phenomenological cell-cycle models.

    PubMed

    Lee, Tae J; Yao, Guang; Bennett, Dorothy C; Nevins, Joseph R; You, Lingchong

    2010-09-21

    The transition of the mammalian cell from quiescence to proliferation is a highly variable process. Over the last four decades, two lines of apparently contradictory, phenomenological models have been proposed to account for such temporal variability. These include various forms of the transition probability (TP) model and the growth control (GC) model, which lack mechanistic details. The GC model was further proposed as an alternative explanation for the concept of the restriction point, which we recently demonstrated as being controlled by a bistable Rb-E2F switch. Here, through a combination of modeling and experiments, we show that these different lines of models in essence reflect different aspects of stochastic dynamics in cell cycle entry. In particular, we show that the variable activation of E2F can be described by stochastic activation of the bistable Rb-E2F switch, which in turn may account for the temporal variability in cell cycle entry. Moreover, we show that temporal dynamics of E2F activation can be recast into the frameworks of both the TP model and the GC model via parameter mapping. This mapping suggests that the two lines of phenomenological models can be reconciled through the stochastic dynamics of the Rb-E2F switch. It also suggests a potential utility of the TP or GC models in defining concise, quantitative phenotypes of cell physiology. This may have implications in classifying cell types or states.

  19. Aberrant Retinoblastoma (RB)-E2F Transcriptional Regulation Defines Molecular Phenotypes of Osteosarcoma*

    PubMed Central

    Scott, Milcah C.; Sarver, Aaron L.; Tomiyasu, Hirotaka; Cornax, Ingrid; Van Etten, Jamie; Varshney, Jyotika; O'Sullivan, M. Gerard; Subramanian, Subbaya; Modiano, Jaime F.

    2015-01-01

    We previously identified two distinct molecular subtypes of osteosarcoma through gene expression profiling. These subtypes are associated with distinct tumor behavior and clinical outcomes. Here, we describe mechanisms that give rise to these molecular subtypes. Using bioinformatic analyses, we identified a significant association between deregulation of the retinoblastoma (RB)-E2F pathway and the molecular subtype with worse clinical outcomes. Xenotransplantation models recapitulated the corresponding behavior for each osteosarcoma subtype; thus, we used cell lines to validate the role of the RB-E2F pathway in regulating the prognostic gene signature. Ectopic RB resets the patterns of E2F regulated gene expression in cells derived from tumors with worse clinical outcomes (molecular phenotype 2) to those comparable with those observed in cells derived from tumors with less aggressive outcomes (molecular phenotype 1), providing a functional association between RB-E2F dysfunction and altered gene expression in osteosarcoma. DNA methyltransferase and histone deacetylase inhibitors similarly reset the transcriptional state of the molecular phenotype 2 cells from a state associated with RB deficiency to one seen with RB sufficiency. Our data indicate that deregulation of RB-E2F pathway alters the epigenetic landscape and biological behavior of osteosarcoma. PMID:26378234

  20. Cytoplasmic E2f4 forms organizing centres for initiation of centriole amplification during multiciliogenesis

    PubMed Central

    Mori, Munemasa; Hazan, Renin; Danielian, Paul S.; Mahoney, John E.; Li, Huijun; Lu, Jining; Miller, Emily S.; Zhu, Xueliang; Lees, Jacqueline A.; Cardoso, Wellington V.

    2017-01-01

    Abnormal development of multiciliated cells is a hallmark of a variety of human conditions associated with chronic airway diseases, hydrocephalus and infertility. Multiciliogenesis requires both activation of a specialized transcriptional program and assembly of cytoplasmic structures for large-scale centriole amplification that generates basal bodies. It remains unclear, however, what mechanism initiates formation of these multiprotein complexes in epithelial progenitors. Here we show that this is triggered by nucleocytoplasmic translocation of the transcription factor E2f4. After inducing a transcriptional program of centriole biogenesis, E2f4 forms apical cytoplasmic organizing centres for assembly and nucleation of deuterosomes. Using genetically altered mice and E2F4 mutant proteins we demonstrate that centriole amplification is crucially dependent on these organizing centres and that, without cytoplasmic E2f4, deuterosomes are not assembled, halting multiciliogenesis. Thus, E2f4 integrates nuclear and previously unsuspected cytoplasmic events of centriole amplification, providing new perspectives for the understanding of normal ciliogenesis, ciliopathies and cancer. PMID:28675157

  1. The E2F3—Oncomir 1 axis is activated in Wilms Tumor

    PubMed Central

    Kort, Eric J.; Farber, Leslie; Tretiakova, Maria; Petillo, David; Furge, Kyle A.; Yang, Ximing J.; Cornelius, Albert; Teh, Bin T.

    2008-01-01

    Oncomir-1 is an oncogenic cluster of microRNAs located on chromosome 13. Previous in vitro studies demonstrated that it is transcriptionally regulated by the transcription factor E2F3. In this report we combine expression profiling of both messenger RNA (mRNA) and micro RNAs (miRNA) in Wilms tumor (WT) samples to provide the first evidence that the E2F3—Oncomir 1 axis, previously identified in cell culture, is deregulated in primary human tumors. Analysis of RNA expression signatures demonstrated that an E2F3 gene signature was activated in all Wilms tumor samples analyzed, in contrast to other kidney tumors. This finding was validated by immunohistochemistry (IHC) on the protein level. Expression of E2F3 was lowest in early stage tumors, and highest in metastatic tissue. Expression profiling of miRNAs in WT showed that expression of each measured member of the Oncomir-1 family was highest in WT relative to other kidney tumor subtypes. Quantitative polymerase chain reaction (PCR) confirmed that these microRNAs were overexpressed in Wilms tumor relative to normal kidney tissue. These results suggest that the E2F3—Oncomir-1 axis is activated in Wilms tumor. Our study also demonstrates the utility of integrated genomics combining gene signature analysis with miRNA expression profiling to identify protein-miRNA interactions that are perturbed in disease states. PMID:18519660

  2. The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine.

    PubMed

    Risatti, G R; Borca, M V; Kutish, G F; Lu, Z; Holinka, L G; French, R A; Tulman, E R; Rock, D L

    2005-03-01

    To identify genetic determinants of classical swine fever virus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2 glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2 glycoprotein in swine virulence.

  3. Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

    SciTech Connect

    Kasim, Vivi, E-mail: vivikasim78@gmail.com; Huang, Can; Zhang, Jing

    2014-07-04

    Highlights: • MDM2 is a novel positive regulator of TAp73 transcriptional activity. • MDM2 colocalizes together and physically interacts with E2F1. • Synergistic cooperation of MDM2 and E2F1 is crucial for TAp73 transcription. • MDM2 regulates TAp73 transcriptional activity in a p53-independent manner. - Abstract: TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. Wemore » further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73.« less

  4. Active sites prediction and binding analysis E1-E2 protein human papillomavirus with biphenylsulfonacetic acid

    NASA Astrophysics Data System (ADS)

    Iryani, I.; Amelia, F.; Iswendi, I.

    2018-04-01

    Cervix cancer triggered by Human papillomavirus infection is the second cause to woman death in worldwide. The binding site of E1-E2 protein of HPV 16 is not known from a 3-D structure yet, so in this study we address this issue to study the structure of E1-E2 protein from Human papillomavirus type 16 and to find its potential binding sites using biphenylsulfonacetic acid as inhibitor. Swiss model was used for 3D structure prediction and PDB: 2V9P (E1 protein) and 2NNU (E2 protein) having 52.32% and 100% identity respectively was selected as a template. The 3D model structure developed of E1 and E2 in the core and allowed regions were 99.2% and 99.5%. The ligand binding sites were predicted using online server meta pocket 2.0 and MOE 2009.10 was used for docking. E1-and E2 protein of HPV-16 has three potential binding site that can interact with the inhibitors. The Docking biphenylsulfonacetic acid using these binding sites shows that ligand interact with the protein through hydrogen bonds on Lys 403, Arg 410, His 551 in the first pocket, on Tyr 32, Leu 99 in the second pocket, and Lys 558m Lys 517 in the third pocket.

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

    PubMed Central

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

    2016-01-01

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

  6. Ripening of the cervix with prostaglandin E2-gel. A randomized study with a new ready-to-use compound of triacetin-prostaglandin-E2-gel.

    PubMed

    Kristoffersen, M; Sande, H A; Sande, O S

    1986-08-01

    A randomized study with a group of patients treated with a new ready-to-use triacetin-prostaglandin E2-gel compared to a non-treated group was conducted. The gel-treated group showed a distinct difference in cervical score after 12 h and ten patients were delivered during this period without further induction attempt compared to none in the control group. There was a significantly lower need for oxytocin stimulation in the treated group (P less than 0.0005), but there was no difference in the cesarean section rate or instrumental delivery rate. No side-effects were seen. This new gel seems effective and safe.

  7. Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

    PubMed

    Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

    2017-11-22

    Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

  8. Computational enzyme design: transitioning from catalytic proteins to enzymes.

    PubMed

    Mak, Wai Shun; Siegel, Justin B

    2014-08-01

    The widespread interest in enzymes stem from their ability to catalyze chemical reactions under mild and ecologically friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biological catalysts capable of performing the desired chemical transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chemistry and force-field based protein molecular modeling have led to the design of novel proteins capable of catalyzing chemical reactions not catalyzed by naturally occurring enzymes. Here we discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward. Published by Elsevier Ltd.

  9. Enzymes in human milk

    PubMed Central

    Dallas, David C.; German, J. Bruce

    2017-01-01

    Milk proteins are a complex and diverse source of biological activities. Beyond their function intact, milk proteins also act as carriers of encrypted functional sequences that when released as peptides exert biological functions, including antimicrobial and immunomodulatory, which could contribute to the infant’s competitive success. Research has now revealed that the release of these functional peptides begins within the mammary gland itself. A complex array of proteases produced in mother’s milk have been shown to be active in the milk, releasing these peptides. Moreover, our recent research demonstrates that these milk proteases continue to digest milk proteins within the infant’s stomach, possibly even to a larger extent than the infant’s own proteases. As the neonate has relatively low digestive capacity, the activity of milk proteases in the infant may provide important assistance to digesting milk proteins. The coordinated release of these encrypted sequences is accomplished by selective proteolytic action provided by an array of native milk proteases and infant-produced enzymes. The task for scientists is now to discover the selective advantages of this protein-protease based peptide release system. PMID:28346930

  10. Enzymes in Human Milk.

    PubMed

    Dallas, David C; German, J Bruce

    2017-01-01

    Milk proteins are a complex and diverse source of biological activities. Beyond their function, intact milk proteins also act as carriers of encrypted functional sequences that, when released as peptides, exert biological functions, including antimicrobial and immunomodulatory activity, which could contribute to the infant's competitive success. Research has now revealed that the release of these functional peptides begins within the mammary gland itself. A complex array of proteases produced in mother's milk has been shown to be active in the milk, releasing these peptides. Moreover, our recent research demonstrates that these milk proteases continue to digest milk proteins within the infant's stomach, possibly even to a larger extent than the infant's own proteases. As the neonate has relatively low digestive capacity, the activity of milk proteases in the infant may provide important assistance to digesting milk proteins. The coordinated release of these encrypted sequences is accomplished by selective proteolytic action provided by an array of native milk proteases and infant-produced enzymes. The task for scientists is now to discover the selective advantages of this protein-protease-based peptide release system. © 2017 Nestec Ltd., Vevey/S. Karger AG, Basel.

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

    DOE PAGES

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

    2016-06-30

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

  12. Release of deuterated (E)-2-nonenal during beer aging from labeled precursors synthesized before boiling.

    PubMed

    Liégeois, Catherine; Meurens, Nicolas; Badot, Camille; Collin, Sonia

    2002-12-18

    Although lipid autoxidation in the boiling kettle is a key determinant of the cardboard flavor of aged beers, recent results show that mashing is another significant source of wort nonenal potential, the well-known indicator of how a beer will release (E)-2-nonenal during storage. Although unstable, deuterated (E)-2-nonenal nitrogen adducts created during mashing can in some cases partially persist in the pitching wort, to release deuterated (E)-2-nonenal during beer aging. In the experiment described here, the relative contributions of mashing and boiling were estimated at 30 and 70%, respectively. The presence of oxygen during mashing and, to a lesser extent, high lipoxygenase activity can intensify the stale cardboard flavor.

  13. Reduced probabilities for E2 transitions between excited collective states of triaxial even–even nuclei

    SciTech Connect

    Nadyrbekov, M. S., E-mail: nodirbekov@inp.uz; Bozarov, O. A.

    Reduced probabilities for intra- and interband E2 transitions in excited collective states of even–even lanthanide and actinide nuclei are analyzed on the basis of a model that admits an arbitrary triaxiality. They are studied in detail in the energy spectra of {sup 154}Sm, {sup 156}Gd, {sup 158}Dy, {sup 162,164}Er, {sup 230,232}Th, and {sup 232,234,236,238}U even–even nuclei. Theoretical and experimental values of the reduced probabilities for the respective E2 transitions are compared. This comparison shows good agreement for all states, including high-spin ones. The ratios of the reduced probabilities for the E2 transitions in question are compared with results following frommore » the Alaga rules. These comparisons make it possible to assess the sensitivity of the probabilities being considered to the presence of quadrupole deformations.« less

  14. e2v CMOS and CCD sensors and systems for astronomy

    NASA Astrophysics Data System (ADS)

    Jorden, P. R.; Jerram, P. A.; Fryer, M.; Stefanov, K. D.

    2017-07-01

    e2v designs and manufactures a wide range of sensors for space and astronomy applications. This includes high performance CCDs for X-ray, visible and near-IR wavelengths. In this paper we illustrate the maturity of CMOS capability for these applications; examples are presented together with performance data. The majority of e2v sensors for these applications are back-thinned for highest spectral response and designed for very low read-out noise; the combination delivers high signal to noise ratio in association with a variety of formats and package designs. The growing e2v capability in delivery of sub-systems and cryogenic cameras is illustrated—including the 1.2 Giga-pixel J-PAS camera system.

  15. The challenges of classical swine fever control: modified live and E2 subunit vaccines.

    PubMed

    Huang, Yu-Liang; Deng, Ming-Chung; Wang, Fun-In; Huang, Chin-Cheng; Chang, Chia-Yi

    2014-01-22

    Classical swine fever (CSF) is an economically important, highly contagious disease of swine worldwide. CSF is caused by classical swine fever virus (CSFV), and domestic pigs and wild boars are its only natural hosts. The two main strategies used to control CSF epidemic are systematic prophylactic vaccination and a non-vaccination stamping-out policy. This review compares the protective efficacy of the routinely used modified live vaccine (MLV) and E2 subunit vaccines and summarizes the factors that influence the efficacy of the vaccines and the challenges that both vaccines face to CSF control. Although MLV provide earlier and more complete protection than E2 subunit vaccines, it has the drawback of not allowing differentiation between infected and vaccinated animals (DIVA). The marker vaccine of E2 protein with companion discriminatory test to detect antibodies against E(rns) allows DIVA and is a promising strategy for future control and eradication of CSF. Maternal derived antibody (MDA) is the critical factor in impairing the efficacy of both MLV and E2 subunit vaccines, so the well-designed vaccination programs of sows and piglets should be considered together. Because of the antigen variation among various genotypes of CSFV, antibodies raised by either MLV or subunit vaccine neutralize genotypically homologous strains better than heterologous ones. However, although this is not a major concern for MLV as the induced immune responses can protect pigs against the challenge of various genotypes of CSFVs, it is critical for E2 subunit vaccines. It is thus necessary to evaluate whether the E2 subunit vaccine can completely protect against the current prevalent strains in the field. An ideal new generation of vaccine should be able to maintain the high protective efficiency of MLV and overcome the problem of antigenic variations while allowing for DIVA. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Structures of (2E,5E)-2-(4-cyanobenzylidene)-5-(4-dimethylaminobenzylidene)cyclopentanone and (2E,5E)-2-benzylidene-5-cinnamylidenecyclopentanone

    NASA Astrophysics Data System (ADS)

    Zoto, Christopher A.; MacDonald, John C.

    2017-10-01

    The X-ray crystal structures of (2E,5E)-2-(4-cyanobenzylidene)-5-(4-dimethylaminobenzylidene)cyclopentanone (I) and (2E,5E)-2-benzylidene-5-cinnamylidenecyclopentanone (II) are presented, compared to the gas phase structures calculated using density functional theory, and discussed in the context of the photophysical behavior exhibited by I and II. Compound I crystallizes in the triclinic space group P 1 bar with a = 6.8743(2) Å, b = 8.8115(2) Å, c = 14.9664(4) Å, α = 77.135(2)°, β = 81.351(2)°, γ = 80.975(2)°, and Z = 2, and exhibits a planar structure. Compound II crystallizes in the monoclinic space group C2/c with a = 33.4281(10) Å, b = 11.9668(4) Å, c = 7.8031(2) Å, β = 92.785(2)°, and Z = 8, and adopts a nonplanar structure in the solid state and calculated structure.

  17. Fatigue Investigation of the E-2B/C Nose Landing Gear

    DTIC Science & Technology

    1975-12-31

    Specimen - Outer Cylinder and Caster Barrel 9 3 E-2B NLG Test Specimen - Drag Brace 10 4 E-2B NLG Drag Brace Attachment Lugs Modification . . 11 5...Attachment Lug Fracture Surface . . 23 ■■■■ ? 16 Tow Link Modification 30 17 Tow Link #2 Failur 31 18 Tow Link #2 Fracture Face 32 19...34-" ’’- .-.„.,.,„...^ ,.., M1| lf^^~i*>-^^-^^ "u- II li ’ 7«™.™»«*" ■ NADC-75369-30 Bach «hock «true assembly consist« of the «xle- caster barrel, outer cylinder

  18. Discovery and Classification of the z=1.86 SLSNe: DES15E2mlf

    NASA Astrophysics Data System (ADS)

    Pan, Y.-C.; Foley, R. J.; Galbany, L.; Gonzalez-Gaitan, S.; Forster, F.; Hamuy, M.; Prieto, J. L.; Yuan, F.; Tucker, B. E.; Lidman, C.; Martini, P.; Gshwend, Julia; Moller, A.; Zhang, B.; Desai, S.; Paech, K.; Smith, R. C.; Schubnell, M.; Kessler, R.; Lasker, J.; Scolnic, D.; Brout, D. J.; Gladney, L.; Sako, M.; Wolf, R. C.; Brown, P. J.; Krisciunas, K.; Suntzeff, N.; Nichol, R.; Papadopoulos, A.; Childress, M.; D'Andrea, C.; Prajs, S.; Smith, M.; Sullivan, M.; Maartens, R.; Gupta, R.; Kovacs, E.; Kuhlmann, S.; Spinka, H.; Ahn, E.; Finley, D. A.; Frieman, J.; Marriner, J.; Wester, W.; Aldering, G.; Kim, A. G.; Thomas, R. C.; Barbary, K.; Bloom, J. S.; Goldstein, D.; Nugent, P.; Perlmutter, S.; Casas, R.; Castander, F. J.

    2015-12-01

    We report the spectroscopic classification of DES15E2mlf as a superluminous supernova (SLSN) discovered by the Dark Energy Survey (ATEL #4668). DES15E2mlf was discovered on 7 November 2015 at R.A. = 00:41:33.40, Decl = -43:27:17.2 with r = 24.1 mag. We obtained spectra using GMOS on Gemini-South (520-990nm) on 06 December 2015 which indicated a redshift of z = 1.86 from Mg II 2800 absorption.

  19. US NDC Modernization Iteration E2 Prototyping Report: User Interface Framework

    SciTech Connect

    Lewis, Jennifer E.; Palmer, Melanie A.; Vickers, James Wallace

    2014-12-01

    During the second iteration of the US NDC Modernization Elaboration phase (E2), the SNL US NDC Modernization project team completed follow-on Rich Client Platform (RCP) exploratory prototyping related to the User Interface Framework (UIF). The team also developed a survey of browser-based User Interface solutions and completed exploratory prototyping for selected solutions. This report presents the results of the browser-based UI survey, summarizes the E2 browser-based UI and RCP prototyping work, and outlines a path forward for the third iteration of the Elaboration phase (E3).

  20. YB-1, the E2F Pathway, and Regulation of Tumor Cell Growth

    PubMed Central

    Samuel, Weini; Cao, Helen; Patel, Rachna; Mehta, Reena; Stern, J. Lewis; Reid, Glen; Woolley, Adele G.; Miller, Lance D.; Black, Michael A; Shelling, Andrew N.; Print, Cristin G.; Braithwaite, Antony W.

    2012-01-01

    Background Y-box binding factor 1 (YB-1) has been associated with prognosis in many tumor types. Reduced YB-1 expression inhibits tumor cell growth, but the mechanism is unclear. Methods YB-1 mRNA levels were compared with tumor grade and histology using microarray data from 771 breast cancer patients and with disease-free survival and distant metastasis–free survival using data from 375 of those patients who did not receive adjuvant therapy. Microarrays were further searched for genes that had correlated expression with YB-1 mRNA. Small interfering RNA (siRNA) was used to study the effects of reduced YB-1 expression on growth of three tumor cell lines (MCF-7 breast, HCT116 colon, and A549 lung cancer cells), on tumorigenesis by A549 cells in nude mice, and on global transcription in the three cancer cell lines. Reporter gene assays were used to determine whether YB-1 siRNAs affected the expression of E2F1, and chromatin immunoprecipitation was used to determine whether YB-1 bound to various E2F promoters as well as E2F1-regulated promoters. All P values were from two-sided tests. Results YB-1 levels were elevated in more aggressive tumors and were strongly associated with poor disease-free survival and distant metastasis–free survival. YB-1 expression was often associated with the expression of genes with E2F sites in their promoters. Cells expressing YB-1 siRNA grew substantially more slowly than control cells and formed tumors less readily in nude mice. Transcripts that were altered in cancer cell lines with YB-1 siRNA included 32 genes that are components of prognostic gene expression signatures. YB-1 regulated expression of an E2F1 promoter–reporter construct in A549 cells (eg, relative E2F1 promoter activity with control siRNA = 4.04; with YB-1 siRNA = 1.40, difference= −2.64, 95% confidence interval = −3.57 to −1.71, P < .001) and bound to the promoters of several well-defined E2F1 target genes. Conclusion YB-1 expression is associated with the

  1. Making the Rate: Enzyme Dynamics

    ERIC Educational Resources Information Center

    Ragsdale, Frances R.

    2004-01-01

    An enzyme exercise to address the problem of students inability to visualize chemical reaction at the molecular level is described. This exercise is designed as a dry lab exercise but can be modified into a classroom activity then can be augmented by a wet lab procedure, thereby providing students with a practical exposure to enzyme function.

  2. Measurement of Enzyme Isotope Effects.

    PubMed

    Kholodar, Svetlana A; Ghosh, Ananda K; Kohen, Amnon

    2017-01-01

    Enzyme isotope effects, or the kinetic effects of "heavy" enzymes, refer to the effect of isotopically labeled protein residues on the enzyme's activity or physical properties. These effects are increasingly employed in the examination of the possible contributions of protein dynamics to enzyme catalysis. One hypothesis assumed that isotopic substitution of all 12 C, 14 N, and nonexchangeable 1 H by 13 C, 15 N, and 2 H, would slow down protein picosecond to femtosecond dynamics without any effect on the system's electrostatics following the Born-Oppenheimer approximation. It was suggested that reduced reaction rates reported for several "heavy" enzymes accords with that hypothesis. However, numerous deviations from the predictions of that hypothesis were also reported. Current studies also attempt to test the role of individual residues by site-specific labeling or by labeling a pattern of residues on activity. It appears that in several systems the protein's fast dynamics are indeed reduced in "heavy" enzymes in a way that reduces the probability of barrier crossing of its chemical step. Other observations, however, indicated that slower protein dynamics are electrostatically altered in isotopically labeled enzymes. Interestingly, these effects appear to be system dependent, thus it might be premature to suggest a general role of "heavy" enzymes' effect on catalysis. © 2017 Elsevier Inc. All rights reserved.

  3. Enzyme catalysis: Evolution made easy

    NASA Astrophysics Data System (ADS)

    Wee, Eugene J. H.; Trau, Matt

    2014-09-01

    Directed evolution is a powerful tool for the development of improved enzyme catalysts. Now, a method that enables an enzyme, its encoding DNA and a fluorescent reaction product to be encapsulated in a gel bead enables the application of directed evolution in an ultra-high-throughput format.

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

    PubMed

    2016-07-19

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

  5. Nanoporous Gold for Enzyme Immobilization.

    PubMed

    Stine, Keith J; Jefferson, Kenise; Shulga, Olga V

    2017-01-01

    Nanoporous gold (NPG) is a material of emerging interest for immobilization of biomolecules, especially enzymes. The material provides a high surface area form of gold that is suitable for physisorption or for covalent modification by self-assembled monolayers. The material can be used as a high surface area electrode and with immobilized enzymes can be used for amperometric detection schemes. NPG can be prepared in a variety of formats from alloys containing between 20 and 50 % atomic composition of gold and less noble element(s) by dealloying procedures. Materials resembling NPG can be prepared by hydrothermal and electrodeposition methods. Related high surface area gold structures have been prepared using templating approaches. Covalent enzyme immobilization can be achieved by first forming a self-assembled monolayer on NPG bearing a terminal reactive functional group followed by conjugation to the enzyme through amide linkages to lysine residues. Enzymes can also be entrapped by physisorption or immobilized by electrostatic interactions.

  6. Positron emitter labeled enzyme inhibitors

    SciTech Connect

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline andmore » L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.« less

  7. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1987-05-22

    This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  8. Positron emitter labeled enzyme inhibitors

    SciTech Connect

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgylinemore » and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.« less

  9. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt

    1990-01-01

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  10. E2P phosphoforms of Na,K-ATPase. I. Comparison of phosphointermediates formed from ATP and Pi by their reactivity toward hydroxylamine and vanadate.

    PubMed

    Fedosova, N U; Cornelius, F; Klodos, I

    1998-09-29

    The properties of Na,K-ATPase phosphoenzymes formed either from ATP in the presence of Mg2+ and Na+ or from Pi in the absence of alkali cations were investigated by biochemical methods and spectrofluorometry employing the styryl dye RH421. We characterized the phosphoenzyme species by their reaction to N-methyl hydroxylamine, which attacks specifically the protein-phosphate bond. We studied reactions of the phospho- and dephospho-enzymes with vanadate, which is a transition-state analogue of phosphate in this enzyme. On the basis of substantial differences in the properties of the phosphoenzyme species formed either from ATP or Pi, especially in their reactivity to N-methyl hydroxylamine, it is suggested that the two phosphoenzyme species are two subconformations of the E2P phosphoform. Analysis of the RH421 fluorescence responses under a variety of experimental conditions and comparing different enzyme sources suggested that the increase of RH421 fluorescence induced by inorganic phosphate in the absence of alkali cations is associated with the formation of the covalent acyl-phosphate bond.

  11. DOE Challenge Home Case Study: e2 Homes – Winter Park, Florida

    SciTech Connect

    none,

    2013-01-01

    This Challenge Home case study describes the first certified DOE Challenge Home as constructed by e2 Homes. Completed in May 2012, the “Wilson Residence” in Winter Park, Florida, is a 4,305-ft2 custom home that scores a HERS 57 without solar and a better than zero net-energy HERS -7 with solar.

  12. Stages of Change – Continuous Measure (URICA-E2): psychometrics of a Norwegian version

    PubMed Central

    Lerdal, Anners; Moe, Britt; Digre, Elin; Harding, Thomas; Kristensen, Frode; Grov, Ellen K; Bakken, Linda N; Eklund, Marthe L; Ruud, Ireen; Rossi, Joseph S

    2009-01-01

    Title Stages of Change – Continuous Measure (URICA-E2): psychometrics of a Norwegian version. Aim This paper is a report of research to translate the English version of the Stages of Change continuous measure questionnaire (URICA-E2) into Norwegian and to test the validity of the questionnaire and its usefulness in predicting behavioural change. Background While the psychometric properties of the Stages of Change categorical measure have been tested extensively, evaluation of the psychometric properties of the continuous questionnaire has not been described elsewhere in the literature. Method Cross-sectional data were collected with a convenience sample of 198 undergraduate nursing students in 2005 and 2006. The English version of URICA-E2 was translated into Norwegian according to standardized procedures. Findings Principal components analysis clearly confirmed five of the dimensions of readiness to change (Precontemplation Non-Believers, Precontemplation Believers, Contemplation, Preparation and Maintenance), while the sixth dimension, Action, showed the lowest Eigenvalue (0·93). Findings from the cluster analysis indicate distinct profiles among the respondents in terms of readiness to change their exercise behaviour. Conclusion The URICA-E2 was for the most part replicated from Reed’s original work. The result of the cluster analysis of the items associated with the factor ‘Action’ suggests that these do not adequately measure the factor. PMID:19032513

  13. 26 CFR 301.6231(e)-2 - Judicial decision not a bar to certain adjustments.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 18 2010-04-01 2010-04-01 false Judicial decision not a bar to certain....6231(e)-2 Judicial decision not a bar to certain adjustments. (a) In general. A court decision with respect to a partner's income tax liability attributable to nonpartnership items shall not be a bar to...

  14. Studying fission neutrons with 2E-2v and 2E

    NASA Astrophysics Data System (ADS)

    Al-Adili, Ali; Jansson, Kaj; Tarrío, Diego; Hambsch, Franz-Josef; Göök, Alf; Oberstedt, Stephan; Olivier Frégeau, Marc; Gustavsson, Cecilia; Lantz, Mattias; Mattera, Andrea; Prokofiev, Alexander V.; Rakopoulos, Vasileios; Solders, Andreas; Vidali, Marzio; Österlund, Michael; Pomp, Stephan

    2018-03-01

    This work aims at measuring prompt-fission neutrons at different excitation energies of the nucleus. Two independent techniques, the 2E-2v and the 2E techniques, are used to map the characteristics of the mass-dependent prompt fission neutron multiplicity, v(A), when the excitation energy is increased. The VERDI 2E-2v spectrometer is being developed at JRC-GEEL. The Fission Fragment (FF) energies are measured using two arrays of 16 silicon (Si) detectors each. The FFs velocities are obtained by time-of-flight, measured between micro-channel plates (MCP) and Si detectors. With MCPs placed on both sides of the fission source, VERDI allows for independent timing measurements for both fragments. 252Cf(sf) was measured and the present results revealed particular features of the 2E-2v technique. Dedicated simulations were also performed using the GEF code to study important aspects of the 2E-2v technique. Our simulations show that prompt neutron emission has a non-negligible impact on the deduced fragment data and affects also the shape of v(A). Geometrical constraints lead to a total-kinetic energy-dependent detection efficiency. The 2E technique utilizes an ionization chamber together with two liquid scintillator detectors. Two measurements have been performed, one of 252Cf(sf) and another one of thermal-neutron induced fission in 235U(n,f). Results from 252Cf(sf) are reported here.

  15. "Expectations to Change" ((E2C): A Participatory Method for Facilitating Stakeholder Engagement with Evaluation Findings

    ERIC Educational Resources Information Center

    Adams, Adrienne E.; Nnawulezi, Nkiru A.; Vandenberg, Lela

    2015-01-01

    From a utilization-focused evaluation perspective, the success of an evaluation is rooted in the extent to which the evaluation was used by stakeholders. This paper details the "Expectations to Change" (E2C) process, an interactive, workshop-based method designed to engage primary users with their evaluation findings as a means of…

  16. The E2 Domains of APP and APLP1 Share a Conserved Mode of Dimerization

    SciTech Connect

    S Lee; Y Xue; J Hulbert

    2011-12-31

    Amyloid precursor protein (APP) is genetically linked to Alzheimer's disease. APP is a type I membrane protein, and its oligomeric structure is potentially important because this property may play a role in its function or affect the processing of the precursor by the secretases to generate amyloid {beta}-peptide. Several independent studies have shown that APP can form dimers in the cell, but how it dimerizes remains controversial. At least three regions of the precursor, including a centrally located and conserved domain called E2, have been proposed to contribute to dimerization. Here we report two new crystal structures of E2, onemore » from APP and the other from APLP1, a mammalian APP homologue. Comparison with an earlier APP structure, which was determined in a different space group, shows that the E2 domains share a conserved and antiparallel mode of dimerization. Biophysical measurements in solution show that heparin binding induces E2 dimerization. The 2.1 {angstrom} resolution electron density map also reveals phosphate ions that are bound to the protein surface. Mutational analysis shows that protein residues interacting with the phosphate ions are also involved in heparin binding. The locations of two of these residues, Arg-369 and His-433, at the dimeric interface suggest a mechanism for heparin-induced protein dimerization.« less

  17. 26 CFR 48.4216(e)-2 - Limitation on aggregate of exclusions and price readjustments.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 16 2010-04-01 2010-04-01 true Limitation on aggregate of exclusions and price... Applicable to Manufacturers Taxes § 48.4216(e)-2 Limitation on aggregate of exclusions and price readjustments. (a) In general. The sum of the amount excluded from taxable price in respect of charges for local...

  18. Deregulation of E2-EPF ubiquitin carrier protein in papillary renal cell carcinoma.

    PubMed

    Roos, Frederik C; Evans, Andrew J; Brenner, Walburgis; Wondergem, Bill; Klomp, Jeffery; Heir, Pardeep; Roche, Olga; Thomas, Christian; Schimmel, Heiko; Furge, Kyle A; Teh, Bin T; Thüroff, Joachim W; Hampel, Christian; Ohh, Michael

    2011-02-01

    Molecular pathways associated with pathogenesis of sporadic papillary renal cell carcinoma (PRCC), the second most common form of kidney cancer, are poorly understood. We analyzed primary tumor specimens from 35 PRCC patients treated by nephrectomy via gene expression analysis and tissue microarrays constructed from an additional 57 paraffin-embedded PRCC samples via immunohistochemistry. Gene products were validated and further studied by Western blot analyses using primary PRCC tumor samples and established renal cell carcinoma cell lines, and potential associations with pathologic variables and survival in 27 patients with follow-up information were determined. We show that the expression of E2-EPF ubiquitin carrier protein, which targets the principal negative regulator of hypoxia-inducible factor (HIF), von Hippel-Lindau protein, for proteasome-dependent degradation, is markedly elevated in the majority of PRCC tumors exhibiting increased HIF1α expression, and is associated with poor prognosis. In addition, we identified multiple hypoxia-responsive elements within the E2-EPF promoter, and for the first time we demonstrated that E2-EPF is a hypoxia-inducible gene directly regulated via HIF1. These findings reveal deregulation of the oxygen-sensing pathway impinging on the positive feedback mechanism of HIF1-mediated regulation of E2-EPF in PRCC. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  19. Deregulation of E2-EPF Ubiquitin Carrier Protein in Papillary Renal Cell Carcinoma

    PubMed Central

    Roos, Frederik C.; Evans, Andrew J.; Brenner, Walburgis; Wondergem, Bill; Klomp, Jeffery; Heir, Pardeep; Roche, Olga; Thomas, Christian; Schimmel, Heiko; Furge, Kyle A.; Teh, Bin T.; Thüroff, Joachim W.; Hampel, Christian; Ohh, Michael

    2011-01-01

    Molecular pathways associated with pathogenesis of sporadic papillary renal cell carcinoma (PRCC), the second most common form of kidney cancer, are poorly understood. We analyzed primary tumor specimens from 35 PRCC patients treated by nephrectomy via gene expression analysis and tissue microarrays constructed from an additional 57 paraffin-embedded PRCC samples via immunohistochemistry. Gene products were validated and further studied by Western blot analyses using primary PRCC tumor samples and established renal cell carcinoma cell lines, and potential associations with pathologic variables and survival in 27 patients with follow-up information were determined. We show that the expression of E2-EPF ubiquitin carrier protein, which targets the principal negative regulator of hypoxia-inducible factor (HIF), von Hippel-Lindau protein, for proteasome-dependent degradation, is markedly elevated in the majority of PRCC tumors exhibiting increased HIF1α expression, and is associated with poor prognosis. In addition, we identified multiple hypoxia-responsive elements within the E2-EPF promoter, and for the first time we demonstrated that E2-EPF is a hypoxia-inducible gene directly regulated via HIF1. These findings reveal deregulation of the oxygen-sensing pathway impinging on the positive feedback mechanism of HIF1-mediated regulation of E2-EPF in PRCC. PMID:21281817

  20. 17 CFR 270.22e-2 - Pricing of redemption requests in accordance with Rule 22c-1.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Pricing of redemption requests in accordance with Rule 22c-1. 270.22e-2 Section 270.22e-2 Commodity and Securities Exchanges....22e-2 Pricing of redemption requests in accordance with Rule 22c-1. An investment company shall not be...

  1. An Integrated Bioinformatics Approach Identifies Elevated Cyclin E2 Expression and E2F Activity as Distinct Features of Tamoxifen Resistant Breast Tumors

    PubMed Central

    Huang, Lei; Zhao, Shuangping; Frasor, Jonna M.; Dai, Yang

    2011-01-01

    Approximately half of estrogen receptor (ER) positive breast tumors will fail to respond to endocrine therapy. Here we used an integrative bioinformatics approach to analyze three gene expression profiling data sets from breast tumors in an attempt to uncover underlying mechanisms contributing to the development of resistance and potential therapeutic strategies to counteract these mechanisms. Genes that are differentially expressed in tamoxifen resistant vs. sensitive breast tumors were identified from three different publically available microarray datasets. These differentially expressed (DE) genes were analyzed using gene function and gene set enrichment and examined in intrinsic subtypes of breast tumors. The Connectivity Map analysis was utilized to link gene expression profiles of tamoxifen resistant tumors to small molecules and validation studies were carried out in a tamoxifen resistant cell line. Despite little overlap in genes that are differentially expressed in tamoxifen resistant vs. sensitive tumors, a high degree of functional similarity was observed among the three datasets. Tamoxifen resistant tumors displayed enriched expression of genes related to cell cycle and proliferation, as well as elevated activity of E2F transcription factors, and were highly correlated with a Luminal intrinsic subtype. A number of small molecules, including phenothiazines, were found that induced a gene signature in breast cancer cell lines opposite to that found in tamoxifen resistant vs. sensitive tumors and the ability of phenothiazines to down-regulate cyclin E2 and inhibit proliferation of tamoxifen resistant breast cancer cells was validated. Our findings demonstrate that an integrated bioinformatics approach to analyze gene expression profiles from multiple breast tumor datasets can identify important biological pathways and potentially novel therapeutic options for tamoxifen-resistant breast cancers. PMID:21789246

  2. Single Cell Analysis to locate the Restriction Point with respect to E2F Expression

    NASA Astrophysics Data System (ADS)

    Pimienta, R.; Johnson, A.

    2011-12-01

    The restriction point is a G1-phase checkpoint that regulates passage through the cell cycle and is misregulated in all known types of cancer. The Rb-E2F switch is thought to be one of the most relevant molecular mechanisms which regulate the restriction point in mammalian cells. However, recent experiments have brought the timing of the restriction point into question. In previous studies, cells were analyzed as populations and this prevented an accurate determination of the restriction point. By creating and analyzing an E2F-GFP reporter in single cells, we can pinpoint the timing of E2F activation and determine whether it coincides with the restriction point. Using calcium phosphate and Fugene,we transfected human embryonic kidney (293T) cells with a CMV-GFP plasmid and an E2F-GFP reporter. Based on our results, it appears that calcium phosphate is more effective than Fugene at transfecting mammalian cells. The calcium phosphate transfection had 9.59% more fluorescent cells than Fugene. However, this result only occurred with the CMV-GFP plasmid and not the E2F-GFP reporter, which was not properly expressed in human embryonic kidney (293T) cells. We will continue troubleshooting to fix this reporter as we proceed with our research. Once the reporter is properly cloned, we will transfect it into retinal pigmented epithelial (RPE1-hTERT) cells using the calcium phosphate method. RPE1-hTERT cells are an immortalized with telomerase and are more close to normal cells than tumor-derived cell lines. Through this research we will better comprehend commitment to the mammalian cell cycle.

  3. [Automated analyzer of enzyme immunoassay].

    PubMed

    Osawa, S

    1995-09-01

    Automated analyzers for enzyme immunoassay can be classified by several points of view: the kind of labeled antibodies or enzymes, detection methods, the number of tests per unit time, analytical time and speed per run. In practice, it is important for us consider the several points such as detection limits, the number of tests per unit time, analytical range, and precision. Most of the automated analyzers on the market can randomly access and measure samples. I will describe the recent advance of automated analyzers reviewing their labeling antibodies and enzymes, the detection methods, the number of test per unit time and analytical time and speed per test.

  4. Genomic structure, expression pattern, and functional characterization of transcription factor E2F-2 from black tiger shrimp (Penaeus monodon)

    PubMed Central

    Zhao, Chao; Qiu, Lihua

    2017-01-01

    Transcription factor E2F-2 is a regulator of cell cycle. Researchers identified E2F-2 genes from yeasts to humans, but few reports investigated E2F-2 gene from black tiger shrimp. In the present study, we cloned E2F-2 gene from black tiger shrimp (Penaeus monodon). Full-length PmE2F-2 complementary DNA sequence measures 3,189 bp with an open reading frame of 1,371 bp. Complete PmE2F-2 genomic sequence (17,305 bp) of P. monodon contains nine exons, which are separated by eight introns. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that PmE2F-2 is highly expressed in hepatopancreas and ovaries of P. monodon. Highest PmE2F-2 expression levels were observed in stage III ovarian development of P. monodon. PmE2F-2 expression levels were significantly augmented in ovaries of P. monodon after 5-hydroxytryptamine injection and eyestalk ablation. RNA interference experiments were conducted to examine PmE2F-2, PmCDK2, and PmCyclin E expression profiles. PmE2F-2 was successfully knocked down in ovaries and hepatopancreas via double-stranded RNA (dsRNA)–E2F-2 injection. In the same organs, PmE2F-2 expression localization and level were investigated through in situ hybridization, which revealed consistent results with those of qRT-PCR. After dsRNA—E2F-2 injection, gonadosomatic index of shrimp was significantly lower than those following dsRNA—GFP and phosphate-buffered solution injections. Therefore, PmE2F-2 may be involved in ovarian maturation in P. monodon. PMID:28558060

  5. A prostaglandin E2 receptor antagonist prevents pregnancies during a preclinical contraceptive trial with female macaques.

    PubMed

    Peluffo, M C; Stanley, J; Braeuer, N; Rotgeri, A; Fritzemeier, K-H; Fuhrmann, U; Buchmann, B; Adevai, T; Murphy, M J; Zelinski, M B; Lindenthal, B; Hennebold, J D; Stouffer, R L

    2014-07-01

    Can administration of a prostaglandin (PG) E2 receptor 2 (PTGER2) antagonist prevent pregnancy in adult female monkeys by blocking periovulatory events in the follicle without altering menstrual cyclicity or general health? This is the first study to demonstrate that a PTGER2 antagonist can serve as an effective non-hormonal contraceptive in primates. The requirement for PGE2 in ovulation and the release of an oocyte surrounded by expanded cumulus cells (cumulus-oocyte expansion; C-OE) was established through the generation of PTGS2 and PTGER2 null-mutant mice. A critical role for PGE2 in primate ovulation is supported by evidence that intrafollicular injection of indomethacin in rhesus monkeys suppressed follicle rupture, whereas co-injection of PGE2 with indomethacin resulted in ovulation. First, controlled ovulation protocols were performed in adult, female rhesus monkeys to analyze the mRNA levels for genes encoding PGE2 synthesis and signaling components in the naturally selected pre-ovulatory follicle at different times after the ovulatory hCG stimulus (0, 12, 24, 36 h pre-ovulation; 36 h post-ovulation, n = 3-4/time point). Second, controlled ovarian stimulation cycles were utilized to obtain multiple cumulus-oocyte complexes (COCs) from rhesus monkeys to evaluate the role of PGE2 in C-OE in vitro (n = 3-4 animals/treatment; ≥3 COCs/animal/treatment). Third, adult cycling female cynomolgus macaques were randomly assigned (n = 10/group) to vehicle (control) or PTGER2 antagonist (BAY06) groups to perform a contraceptive trial. After the first treatment cycle, a male of proven fertility was introduced into each group and they remained housed together for the duration of the 5-month contraceptive trial that was followed by a post-treatment reversibility trial. Quantitative real-time PCR, COC culture and expansion, immunofluorescence/confocal microscopy, enzyme immunoassay, contraceptive trial, ultrasonography, complete blood counts, serum biochemistry tests

  6. A prostaglandin E2 receptor antagonist prevents pregnancies during a preclinical contraceptive trial with female macaques

    PubMed Central

    Peluffo, M.C.; Stanley, J.; Braeuer, N.; Rotgeri, A.; Fritzemeier, K.-H.; Fuhrmann, U.; Buchmann, B.; Adevai, T.; Murphy, M.J.; Zelinski, M.B.; Lindenthal, B.; Hennebold, J.D.; Stouffer, R.L.

    2014-01-01

    STUDY QUESTION Can administration of a prostaglandin (PG) E2 receptor 2 (PTGER2) antagonist prevent pregnancy in adult female monkeys by blocking periovulatory events in the follicle without altering menstrual cyclicity or general health? SUMMARY ANSWER This is the first study to demonstrate that a PTGER2 antagonist can serve as an effective non-hormonal contraceptive in primates. WHAT IS KNOWN ALREADY The requirement for PGE2 in ovulation and the release of an oocyte surrounded by expanded cumulus cells (cumulus–oocyte expansion; C-OE) was established through the generation of PTGS2 and PTGER2 null-mutant mice. A critical role for PGE2 in primate ovulation is supported by evidence that intrafollicular injection of indomethacin in rhesus monkeys suppressed follicle rupture, whereas co-injection of PGE2 with indomethacin resulted in ovulation. STUDY DESIGN, SIZE, DURATION First, controlled ovulation protocols were performed in adult, female rhesus monkeys to analyze the mRNA levels for genes encoding PGE2 synthesis and signaling components in the naturally selected pre-ovulatory follicle at different times after the ovulatory hCG stimulus (0, 12, 24, 36 h pre-ovulation; 36 h post-ovulation, n = 3–4/time point). Second, controlled ovarian stimulation cycles were utilized to obtain multiple cumulus–oocyte complexes (COCs) from rhesus monkeys to evaluate the role of PGE2 in C-OE in vitro (n = 3–4 animals/treatment; ≥3 COCs/animal/treatment). Third, adult cycling female cynomolgus macaques were randomly assigned (n = 10/group) to vehicle (control) or PTGER2 antagonist (BAY06) groups to perform a contraceptive trial. After the first treatment cycle, a male of proven fertility was introduced into each group and they remained housed together for the duration of the 5-month contraceptive trial that was followed by a post-treatment reversibility trial. PARTICIPANTS/MATERIALS, SETTING, METHODS Quantitative real-time PCR, COC culture and expansion, immunofluorescence

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

    PubMed Central

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

    2006-01-01

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

  8. The origins of enzyme kinetics.

    PubMed

    Cornish-Bowden, Athel

    2013-09-02

    The equation commonly called the Michaelis-Menten equation is sometimes attributed to other authors. However, although Victor Henri had derived the equation from the correct mechanism, and Adrian Brown before him had proposed the idea of enzyme saturation, it was Leonor Michaelis and Maud Menten who showed that this mechanism could also be deduced on the basis of an experimental approach that paid proper attention to pH and spontaneous changes in the product after formation in the enzyme-catalysed reaction. By using initial rates of reaction they avoided the complications due to substrate depletion, product accumulation and progressive inactivation of the enzyme that had made attempts to analyse complete time courses very difficult. Their methodology has remained the standard approach to steady-state enzyme kinetics ever since. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. Watching Individual Enzymes at Work

    NASA Astrophysics Data System (ADS)

    Blank, Kerstin; Rocha, Susana; De Cremer, Gert; Roeffaers, Maarten B. J.; Uji-i, Hiroshi; Hofkens, Johan

    Single-molecule fluorescence experiments are a powerful tool to analyze reaction mechanisms of enzymes. Because of their unique potential to detect heterogeneities in space and time, they have provided unprecedented insights into the nature and mechanisms of conformational changes related to the catalytic reaction. The most important finding from experiments with single enzymes is the generally observed phenomenon that the catalytic rate constants fluctuate over time (dynamic disorder). These fluctuations originate from conformational changes occurring on time scales, which are similar to or slower than that of the catalytic reaction. Here, we summarize experiments with enzymes that show dynamic disorder and introduce new experimental strategies showing how single-molecule fluorescence experiments can be applied to address other open questions in medical and industrial enzymology, such as enzyme inactivation processes, reactant transfer in cascade reactions, and the mechanisms of interfacial catalysis.

  10. Molecular Characterization of LubX: Functional Divergence of the U-Box Fold by Legionella pneumophila.

    PubMed

    Quaile, Andrew T; Urbanus, Malene L; Stogios, Peter J; Nocek, Boguslaw; Skarina, Tatiana; Ensminger, Alexander W; Savchenko, Alexei

    2015-08-04

    LubX is part of the large arsenal of effectors in Legionella pneumophila that are translocated into the host cytosol during infection. Despite such unique features as the presence of two U-box motifs and its targeting of another effector SidH, the molecular basis of LubX activity remains poorly understood. Here we show that the N terminus of LubX is able to activate an extended number of ubiquitin-conjugating (E2) enzymes including UBE2W, UBEL6, and all tested members of UBE2D and UBE2E families. Crystal structures of LubX alone and in complex with UBE2D2 revealed drastic molecular diversification between the two U-box domains, with only the N-terminal U-box retaining E2 recognition features typical for its eukaryotic counterparts. Extensive mutagenesis followed by functional screening in a yeast model system captured functionally important LubX residues including Arg121, critical for interactions with SidH. Combined, these data provide a new molecular insight into the function of this unique pathogenic factor. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. The Spindle Assembly Checkpoint Is Not Essential for Viability of Human Cells with Genetically Lowered APC/C Activity.

    PubMed

    Wild, Thomas; Larsen, Marie Sofie Yoo; Narita, Takeo; Schou, Julie; Nilsson, Jakob; Choudhary, Chunaram

    2016-03-01

    The anaphase-promoting complex/cyclosome (APC/C) and the spindle assembly checkpoint (SAC), which inhibits the APC/C, are essential determinants of mitotic timing and faithful division of genetic material. Activation of the APC/C is known to depend on two APC/C-interacting E2 ubiquitin-conjugating enzymes-UBE2C and UBE2S. We show that APC/C activity in human cells is tuned by the combinatorial use of three E2s, namely UBE2C, UBE2S, and UBE2D. Genetic deletion of UBE2C and UBE2S, individually or in combination, leads to discriminative reduction in APC/C function and sensitizes cells to UBE2D depletion. Reduction of APC/C activity results in loss of switch-like metaphase-to-anaphase transition and, strikingly, renders cells insensitive to chemical inhibition of MPS1 and genetic ablation of MAD2, both of which are essential for the SAC. These results provide insights into the regulation of APC/C activity and demonstrate that the essentiality of the SAC is imposed by the strength of the APC/C. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Enzymes: principles and biotechnological applications

    PubMed Central

    Robinson, Peter K.

    2015-01-01

    Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed. PMID:26504249

  13. Heat Stable Enzymes from Thermophiles

    DTIC Science & Technology

    1998-02-01

    final product and is somewhat messy to work with. Therefore, alternatives were tested. However, no combination of corn syrup , alternative sugars and...INTRODUCTION 9 CLONING OF ALKALINE PHOSPHATASE GENE AND PRODUCTION OF HIGH SPECIFIC ACTIVITY ENZYME 9 Cloning into E. coil and expression of high activity...JKR209, into an alternative, better producing organism. CLONING OF ALKALINE PHOSPHATASE GENE AND PRODUCTION OF HIGH SPECIFIC ACTIVITY ENZYME Cloning into

  14. Thymoquinone suppresses migration of LoVo human colon cancer cells by reducing prostaglandin E2 induced COX-2 activation.

    PubMed

    Hsu, Hsi-Hsien; Chen, Ming-Cheng; Day, Cecilia Hsuan; Lin, Yueh-Min; Li, Shin-Yi; Tu, Chuan-Chou; Padma, Viswanadha Vijaya; Shih, Hui-Nung; Kuo, Wei-Wen; Huang, Chih-Yang

    2017-02-21

    To identify potential anti-cancer constituents in natural extracts that inhibit cancer cell growth and migration. Our experiments used high dose thymoquinone (TQ) as an inhibitor to arrest LoVo (a human colon adenocarcinoma cell line) cancer cell growth, which was detected by cell proliferation assay and immunoblotting assay. Low dose TQ did not significantly reduce LoVo cancer cell growth. Cyclooxygenase 2 (COX-2) is an enzyme that is involved in the conversion of arachidonic acid into prostaglandin E2 (PGE2) in humans. PGE2 can promote COX-2 protein expression and tumor cell proliferation and was used as a control. Our results showed that 20 μmol/L TQ significantly reduced human LoVo colon cancer cell proliferation. TQ treatment reduced the levels of p-PI3K, p-Akt, p-GSK3β, and β-catenin and thereby inhibited the downstream COX-2 expression. Results also showed that the reduction in COX-2 expression resulted in a reduction in PGE2 levels and the suppression of EP2 and EP4 activation. Further analysis showed that TG treatment inhibited the nuclear translocation of β-catenin in LoVo cancer cells. The levels of the cofactors LEF-1 and TCF-4 were also decreased in the nucleus following TQ treatment in a dose-dependent manner. Treatment with low dose TQ inhibited the COX-2 expression at the transcriptional level and the regulation of COX-2 expression efficiently reduced LoVo cell migration. The results were further verified in vivo by confirming the effects of TQ and/or PGE2 using tumor xenografts in nude mice. TQ inhibits LoVo cancer cell growth and migration, and this result highlights the therapeutic advantage of using TQ in combination therapy against colorectal cancer.

  15. de novo computational enzyme design.

    PubMed

    Zanghellini, Alexandre

    2014-10-01

    Recent advances in systems and synthetic biology as well as metabolic engineering are poised to transform industrial biotechnology by allowing us to design cell factories for the sustainable production of valuable fuels and chemicals. To deliver on their promises, such cell factories, as much as their brick-and-mortar counterparts, will require appropriate catalysts, especially for classes of reactions that are not known to be catalyzed by enzymes in natural organisms. A recently developed methodology, de novo computational enzyme design can be used to create enzymes catalyzing novel reactions. Here we review the different classes of chemical reactions for which active protein catalysts have been designed as well as the results of detailed biochemical and structural characterization studies. We also discuss how combining de novo computational enzyme design with more traditional protein engineering techniques can alleviate the shortcomings of state-of-the-art computational design techniques and create novel enzymes with catalytic proficiencies on par with natural enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Second-order Born calculation of coplanar symmetric (e, 2e) process on Mg

    NASA Astrophysics Data System (ADS)

    Zhang, Yong-Zhi; Wang, Yang; Zhou, Ya-Jun

    2014-06-01

    The second-order distorted wave Born approximation (DWBA) method is employed to investigate the triple differential cross sections (TDCS) of coplanar doubly symmetric (e, 2e) collisions for magnesium at excess energies of 6 eV-20 eV. Comparing with the standard first-order DWBA calculations, the inclusion of the second-order Born term in the scattering amplitude improves the degree of agreement with experiments, especially for backward scattering region of TDCS. This indicates that the present second-order Born term is capable to give a reasonable correction to DWBA model in studying coplanar symmetric (e, 2e) problems of two-valence-electron target in low energy range.

  17. RANTES release by human airway smooth muscle: effects of prostaglandin E(2) and fenoterol.

    PubMed

    Lazzeri, N; Belvisi, M G; Patel, H J; Chung, K F; Yacoub, M H; Mitchell, J A

    2001-12-21

    In human airway smooth muscle cells, the levels of RANTES were increased upon stimulation with interleukin-1beta together with tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1) for each). In this study, we have assessed the effects of prostaglandin E(2) and the beta(2)-adrenoceptor agonist, fenoterol on RANTES (regulated upon activation, normal T cell expressed and secreted) release by these cells. The levels of RANTES released by human airway smooth muscle cells were measured after 24 h of treatment. Prostaglandin E(2) and fenoterol, only in presence of a cyclo-oxygenase inhibitor indomethacin (10(-6) M), provoked a concentration-dependent reduction in RANTES release. These data suggest that, in settings where cyclo-oxygenase activity is low, both drugs may relieve the symptoms of airway diseases by reducing RANTES production.

  18. The selective estrogen enzyme modulators in breast cancer: a review.

    PubMed

    Pasqualini, Jorge R

    2004-06-07

    It is well established that increased exposure to estradiol (E(2)) is an important risk factor for the genesis and evolution of breast tumors, most of which (approximately 95-97%) in their early stage are estrogen-sensitive. However, two thirds of breast cancers occur during the postmenopausal period when the ovaries have ceased to be functional. Despite the low levels of circulating estrogens, the tissular concentrations of these hormones are significantly higher than those found in the plasma or in the area of the breast considered as normal tissue, suggesting a specific tumoral biosynthesis and accumulation of these hormones. Several factors could be implicated in this process, including higher uptake of steroids from plasma and local formation of the potent E(2) by the breast cancer tissue itself. This information extends the concept of 'intracrinology' where a hormone can have its biological response in the same organ where it is produced. There is substantial information that mammary cancer tissue contains all the enzymes responsible for the local biosynthesis of E(2) from circulating precursors. Two principal pathways are implicated in the last steps of E(2) formation in breast cancer tissues: the 'aromatase pathway' which transforms androgens into estrogens, and the 'sulfatase pathway' which converts estrone sulfate (E(1)S) into E(1) by the estrone-sulfatase. The final step of steroidogenesis is the conversion of the weak E(1) to the potent biologically active E(2) by the action of a reductive 17beta-hydroxysteroid dehydrogenase type 1 activity (17beta-HSD-1). Quantitative evaluation indicates that in human breast tumor E(1)S 'via sulfatase' is a much more likely precursor for E(2) than is androgens 'via aromatase'. Human breast cancer tissue contains all the enzymes (estrone sulfatase, 17beta-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of E(2) biosynthesis. This tissue also contains sulfotransferase for the formation of the

  19. Spectroscopic ellipsometry study on E2 peak splitting of Si-Ge short period superlattices

    NASA Astrophysics Data System (ADS)

    Kim, Y. D.; Klein, M. V.; Baribeau, J.-M.; Hwang, S. H.; Whang, K. W.; Yoon, E.

    1997-06-01

    We report spectroscopic ellipsometry (SE) studies on (Si)2(Ge)12, (Si)6(Ge)2, and (Si)12(Ge)2 short period superlattices (SLs) whose optical response has not been reported yet. Multilayer calculations enabled us to determine the dielectric response of the superlattice layers. We report the clear observation of splitting of the E2 peak in (Si)m(Ge)n superlattices contrary to the previous SE report that the separation was observed only in larger period SLs.

  20. Advanced Stirling Convertor (ASC-E2) Performance Testing at NASA Glenn Research Center

    NASA Technical Reports Server (NTRS)

    Oriti, Salvatore; Wilson, Scott

    2011-01-01

    The National Aeronautics and Space Administration (NASA) Glenn Research Center (GRC) has been supporting development of the Advanced Stirling Radioisotope Generator (ASRG) since 2006. A key element of the ASRG Project is providing life, reliability, and performance testing of the Advanced Stirling Convertor (ASC). For this purpose, four pairs of ASCs capable of operating to 850 C and designated with the model number ASC-E2, were delivered by Sunpower of Athens, OH, to GRC in 2010. The ASC-E2s underwent a series of tests that included workmanship vibration testing, performance mapping, and extended operation. Workmanship vibration testing was performed following fabrication of each convertor to verify proper hardware build. Performance mapping consisted of operating each convertor at various conditions representing the range expected during a mission. Included were conditions representing beginning-of-mission (BOM), end-of-mission (EOM), and fueling. This same series of tests was performed by Sunpower prior to ASC-E2 delivery. The data generated during the GRC test were compared to performance before delivery. Extended operation consisted of a 500-hour period of operation with conditions maintained at the BOM point. This was performed to demonstrate steady convertor performance following performance mapping. Following this initial 500-hour period, the ASC-E2s will continue extended operation, controller development and special durability testing, during which the goal is to accumulate tens of thousands of hours of operation. Data collected during extended operation will support reliability analysis. Performance data from these tests is summarized in this paper.

  1. Advanced Stirling Convertor (ASC-E2) Performance Testing at NASA Glenn Research Center

    NASA Technical Reports Server (NTRS)

    Oriti, Salvatore; Wilson, Scott

    2011-01-01

    The National Aeronautics and Space Administration (NASA) Glenn Research Center (GRC) has been supporting development of the Advanced Stirling Radioisotope Generator (ASRG) since 2006. A key element of the ASRG Project is providing life, reliability, and performance testing of the Advanced Stirling Convertor (ASC). For this purpose, four pairs of ASCs capable of operating to 850 C and designated with the model number ASC-E2, were delivered by Sunpower of Athens, Ohio, to GRC in 2010. The ASC-E2s underwent a series of tests that included workmanship vibration testing, performance mapping, and extended operation. Workmanship vibration testing was performed following fabrication of each convertor to verify proper hardware build. Performance mapping consisted of operating each convertor at various conditions representing the range expected during a mission. Included were conditions representing beginning-of-mission (BOM), end-of-mission (EOM), and fueling. This same series of tests was performed by Sunpower prior to ASC-E2 delivery. The data generated during the GRC test were compared to performance before delivery. Extended operation consisted of a 500-hr period of operation with conditions maintained at the BOM point. This was performed to demonstrate steady convertor performance following performance mapping. Following this initial 500-hr period, the ASC-E2s will continue extended operation, controller development and special durability testing, during which the goal is to accumulate tens of thousands of hours of operation. Data collected during extended operation will support reliability analysis. Performance data from these tests is summarized in this paper.

  2. Probing the E2 properties of the scissors mode with real photons

    NASA Astrophysics Data System (ADS)

    Beck, Tobias; Pietralla, Norbert; Beller, Jacob; Derya, Vera; Löher, Bastian; Savran, Deniz; Tornow, Werner; Werner, Volker; Zilges, Andreas

    2018-05-01

    The E2/M1 multipole mixing ratio δ1→2 of the 1+ sc → 2+ 1 γ-ray transition of 156Gd and 164Dy has been measured using the linearly polarized photon beams of the HIγS facility. The employed method of photonscattering experiments in combination with polarized, quasi-monochromatic beams and a dedicated detector setup is highly sensitive to the electric quadrupole-decay properties of the scissors mode.

  3. Electrophile-modified lipoic derivatives of PDC-E2 elicits anti-mitochondrial antibody reactivity.

    PubMed

    Naiyanetr, Phornnop; Butler, Jeffrey D; Meng, Liping; Pfeiff, Janice; Kenny, Thomas P; Guggenheim, Kathryn G; Reiger, Roman; Lam, Kit; Kurth, Mark J; Ansari, Aftab A; Coppel, Ross L; López-Hoyos, Marcos; Gershwin, M Eric; Leung, Patrick S C

    2011-11-01

    Our laboratory has hypothesized that xenobiotic modification of the native lipoyl moiety of the major mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), may lead to loss of self-tolerance in primary biliary cirrhosis (PBC). This thesis is based on the finding of readily detectable levels of immunoreactivity of PBC sera against extensive panels of protein microarrays containing mimics of the inner lipoyl domain of PDC-E2 and subsequent quantitative structure-activity relationships (QSARs). Importantly, we have demonstrated that murine immunization with one such mimic, 2-octynoic acid coupled to bovine serum albumin (BSA), induces anti-mitochondrial antibodies (AMAs) and cholangitis. Based upon these data, we have focused on covalent modifications of the lipoic acid disulfide ring and subsequent analysis of such xenobiotics coupled to a 15mer of PDC-E2 for immunoreactivity against a broad panel of sera from patients with PBC and controls. Our results demonstrate that AMA-positive PBC sera demonstrate marked reactivity against 6,8-bis(acetylthio)octanoic acid, implying that chemical modification of the lipoyl ring, i.e. disruption of the S-S disulfide, renders lipoic acid to its reduced form that will promote xenobiotic modification. This observation is particularly significant in light of the function of the lipoyl moiety in electron transport of which the catalytic disulfide constantly opens and closes and, thus, raises the intriguing thesis that common electrophilic agents, i.e. acetaminophen or non-steroidal anti-inflammatory drugs (NSAIDs), may lead to xenobiotic modification in genetically susceptible individuals that results in the generation of AMAs and ultimately clinical PBC. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Effect of Higher Order Solvation and Temperature on SN2 and E2 Reactivity (Postprint)

    DTIC Science & Technology

    2014-07-05

    effects in microsolvated gas-phase E2 reactions, J. Am. Soc. Mass Spectrom. 18 (6) (2007) 1046–1051. [16] J.V. Seeley , R.A. Morris, A.A. Viggiano...17] J.V. Seeley , et al., Temperature dependence of the rate constants and branching ratios for the reactions of Cl(D2O)1–3 with CH3Br and thermal

  5. Salivary Prostaglandin E2: Role in Tick-Induced Allergy to Red Meat.

    PubMed

    Cabezas-Cruz, Alejandro; Mateos-Hernández, Lourdes; Chmelař, Jindrǐch; Villar, Margarita; de la Fuente, José

    2017-07-01

    Tick-induced allergy to red meat is associated with anti-α-Gal IgE antibody levels. We propose that tick salivary prostaglandin E2 triggers antibody class switching in mature B cells, increasing the levels of anti-α-Gal IgE antibodies. Immune tolerance to α-Gal in blood type B individuals might reduce the risk to this allergy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Genetic variation in the prostaglandin E2 pathway is associated with primary graft dysfunction.

    PubMed

    Diamond, Joshua M; Akimova, Tatiana; Kazi, Altaf; Shah, Rupal J; Cantu, Edward; Feng, Rui; Levine, Matthew H; Kawut, Steven M; Meyer, Nuala J; Lee, James C; Hancock, Wayne W; Aplenc, Richard; Ware, Lorraine B; Palmer, Scott M; Bhorade, Sangeeta; Lama, Vibha N; Weinacker, Ann; Orens, Jonathan; Wille, Keith; Crespo, Maria; Lederer, David J; Arcasoy, Selim; Demissie, Ejigayehu; Christie, Jason D

    2014-03-01

    Biologic pathways with significant genetic conservation across human populations have been implicated in the pathogenesis of primary graft dysfunction (PGD). The evaluation of the role of recipient genetic variation in PGD has thus far been limited to single, candidate gene analyses. We sought to identify genetic variants in lung transplant recipients that are responsible for increased risk of PGD using a two-phase large-scale genotyping approach. Phase 1 was a large-scale candidate gene association study of the multicenter, prospective Lung Transplant Outcomes Group cohort. Phase 2 included functional evaluation of selected variants and a bioinformatics screening of variants identified in phase 1. After genetic data quality control, 680 lung transplant recipients were included in the analysis. In phase 1, a total of 17 variants were significantly associated with PGD, four of which were in the prostaglandin E2 family of genes. Among these were a coding variant in the gene encoding prostaglandin E2 synthase (PTGES2; P = 9.3 × 10(-5)) resulting in an arginine to histidine substitution at amino acid position 298, and three variants in a block containing the 5' promoter and first intron of the PTGER4 gene (encoding prostaglandin E2 receptor subtype 4; all P < 5 × 10(-5)). Functional evaluation in regulatory T cells identified that rs4434423A in the PTGER4 gene was associated with differential suppressive function of regulatory T cells. Further research aimed at replication and additional functional insight into the role played by genetic variation in prostaglandin E2 synthetic and signaling pathways in PGD is warranted.

  7. Genetic Variation in the Prostaglandin E2 Pathway Is Associated with Primary Graft Dysfunction

    PubMed Central

    Akimova, Tatiana; Kazi, Altaf; Shah, Rupal J.; Cantu, Edward; Feng, Rui; Levine, Matthew H.; Kawut, Steven M.; Meyer, Nuala J.; Lee, James C.; Hancock, Wayne W.; Aplenc, Richard; Ware, Lorraine B.; Palmer, Scott M.; Bhorade, Sangeeta; Lama, Vibha N.; Weinacker, Ann; Orens, Jonathan; Wille, Keith; Crespo, Maria; Lederer, David J.; Arcasoy, Selim; Demissie, Ejigayehu; Christie, Jason D.

    2014-01-01

    Rationale: Biologic pathways with significant genetic conservation across human populations have been implicated in the pathogenesis of primary graft dysfunction (PGD). The evaluation of the role of recipient genetic variation in PGD has thus far been limited to single, candidate gene analyses. Objectives: We sought to identify genetic variants in lung transplant recipients that are responsible for increased risk of PGD using a two-phase large-scale genotyping approach. Methods: Phase 1 was a large-scale candidate gene association study of the multicenter, prospective Lung Transplant Outcomes Group cohort. Phase 2 included functional evaluation of selected variants and a bioinformatics screening of variants identified in phase 1. Measurements and Main Results: After genetic data quality control, 680 lung transplant recipients were included in the analysis. In phase 1, a total of 17 variants were significantly associated with PGD, four of which were in the prostaglandin E2 family of genes. Among these were a coding variant in the gene encoding prostaglandin E2 synthase (PTGES2; P = 9.3 × 10−5) resulting in an arginine to histidine substitution at amino acid position 298, and three variants in a block containing the 5′ promoter and first intron of the PTGER4 gene (encoding prostaglandin E2 receptor subtype 4; all P < 5 × 10−5). Functional evaluation in regulatory T cells identified that rs4434423A in the PTGER4 gene was associated with differential suppressive function of regulatory T cells. Conclusions: Further research aimed at replication and additional functional insight into the role played by genetic variation in prostaglandin E2 synthetic and signaling pathways in PGD is warranted. PMID:24467603

  8. Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

    SciTech Connect

    Kanginakudru, Sriramana, E-mail: skangina@iu.edu; DeSmet, Marsha, E-mail: mdesmet@iupui.edu; Thomas, Yanique, E-mail: ysthomas@umail.iu.edu

    2015-04-15

    The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reducedmore » viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication.« less

  9. E2FM: an encrypted and compressed full-text index for collections of genomic sequences.

    PubMed

    Montecuollo, Ferdinando; Schmid, Giovannni; Tagliaferri, Roberto

    2017-09-15

    Next Generation Sequencing (NGS) platforms and, more generally, high-throughput technologies are giving rise to an exponential growth in the size of nucleotide sequence databases. Moreover, many emerging applications of nucleotide datasets-as those related to personalized medicine-require the compliance with regulations about the storage and processing of sensitive data. We have designed and carefully engineered E 2 FM -index, a new full-text index in minute space which was optimized for compressing and encrypting nucleotide sequence collections in FASTA format and for performing fast pattern-search queries. E 2 FM -index allows to build self-indexes which occupy till to 1/20 of the storage required by the input FASTA file, thus permitting to save about 95% of storage when indexing collections of highly similar sequences; moreover, it can exactly search the built indexes for patterns in times ranging from few milliseconds to a few hundreds milliseconds, depending on pattern length. Source code is available at https://github.com/montecuollo/E2FM . ferdinando.montecuollo@unicampania.it. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  10. E2F4 Promotes Neuronal Regeneration and Functional Recovery after Spinal Cord Injury in Zebrafish

    PubMed Central

    Sasagawa, Shota; Nishimura, Yuhei; Hayakawa, Yuka; Murakami, Soichiro; Ashikawa, Yoshifumi; Yuge, Mizuki; Okabe, Shiko; Kawaguchi, Koki; Kawase, Reiko; Tanaka, Toshio

    2016-01-01

    Mammals exhibit poor recovery after spinal cord injury (SCI), whereas non-mammalian vertebrates exhibit significant spontaneous recovery after SCI. The mechanisms underlying this difference have not been fully elucidated; therefore, the purpose of this study was to investigate these mechanisms. Using comparative transcriptome analysis, we demonstrated that genes related to cell cycle were significantly enriched in the genes specifically dysregulated in zebrafish SCI. Most of the cell cycle-related genes dysregulated in zebrafish SCI were down-regulated, possibly through activation of e2f4. Using a larval zebrafish model of SCI, we demonstrated that the recovery of locomotive function and neuronal regeneration after SCI were significantly inhibited in zebrafish treated with an E2F4 inhibitor. These results suggest that activation of e2f4 after SCI may be responsible, at least in part, for the significant recovery in zebrafish. This provides novel insight into the lack of recovery after SCI in mammals and informs potential therapeutic strategies. PMID:27242526

  11. A novel E2 box-GATA element modulates Cdc6 transcription during human cells polyploidization

    PubMed Central

    Vilaboa, Nuria; Bermejo, Rodrigo; Martinez, Pilar; Bornstein, Rafael; Calés, Carmela

    2004-01-01

    Cdc6 is a key regulator of the strict alternation of S and M phases during the mitotic cell cycle. In mammalian and plant cells that physiologically become polyploid, cdc6 is transcriptionally and post-translationally regulated. We have recently reported that Cdc6 levels are maintained in megakaryoblastic HEL cells, but severely downregulated by ectopic expression of transcriptional repressor Drosophila melanogaster escargot. Here, we show that cdc6 promoter activity is upregulated during megakaryocytic differentiation of HEL endoreplicating cells, and that Escargot interferes with such activation. Transactivation experiments showed that a 1.7 kb region located at 2800 upstream cdc6 transcription initiation site behaved as a potent enhancer in endoreplicating cells only. This activity was mainly dependent on a novel cis-regulatory element composed by an E2 box overlapping a GATA motif. Ectopic Escargot could bind this regulatory element in vitro and endogenous GATA-1 and E2A formed specific complexes in megakaryoblastic cells as well as in primary megakaryocytes. Chromatin Immunoprecipitation analysis revealed that both transcription factors were occupying the E2 box/GATA site in vivo. Altogether, these data suggest that cdc6 expression could be actively maintained during megakaryocytic differentiation through transcriptional mechanisms involving specific cis- and trans-regulatory elements. PMID:15590906

  12. CMIP5 Historical Simulations (1850-2012) with GISS ModelE2

    NASA Technical Reports Server (NTRS)

    Miller, Ronald Lindsay; Schmidt, Gavin A.; Nazarenko, Larissa S.; Tausnev, Nick; Bauer, Susanne E.; DelGenio, Anthony D.; Kelley, Max; Lo, Ken K.; Ruedy, Reto; Shindell, Drew T.; hide

    2014-01-01

    Observations of climate change during the CMIP5 extended historical period (1850-2012) are compared to trends simulated by six versions of the NASA Goddard Institute for Space Studies ModelE2 Earth System Model. The six models are constructed from three versions of the ModelE2 atmospheric general circulation model, distinguished by their treatment of atmospheric composition and the aerosol indirect effect, combined with two ocean general circulation models, HYCOM and Russell. Forcings that perturb the model climate during the historical period are described. Five-member ensemble averages from each of the six versions of ModelE2 simulate trends of surface air temperature, atmospheric temperature, sea ice and ocean heat content that are in general agreement with observed trends, although simulated warming is slightly excessive within the past decade. Only simulations that include increasing concentrations of long-lived greenhouse gases match the warming observed during the twentieth century. Differences in twentieth-century warming among the six model versions can be attributed to differences in climate sensitivity, aerosol and ozone forcing, and heat uptake by the deep ocean. Coupled models with HYCOM export less heat to the deep ocean, associated with reduced surface warming in regions of deepwater formation, but greater warming elsewhere at high latitudes along with reduced sea ice. All ensembles show twentieth-century annular trends toward reduced surface pressure at southern high latitudes and a poleward shift of the midlatitude westerlies, consistent with observations.

  13. Conservation of direct dynamics in sterically hindered SN2/E2 reactions.

    PubMed

    Carrascosa, Eduardo; Meyer, Jennifer; Michaelsen, Tim; Stei, Martin; Wester, Roland

    2018-01-21

    Nucleophilic substitution (S N 2) and base-induced elimination (E2), two indispensable reactions in organic synthesis, are commonly assumed to proceed under stereospecific conditions. Understanding the way in which the reactants pre-orient in these reactions, that is its stereodynamics, is essential in order to achieve a detailed atomistic picture and control over such processes. Using crossed beam velocity map imaging, we study the effect of steric hindrance in reactions of Cl - and CN - with increasingly methylated alkyl iodides by monitoring the product ion energy and scattering angle. For both attacking anions the rebound mechanism, indicative of a direct S N 2 pathway, is found to contribute to the reaction at high relative collision energies despite being increasingly hindered. An additional forward scattering mechanism, ascribed to a direct E2 reaction, also contributes at these energies. Inspection of the product energy distributions confirms the direct and fast character of both mechanisms as opposed to an indirect reaction mechanism which leads to statistical energy redistribution in the reaction complex. This work demonstrates that nonstatistical dynamics and energetics govern S N 2 and E2 pathways even in sterically hindered exchange reaction systems.

  14. Conservation of direct dynamics in sterically hindered SN2/E2 reactions

    PubMed Central

    Carrascosa, Eduardo; Meyer, Jennifer; Michaelsen, Tim; Stei, Martin

    2017-01-01

    Nucleophilic substitution (SN2) and base-induced elimination (E2), two indispensable reactions in organic synthesis, are commonly assumed to proceed under stereospecific conditions. Understanding the way in which the reactants pre-orient in these reactions, that is its stereodynamics, is essential in order to achieve a detailed atomistic picture and control over such processes. Using crossed beam velocity map imaging, we study the effect of steric hindrance in reactions of Cl– and CN– with increasingly methylated alkyl iodides by monitoring the product ion energy and scattering angle. For both attacking anions the rebound mechanism, indicative of a direct SN2 pathway, is found to contribute to the reaction at high relative collision energies despite being increasingly hindered. An additional forward scattering mechanism, ascribed to a direct E2 reaction, also contributes at these energies. Inspection of the product energy distributions confirms the direct and fast character of both mechanisms as opposed to an indirect reaction mechanism which leads to statistical energy redistribution in the reaction complex. This work demonstrates that nonstatistical dynamics and energetics govern SN2 and E2 pathways even in sterically hindered exchange reaction systems. PMID:29629138

  15. Studies on orientation and rotation parameters of 4179 Toutatis from Chang'e-2 mission

    NASA Astrophysics Data System (ADS)

    Zhao, Yuhui; Ji, Jianghui; Hu, Shoucun

    The ginger-shaped near-Earth asteroid 4179 Toutatis is close to a 4:1 orbital resonance with the Earth and has made close Earth flybys approximately every four years in the recent 20 years. China’s lunar probe Chang’e-2 achieved a successful flyby the Toutatis on 13th Dec 2012 during its most recent flyby of Earth. During the mission, a series of image with high resolution has been obtained. Combined with the radar model of Toutatis, these figures show the attitude of the asteroid from the camera’s point of view and the orientation of it is then deduced based on the attitude of the camera and the relative position between 4179 Toutatis and Chang'e-2 in our works. According to the previous ground-based observations and works on the rotation parameters of Toutatis, this paper studies the rotating rate of the asteroid in accordance with the imaging result of Toutatis by Chang’e-2 and puts forward a correction to the spin rate parameters.

  16. Engineering Cellulase Enzymes for Bioenergy

    NASA Astrophysics Data System (ADS)

    Atreya, Meera Elizabeth

    Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

  17. Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

    PubMed Central

    Xie, Chuan-Ming; Wei, Wenyi; Sun, Yi

    2013-01-01

    Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainly controlled by the ubiquitin—proteasome system (UPS), which consists of two distinct steps: (1) ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and (2) subsequent degradation by the 26S proteasome. Among all E3 ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such as cancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore, altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis. PMID:23522382

  18. E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

    SciTech Connect

    Fang, Zejun; Gong, Chaoju; Liu, Hong

    2015-08-21

    As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression ofmore » E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration

  19. Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses.

    PubMed

    Azad, Kimi; Banerjee, Manidipa; Johnson, John E

    2017-09-29

    Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

  20. Regulation of 11 beta-hydroxysteroid dehydrogenase enzymes in the rat kidney by estradiol.

    PubMed

    Gomez-Sanchez, Elise P; Ganjam, Venkataseshu; Chen, Yuan Jian; Liu, Ying; Zhou, Ming Yi; Toroslu, Cigdem; Romero, Damian G; Hughson, Michael D; de Rodriguez, Angela; Gomez-Sanchez, Celso E

    2003-08-01

    The 11beta-hydroxysteroid dehydrogenase (11betaHSD) type 1 (11betaHSD1) enzyme is an NADP+-dependent oxidoreductase, usually reductase, of major glucocorticoids. The NAD+-dependent type 2 (11betaHSD2) enzyme is an oxidase that inactivates cortisol and corticosterone, conferring extrinsic specificity of the mineralocorticoid receptor for aldosterone. We reported that addition of a reducing agent to renal homogenates results in the monomerization of 11betaHSD2 dimers and a significant increase in NAD+-dependent corticosterone conversion. Estrogenic effects on expression, dimerization, and activity of the kidney 11betaHSD1 and -2 enzymes are described herein. Renal 11betaHSD1 mRNA and protein expressions were decreased to very low levels by estradiol (E2) treatment of both intact and castrated male rats; testosterone had no effect. NADP+-dependent enzymatic activity of renal homogenates from E2-treated rats measured under nonreducing conditions was less than that of homogenates from intact animals. Addition of 10 mM DTT to aliquots from these same homogenates abrogated the difference in NADP+-dependent activity between E2-treated and control rats. In contrast, 11betaHSD2 mRNA and protein expressions were significantly increased by E2 treatment. There was a marked increase in the number of juxtamedullary proximal tubules stained by the antibody against 11betaHSD2 after the administration of E2. Notwithstanding, neither the total corticosterone and 11-dehydrocorticosterone excreted in the urine nor their ratio differed between E2- and vehicle-treated rats. NAD+-dependent enzymatic activity in the absence or presence of a reducing agent demonstrated that the increase in 11betaHSD2 protein was not associated with an increase in in vitro activity unless the dimers were reduced to monomers.

  1. Nonclassical Kinetics of Clonal yet Heterogeneous Enzymes.

    PubMed

    Park, Seong Jun; Song, Sanggeun; Jeong, In-Chun; Koh, Hye Ran; Kim, Ji-Hyun; Sung, Jaeyoung

    2017-07-06

    Enzyme-to-enzyme variation in the catalytic rate is ubiquitous among single enzymes created from the same genetic information, which persists over the lifetimes of living cells. Despite advances in single-enzyme technologies, the lack of an enzyme reaction model accounting for the heterogeneous activity of single enzymes has hindered a quantitative understanding of the nonclassical stochastic outcome of single enzyme systems. Here we present a new statistical kinetics and exactly solvable models for clonal yet heterogeneous enzymes with possibly nonergodic state dynamics and state-dependent reactivity, which enable a quantitative understanding of modern single-enzyme experimental results for the mean and fluctuation in the number of product molecules created by single enzymes. We also propose a new experimental measure of the heterogeneity and nonergodicity for a system of enzymes.

  2. The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.

    PubMed

    Miles, Jennifer A; Frost, Mark G; Carroll, Eilis; Rowe, Michelle L; Howard, Mark J; Sidhu, Ateesh; Chaugule, Viduth K; Alpi, Arno F; Walden, Helen

    2015-08-21

    The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL*

    PubMed Central

    Miles, Jennifer A.; Frost, Mark G.; Carroll, Eilis; Rowe, Michelle L.; Howard, Mark J.; Sidhu, Ateesh; Chaugule, Viduth K.; Alpi, Arno F.; Walden, Helen

    2015-01-01

    The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo. PMID:26149689

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. 40 CFR Table E-2 to Subpart E of... - Spectral Energy Distribution and Permitted Tolerance for Conducting Radiative Tests

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  7. Subcellular localization of pituitary enzymes

    NASA Technical Reports Server (NTRS)

    Smith, R. E.

    1970-01-01

    A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

  8. Micromotors Powered by Enzyme Catalysis.

    PubMed

    Dey, Krishna K; Zhao, Xi; Tansi, Benjamin M; Méndez-Ortiz, Wilfredo J; Córdova-Figueroa, Ubaldo M; Golestanian, Ramin; Sen, Ayusman

    2015-12-09

    Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymatic reactions and their directed movement in substrate concentration gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the respective substrates, using optical microscopy and dynamic light scattering analysis. The diffusion of the particles was found to increase in a substrate concentration dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture.

  9. Supramolecular catalysis beyond enzyme mimics.

    PubMed

    Meeuwissen, Jurjen; Reek, Joost N H

    2010-08-01

    Supramolecular catalysis - the assembly of catalyst species by harnessing multiple weak intramolecular interactions - has, until recently, been dominated by enzyme-inspired approaches. Such approaches often attempt to create an enzyme-like 'active site' and have concentrated on reactions similar to those catalysed by enzymes themselves. Here, we discuss the application of supramolecular assembly to the more traditional transition metal catalysis and to small-molecule organocatalysis. The modularity of self-assembled multicomponent catalysts means that a relatively small pool of catalyst components can provide rapid access to a large number of catalysts that can be evaluated for industrially relevant reactions. In addition, we discuss how catalyst-substrate interactions can be tailored to direct substrates along particular reaction paths and selectivities.

  10. E2f1 mediates high glucose-induced neuronal death in cultured mouse retinal explants.

    PubMed

    Wang, Yujiao; Zhou, Yi; Xiao, Lirong; Zheng, Shijie; Yan, Naihong; Chen, Danian

    2017-10-02

    Diabetic retinopathy (DR) is the most common complication of diabetes and remains one of the major causes of blindness in the world; infants born to diabetic mothers have higher risk of developing retinopathy of prematurity (ROP). While hyperglycemia is a major risk factor, the molecular and cellular mechanisms underlying DR and diabetic ROP are poorly understood. To explore the consequences of retinal cells under high glucose, we cultured wild type or E2f1 -/- mouse retinal explants from postnatal day 8 with normal glucose, high osmotic or high glucose media. Explants were also incubated with cobalt chloride (CoCl 2 ) to mimic the hypoxic condition. We showed that, at 7 days post exposure to high glucose, retinal explants displayed elevated cell death, ectopic cell division and intact retinal vascular plexus. Cell death mainly occurred in excitatory neurons, such as ganglion and bipolar cells, which were also ectopically dividing. Many Müller glial cells reentered the cell cycle; some had irregular morphology or migrated to other layers. High glucose inhibited the hyperoxia-induced blood vessel regression of retinal explants. Moreover, inactivation of E2f1 rescued high glucose-induced ectopic division and cell death of retinal neurons, but not ectopic cell division of Müller glial cells and vascular phenotypes. This suggests that high glucose has direct but distinct effects on retinal neurons, glial cells and blood vessels, and that E2f1 mediates its effects on retinal neurons. These findings shed new light onto mechanisms of DR and the fetal retinal abnormalities associated with maternal diabetes, and suggest possible new therapeutic strategies.

  11. [Induced abortion using prostaglandin E2 and F2alpha gel].

    PubMed

    Lippert, T H; Modly, T

    1974-01-01

    In this study of 20 patients in the 13th-17th week of pregnancy abortion was induced with intrauterine, extraamniotic application of prostaglandins (PG) E2 or F2 in gel form. The gel composition was as follows: 4% tylose MH 300, 2% glycerine, 1% chlorhexidine digluconate, 83% sterile distilled water and 10% PG stock solution. Both PGE2 and PGF2 gels were used. Final concentration was 2.5 mg E2 or 2.5 mg F2 per g of gel. Gel was applied via transcervical, extraamniotic polyethylene catheter every 2-3 hours. Results: PGE2-gel was used in 14 cases. After 3-4 applications both fetus and placenta were expelled. Average dose used was 4.6 mg E2/patient. First contractions started in 30 minutes; induction to expulsion time was 11 hours 35 minutes. F2-gel given to 6 patients resulted in expulsion of the fetus in all cases but placenta needed removal by curettage in 4 patients. Average dose per patient was 17.7 mg of F2; first contractions in 30 minutes, average expulsion time 17 hours 38 minutes. With both PGs there were painful contractions which were controlled with a combination of pentazocine and Valium. PGE2 caused vomiting in 5 patients. No increased bleeding or postabortion infection occurred. Follow-up curettage was done in all patients to ensure removal of all tissues. Overall evaluation of the PG-gels was considered good. PG stability in gel form is good; during 8 months of preservation in sterile aluminum tubes at -25 degrees Celsius no decline in clinical effectiveness was noted. The gel application is less expensive than the slow-injection pump method.

  12. Electronic, magnetic, transport, and thermal properties of single-crystalline UF e2A l10

    NASA Astrophysics Data System (ADS)

    Troć, R.; Samsel-Czekała, M.; Talik, E.; Wawryk, R.; Gajek, Z.; Pasturel, M.

    2015-09-01

    The valence and core-level x-ray photoemission spectra (XPS), performed on an UF e2A l10 single crystal, were measured using the Al Kα radiation. The results of valence XPS show practically two separate regions of spectral intensity, one just at the Fermi level (EF) and the other one being a wide content with its maximum at about 0.8 eV below EF. These give rise to two electronic configurations of the 5 f states in the studied aluminide, itinerant and localized ones, i.e., their dual character. In such a situation the corresponding valence spectra, calculated within the local density approximation (LDA), well explain the former configuration, being responsible for a metallic behavior of the studied compound. Moreover, this behavior is confirmed clearly also by our results of magnetotransport measurements. On the other hand, the obtained magnetic susceptibility, specific heat, electrical resistivity, and thermoelectric power data support very well the local character of the 5 f2 -electron configuration of the U4 + ion in UF e2A l10 having the orthorhombic and cage-type crystal structure. Based on that configuration, the magnetic and thermal characteristics of the compound were modeled by the effective crystal field (CF) potential in the intermediate coupling scheme using initial parameters obtained by the angular overlap model (AOM). The obtained final CF parameters yielded the CF level scheme, composed of only singlets, proper for orthorhombic symmetry. Such a set of singlets reproduces in a satisfactory way both the strongly anisotropic temperature variations of the magnetic susceptibility, measured along the three main crystallographic directions, as well as the Schottky anomaly, evaluated using specific heat results of isomorphic ThF e2A l10 as a phonon reference. Also, the strongly anisotropic behavior of the Seebeck coefficient and its low temperature maxima observed for the compound studied here have been explained roughly by the CF effect.

  13. S. pombe Uba1-Ubc15 Structure Reveals a Novel Regulatory Mechanism of Ubiquitin E2 Activity.

    PubMed

    Lv, Zongyang; Rickman, Kimberly A; Yuan, Lingmin; Williams, Katelyn; Selvam, Shanmugam Panneer; Woosley, Alec N; Howe, Philip H; Ogretmen, Besim; Smogorzewska, Agata; Olsen, Shaun K

    2017-02-16

    Ubiquitin (Ub) E1 initiates the Ub conjugation cascade by activating and transferring Ub to tens of different E2s. How Ub E1 cooperates with E2s that differ substantially in their predicted E1-interacting residues is unknown. Here, we report the structure of S. pombe Uba1 in complex with Ubc15, a Ub E2 with intrinsically low E1-E2 Ub thioester transfer activity. The structure reveals a distinct Ubc15 binding mode that substantially alters the network of interactions at the E1-E2 interface compared to the only other available Ub E1-E2 structure. Structure-function analysis reveals that the intrinsically low activity of Ubc15 largely results from the presence of an acidic residue at its N-terminal region. Notably, Ub E2 N termini are serine/threonine rich in many other Ub E2s, leading us to hypothesize that phosphorylation of these sites may serve as a novel negative regulatory mechanism of Ub E2 activity, which we demonstrate biochemically and in cell-based assays. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    DOE PAGES

    Nazarenko, L.; Schmidt, G. A.; Miller, R. L.; ...

    2015-02-24

    We examine the anthropogenically forced climate response for the 21st century representative concentration pathway (RCP) emission scenarios and their extensions for the period 2101–2500. The experiments were performed with ModelE2, a new version of the NASA Goddard Institute for Space Sciences (GISS) coupled general circulation model that includes three different versions for the atmospheric composition components: a noninteractive version (NINT) with prescribed composition and a tuned aerosol indirect effect (AIE), the TCAD version with fully interactive aerosols, whole-atmosphere chemistry, and the tuned AIE, and the TCADI version which further includes a parameterized first indirect aerosol effect on clouds. Each atmosphericmore » version is coupled to two different ocean general circulation models: the Russell ocean model (GISS-E2-R) and HYCOM (GISS-E2-H). By 2100, global mean warming in the RCP scenarios ranges from 1.0 to 4.5° C relative to 1850–1860 mean temperature in the historical simulations. In the RCP2.6 scenario, the surface warming in all simulations stays below a 2 °C threshold at the end of the 21st century. For RCP8.5, the range is 3.5–4.5° C at 2100. Decadally averaged sea ice area changes are highly correlated to global mean surface air temperature anomalies and show steep declines in both hemispheres, with a larger sensitivity during winter months. By the year 2500, there are complete recoveries of the globally averaged surface air temperature for all versions of the GISS climate model in the low-forcing scenario RCP2.6. TCADI simulations show enhanced warming due to greater sensitivity to CO₂, aerosol effects, and greater methane feedbacks, and recovery is much slower in RCP2.6 than with the NINT and TCAD versions. All coupled models have decreases in the Atlantic overturning stream function by 2100. In RCP2.6, there is a complete recovery of the Atlantic overturning stream function by the year 2500 while with scenario RCP8.5, the

  15. (e,2e) and (Î3,2e) Processes: Open and Closed Questions

    NASA Astrophysics Data System (ADS)

    An important breakthrough has been achieved recently in the description of (e,2e) and (Î3,2e) processes with the development of new ab-initio theories: the external complex scaling theory (ECS), the time dependent close coupling theory (TDCC), and the hyperspherical R-matrix theory with semiclassical outgoing waves (HRM-SOW). The principles of these various theories are summarized, their relations are considered, and their achievements are discussed with respect to the available experimental data regarding electron impact ionization of H and photo double ionization of He. Possible directions for future work are outlined.

  16. X-ray spectroscopy of E2 and M3 transitions in Ni-like W

    SciTech Connect

    Clementson, J.; Beiersdorfer, P.; Gu, M. F.

    2010-01-15

    The electric quadrupole (E2) and magnetic octupole (M3) ground-state transitions in Ni-like W{sup 46+} have been measured using high-resolution crystal spectroscopy at the LLNL electron-beam ion trap facility. The lines fall in the soft x-ray region near 7.93 A and were originally observed as an unresolved feature in tokamak plasmas. Using flat ammonium dihydrogen phosphate and quartz crystals, the wavelengths, intensities, and polarizations of the two lines have been measured for various electron-beam energies and compared to intensity and polarization calculations performed using the Flexible Atomic Code (FAC).

  17. X-ray Spectroscopy of E2 and M3 Transitions in Ni-like W

    SciTech Connect

    Clementson, J; Beiersdorfer, P; Gu, M F

    2009-11-09

    The electric quadrupole (E2) and magnetic octupole (M3) ground state transitions in Ni-like W{sup 46+} have been measured using high-resolution crystal spectroscopy at the Livermore electron beam ion trap facility. The lines fall in the soft x-ray region near 7.93 {angstrom} and were originally observed as an unresolved feature in tokamak plasmas. Using flat ADP and quartz crystals the wavelengths, intensities, and polarizations of the two lines have been measured for various electron beam energies and compared to intensity and polarization calculations performed using the Flexible Atomic Code (FAC).

  18. Internal friction in enzyme reactions.

    PubMed

    Rauscher, Anna; Derényi, Imre; Gráf, László; Málnási-Csizmadia, András

    2013-01-01

    The empirical concept of internal friction was introduced 20 years ago. This review summarizes the results of experimental and theoretical studies that help to uncover the nature of internal friction. After the history of the concept, we describe the experimental challenges in measuring and interpreting internal friction based on the viscosity dependence of enzyme reactions. We also present speculations about the structural background of this viscosity dependence. Finally, some models about the relationship between the energy landscape and internal friction are outlined. Alternative concepts regarding the viscosity dependence of enzyme reactions are also discussed. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

  19. Nickel-based Enzyme Systems*

    PubMed Central

    Ragsdale, Stephen W.

    2009-01-01

    Of the eight known nickel enzymes, all but glyoxylase I catalyze the use and/or production of gases central to the global carbon, nitrogen, and oxygen cycles. Nickel appears to have been selected for its plasticity in coordination and redox chemistry and is able to cycle through three redox states (1+, 2+, 3+) and to catalyze reactions spanning ∼1.5 V. This minireview focuses on the catalytic mechanisms of nickel enzymes, with an emphasis on the role(s) of the metal center. The metal centers vary from mononuclear to complex metal clusters and catalyze simple hydrolytic to multistep redox reactions. PMID:19363030

  20. Potential drugs which activate nuclear factor E2-related factor 2 signaling to prevent diabetic cardiovascular complications: A focus on fumaric acid esters.

    PubMed

    Zhou, Shanshan; Jin, Jingpeng; Bai, Tao; Sachleben, Leroy R; Cai, Lu; Zheng, Yang

    2015-08-01

    Diabetes and its cardiovascular complications have been a major public health issue. These complications are mainly attributable to a severe imbalance between free radical and reactive oxygen species production and the antioxidant defense systems. Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that controls the basal and inducible expression of a battery of antioxidant enzyme genes and other cyto-protective phase II detoxifying enzymes. As a result, Nrf2 has gained great attention as a promising drug target for preventing diabetic cardiovascular complications. And while animal studies have shown that several Nrf2 activators manifest a potential to efficiently prevent the diabetic complications, their use in humans has not been approved due to the lack of substantial evidence regarding safety and efficacy of the Nrf2 activation. We provide here a brief review of a few clinically-used drugs that can up-regulate Nrf2 with the potential of extending their usage to diabetic patients for the prevention of cardiovascular complications and conclude with a closer inspection of dimethyl fumarate and its mimic members. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Taking the Mystery Out of Enzymes.

    ERIC Educational Resources Information Center

    DeYoung, H. Garrett

    1984-01-01

    Discusses structure and function of enzymes, design of new enzymes and enzyme substitutes, and enzyme uses in industry, medicine, and wastewater treatment. The latter is a low-cost method which can remove as much as 99 percent of toxic substances found in many industrial wastewater streams. (JN)

  2. The X-ray Mirrors for the Astro-E2 Mission

    NASA Technical Reports Server (NTRS)

    Chan, Kai-Wing; Soong, Yang; Serlemitsos, Peter J.; White, Nicholas E. (Technical Monitor)

    2002-01-01

    The X-Ray telescopes (XRT) for the US/Japan collaborative mission Astro-E2 will be of the same basic design as those built for the original Astro-E mission which failed to reach orbit in Feb. 2000. The NASA/GSFC X-ray Astrophysics Branch will again provide the five lightweight, broad-band mirrors for the mission. X-ray calibrations of the mirrors delivered for the original Astro-E instrument showed spatial resolutions characterized by Half-Power Diameters (HPD) in the range of 1.8 - 2.2 minutes of arc, essentially independent of photon energy in the soft X-ray band. For the mission Astro-E2, both funding constraints and management decisions drastically limit any design modifications, so reflector fabrication and assembly procedures have remained largely unchanged. Nevertheless, in view of the importance in scientific return of attaining even a modest improvement in the spatial resolution of these mirrors, we have carefully considered the various sources of spatial error and, whenever possible, incorporated promising modifications. In this paper, we discuss our current understanding of the various error components as well as the small changes we have been able to implement.

  3. Detection of ethylene glycol - toward W51/e2 and G34.3+0.02

    NASA Astrophysics Data System (ADS)

    Lykke, Julie M.; Favre, Cécile

    2014-07-01

    Ethylene glycol (HOCH2CH2OH), also commenly known as antifreeze, is the reduced alcohol version of glycolaldehyde (CH2OHCHO). Glycoladehyde - the simplest possible aldehyde sugar (Marstokk and Møllendal 1973) - is the first intermediate step in the path toward forming more complex and biologically relevant molecules through the the formose reaction, which begins with formaldehyde (H2CO) and ends with the formation of sugars and ultimately ribose, the backbone of RNA (e.g., Larralde et al. 1995). The presence of glycolaldehyde is therefore an important indication that processes leading to biologically relevant molecules are taking place. It is however, still unclear as to how glycolaldehyde and ethylene glycol are formed in the ISM. It has been proposed that they share a common formation pathway through UV-irradiation of methanol (CH3OH) ices mixed with CO (Öberg et al. 2009). So far, ethylene glycol, in its lower energy con-former (g’Ga(CH2OH)2), has been detected toward SgrB2 (N) by Hollis et al. (2002), tentatively toward IRAS 16293-2422 (Jørgensen et al. 2012) and marginally by Kalenskii and Johansson (2010) toward W51 e1/e2. Here we present a firm detection of ethylene glycol toward W51/e2 as well as a first detection toward G34.3+0.02 at 1mm and 3mm using the IRAM 30m telescope.

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

    DOE PAGES

    Iskra, Ł. W.; Broda, R.; Janssens, R. V.F.; ...

    2015-01-01

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

  5. Validating computational predictions of night-time ventilation in Stanford's Y2E2 building

    NASA Astrophysics Data System (ADS)

    Chen, Chen; Lamberti, Giacomo; Gorle, Catherine

    2017-11-01

    Natural ventilation can significantly reduce building energy consumption, but robust design is a challenging task. We previously presented predictions of natural ventilation performance in Stanford's Y2E2 building using two models with different levels of fidelity, embedded in an uncertainty quantification framework to identify the dominant uncertain parameters and predict quantified confidence intervals. The results showed a slightly high cooling rate for the volume-averaged temperature, and the initial thermal mass temperature and window discharge coefficients were found to have an important influence on the results. To further investigate the potential role of these parameters on the observed discrepancies, the current study is performing additional measurements in the Y2E2 building. Wall temperatures are recorded throughout the nightflush using thermocouples; flow rates through windows are measured using hotwires; and spatial variability in the air temperature is explored. The measured wall temperatures are found the be within the range of our model assumptions, and the measured velocities agree reasonably well with our CFD predications. Considerable local variations in the indoor air temperature have been recorded, largely explaining the discrepancies in our earlier validation study. Future work will therefore focus on a local validation of the CFD results with the measurements. Center for Integrated Facility Engineering (CIFE).

  6. Domain structure and reorientation in CoF e2O4

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  7. Ultrafast dynamics of an unoccupied surface resonance state in B i2T e2Se

    NASA Astrophysics Data System (ADS)

    Munisa, Nurmamat; Krasovskii, E. E.; Ishida, Y.; Sumida, K.; Chen, Jiahua; Yoshikawa, T.; Chulkov, E. V.; Kokh, K. A.; Tereshchenko, O. E.; Shin, S.; Kimura, Akio

    2018-03-01

    Electronic structure and electron dynamics in the ternary topological insulator B i2T e2Se are studied with time- and angle-resolved photoemission spectroscopy using optical pumping. An unoccupied surface resonance split off from the bulk conduction band previously indirectly observed in scanning tunneling measurements is spectroscopically identified. Furthermore, an unoccupied topological surface state (TSS) is found, which is serendipitously located at about 1.5 eV above the occupied TSS, thereby facilitating direct optical transitions between the two surface states at ℏ ω =1.5 eV in an n -type topological insulator. An appreciable nonequilibrium population of the bottom of the bulk conduction band is observed for longer than 15 ps after the pump pulse. This leads to a long recovery time of the lower TSS, which is constantly populated by the electrons coming from the bulk conduction band. Our results demonstrate B i2T e2Se to be an ideal platform for designing future optoelectronic devices based on topological insulators.

  8. Global Carbon Cycle Modeling in GISS ModelE2 GCM

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

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

  9. The Classification and Evolution of Enzyme Function

    PubMed Central

    Martínez Cuesta, Sergio; Rahman, Syed Asad; Furnham, Nicholas; Thornton, Janet M.

    2015-01-01

    Enzymes are the proteins responsible for the catalysis of life. Enzymes sharing a common ancestor as defined by sequence and structure similarity are grouped into families and superfamilies. The molecular function of enzymes is defined as their ability to catalyze biochemical reactions; it is manually classified by the Enzyme Commission and robust approaches to quantitatively compare catalytic reactions are just beginning to appear. Here, we present an overview of studies at the interface of the evolution and function of enzymes. PMID:25986631

  10. Thermodynamics of Enzyme-Catalyzed Reactions Database

    National Institute of Standards and Technology Data Gateway

    SRD 74 Thermodynamics of Enzyme-Catalyzed Reactions Database (Web, free access)   The Thermodynamics of Enzyme-Catalyzed Reactions Database contains thermodynamic data on enzyme-catalyzed reactions that have been recently published in the Journal of Physical and Chemical Reference Data (JPCRD). For each reaction the following information is provided: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the data and an evaluation thereof.

  11. Curious Cases of the Enzymes.

    PubMed

    Ulusu, Nuriye Nuray

    2015-07-01

    Life as we know it heavily relies on biological catalysis, in fact, in a very nonromantic version of it, life could be considered as a series of chemical reactions, regulated by the guarding principles of thermodynamics. In ancient times, a beating heart was a good sign of vitality, however, to me, it is actually the presence of active enzymes that counts… Though we do not usually pay attention, the history of enzymology is as old as humanity itself, and dates back to the ancient times. This paper is dedicated to these early moments of this remarkable science that touched our lives in the past and will make life a lot more efficient for humanity in the future. There was almost always a delicate, fundamentally essential relationship between mankind and the enzymes. Challenged by a very alien and hostile Nature full of predators, prehistoric men soon discovered the medicinal properties of the plants, through trial and error. In fact, they accidently discovered the enzyme inhibitors and thus, in crude terms, kindled a sparkling area of research. These plant-derivatives that acted as enzyme inhibitors helped prehistoric men in their pursuit of survival and protection from predators; in hunting and fishing… Later in history, while the underlying purposes of survival and increasing the quality of life stayed intact, the ways and means of enzymology experienced a massive transformation, as the 'trial and error' methodology of the ancients is now replaced with rational scientific theories.

  12. Biological abatement of enzyme inhibitors

    USDA-ARS?s Scientific Manuscript database

    Lignocellulose pretreatments release phenolic compounds that cause enzyme inhibition and deactivation. Bio-abatement, the biological removal of furfurals, acetic acid and phenolics, may utilize fungal fermentation to metabolize these compounds to CO2, water, cell mass, and heat. Our work with Coni...

  13. Phage lytic enzymes targeting streptococci

    USDA-ARS?s Scientific Manuscript database

    Streptococcal pathogens contribute to a wide variety of human and livestock diseases. There is a need for new antimicrobials to replace over-used conventional antibiotics. Bacteriophage (viruses that infect bacteria) endolysins (enzymes that help degrade the bacterial cell wall) are ideal candidat...

  14. Rennin--a Neglected Enzyme?

    ERIC Educational Resources Information Center

    Gill, John; Saunders, Terry

    1987-01-01

    Presents investigations to explore the substrate specificity, pH, concentration, and temperature relations of an enzyme with only inexpensive commercial rennet and basic laboratory equipment. Describes how the activities were carried out with a group of 15-year-old students. (CW)

  15. Insolubilized enzymes for food synthesis

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1972-01-01

    Cellulose matrix with numerous enzyme-coated silica particles of colloidal size permanently bound at various sites within matrix was produced that has high activity and possesses requisite physical characteristics for filtration or column operations. Product also allows coupling step in synthesis of edible food to proceed under mild conditions.

  16. The enzymes associated with denitrification

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Tomlinson, G. A.

    1988-01-01

    The enzymes involved in the reduction of nitrogenous oxides are thought to be intermediates in denitrification processes. This review examines the roles of nitrate reductase, nitrite reductases, nitric oxide reductase, mechanisms of N-N bond formation, and nitrous oxide reductases.

  17. Rapid-Equilibrium Enzyme Kinetics

    ERIC Educational Resources Information Center

    Alberty, Robert A.

    2008-01-01

    Rapid-equilibrium rate equations for enzyme-catalyzed reactions are especially useful because if experimental data can be fit by these simpler rate equations, the Michaelis constants can be interpreted as equilibrium constants. However, for some reactions it is necessary to use the more complicated steady-state rate equations. Thermodynamics is…

  18. Lipolytic enzymes in Mycobacterium tuberculosis.

    PubMed

    Côtes, K; Bakala N'goma, J C; Dhouib, R; Douchet, I; Maurin, D; Carrière, F; Canaan, S

    2008-04-01

    Mycobacterium tuberculosis is a bacterial pathogen that can persist for decades in an infected patient without causing a disease. In vivo, the tubercle bacillus present in the lungs store triacylglycerols in inclusion bodies. The same process can be observed in vitro when the bacteria infect adipose tissues. Indeed, before entering in the dormant state, bacteria accumulate lipids originating from the host cell membrane degradation and from de novo synthesis. During the reactivation phase, these lipids are hydrolysed and the infection process occurs. The degradation of both extra and intracellular lipids can be directly related to the presence of lipolytic enzymes in mycobacteria, which have been ignored during a long period particularly due to the difficulties to obtain a high expression level of these enzymes in M. tuberculosis. The completion of the M. tuberculosis genome offered new opportunity to this kind of study. The aim of this review is to focus on the recent results obtained in the field of mycobacterium lipolytic enzymes and although no experimental proof has been shown in vivo, it is tempting to speculate that these enzymes could be involved in the virulence and pathogenicity processes.

  19. Structural aspects of denitrifying enzymes.

    PubMed

    Moura, I; Moura, J J

    2001-04-01

    The reduction of nitrate to nitrogen gas via nitrite, nitric oxide and nitrous oxide is the metabolic pathway usually known as denitrification, a key step in the nitrogen cycle. As observed for other elemental cycles, a battery of enzymes are utilized, namely the reductases for nitrate, nitrite, nitric oxide and nitrous oxide, as well as multiple electron donors that interact with these enzymes, in order to carry out the stepwise reactions that involve key intermediates. Because of the importance of this pathway (of parallel importance to the nitrogen-fixation pathway), efforts are underway to understand the structures of the participating enzymes and to uncover mechanistic aspects. Three-dimensional structures have been solved for the majority of these enzymes in the past few years, revealing the architecture of the active metal sites as well as global structural aspects, and possible mechanistic aspects. In addition, the recognition of specific electron-transfer partners raises important questions regarding specific electron-transfer pathways, partner recognition and control of metabolism.

  20. Novel approach to differentiate subclades of varicella-zoster virus genotypes E1 and E2 in Germany.

    PubMed

    Schmidt-Chanasit, Jonas; Olschläger, Stephan; Bialonski, Alexandra; Heinemann, Patrick; Bleymehl, Karoline; Gross, Gerd; Günther, Stephan; Ulrich, Rainer G; Doerr, Hans Wilhelm

    2009-11-01

    Varicella-zoster virus (VZV) is the causative agent of chicken pox (varicella) in children and reactivation of VZV in elderly or immunocompromised persons can cause shingles (zoster). A subclade differentiation of the most prevalent VZV genotypes E1 and E2 in Germany was not possible with the current genotyping methods in use, but is highly important to understand the VZV molecular evolution in more detail and especially to follow up the routes of infection. Therefore the objective of this study was to develop a simple PCR-based method for differentiation of E1 and E2 subclades. Viral DNA was isolated from vesicle fluid samples of six selected German zoster patients and used to amplify nine complete open reading frames (ORFs) of the VZV genome by different PCR assays. Phylogenetic analysis was performed by a Bayesian approach. Based on the analysis of a total of nine ORFs, a 7482 bp stretch consisting of ORFs 5, 37 and 62 contained informative sites for identification of novel subclades E1a, E2a and E2b for VZV genotypes E1 and E2. Specific single nucleotide polymorphisms (SNPs) were demonstrated for subclades E2a and E2b within the ORFs 5, 37 and 62, whereas a subclade E1a-specific SNP was found in ORF 56. The classification of E1 and E2 subclades may facilitate a more exact and in-depth monitoring of the molecular evolution of VZV in Germany in the future.

  1. Interaction of CSFV E2 protein with swine host factors as detected by yeast two-hybrid system

    USDA-ARS?s Scientific Manuscript database

    E2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV). E2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. Howev...

  2. 75 FR 79264 - E-2 Nonimmigrant Status for Aliens in the Commonwealth of the Northern Mariana Islands With Long...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-20

    ... Information to the proposed rule discussed the fact that E-2 CNMI Investor status could be granted directly to... authorization to collect biometric information from applicants for E-2 CNMI Investor status, with the applicant... Mariana Islands With Long-Term Investor Status AGENCY: U.S. Citizenship and Immigration Services, DHS...

  3. Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA.

    PubMed

    Kim, Sanghyun; Zbaida, David; Elbaum, Michael; Leh, Hervé; Nogues, Claude; Buckle, Malcolm

    2015-07-27

    VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein-protein interactions. In order to isolate the protein-DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1-VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1-VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. The association of GSK3 beta with E2F1 facilitates nerve growth factor-induced neural cell differentiation.

    PubMed

    Zhou, Fangfang; Zhang, Long; Wang, Aijun; Song, Bo; Gong, Kai; Zhang, Lihai; Hu, Min; Zhang, Xiufang; Zhao, Nanming; Gong, Yandao

    2008-05-23

    It is widely acknowledged that E2F1 and GSK3beta are both involved in the process of cell differentiation. However, the relationship between E2F1 and GSK3beta in cell differentiation has yet to be discovered. Here, we provide evidence that in the differentiation of PC12 cells induced by nerve growth factor (NGF), GSK3beta was increased at both the mRNA and protein levels, whereas E2F1 at these two levels was decreased. Both wild-type GSK3beta and its kinase-defective mutant GSK3beta KM can inhibit E2F1 by promoting its ubiquitination through physical interaction. In addition, the colocalization of GSK3beta and E2F1 and their subcellular distribution, regulated by NGF, were observed in the process of PC12 differentiation. At the tissue level, GSK3beta colocalized and interacted with E2F1 in mouse hippocampus. Furthermore, GSK3beta facilitated neurite outgrowth by rescuing the promoter activities of Cdk inhibitors p21 and p15 from the inhibition caused by E2F1. To summarize, our findings suggest that GSK3beta can promote the ubiquitination of E2F1 via physical interaction and thus inhibit its transcription activity in a kinase activity independent manner, which plays an important role in the NGF-induced PC12 differentiation.

  5. A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites.

    PubMed Central

    Sellers, W R; Rodgers, J W; Kaelin, W G

    1995-01-01

    An intact T/E1A-binding domain (the pocket) is necessary, but not sufficient, for the retinoblastoma protein (RB) to bind to DNA-protein complexes containing E2F and for RB to induce a G1/S block. Indirect evidence suggests that the binding of RB to E2F may, in addition to inhibiting E2F transactivation function, generate a complex capable of functioning as a transrepressor. Here we show that a chimera in which the E2F1 transactivation domain was replaced with the RB pocket could, in a DNA-binding and pocket-dependent manner, mimic the ability of RB to repress transcription and induce a cell cycle arrest. In contrast, a transdominant negative E2F1 mutant that is capable of blocking E2F-dependent transactivation did not. Fusion of the RB pocket to a heterologous DNA-binding domain unrelated to E2F likewise generated a transrepressor protein when scored against a suitable reporter. These results suggest that growth suppression by RB is due, at least in part, to transrepression mediated by the pocket domain bound to certain promoters via E2F. Images Fig. 4 Fig. 5 PMID:8524800

  6. 77 FR 21782 - International Conference on Harmonisation; Draft Guidance for Industry on E2C(R2) Periodic...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-11

    ... electronic submission of individual case safety reports to regulatory authorities, automated data mining... Safety Data Management: Periodic Safety Update Reports for Marketed Drugs'' (E2C guidance) and ``Addendum to E2C Clinical Safety Data Management: Periodic Safety Update Reports for Marketed Drugs'' (addendum...

  7. 26 CFR 1.927(e)-2T - Temporary regulations; effect of boycott participation on FSC and small FSC benefits.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... small FSC benefits. (a) International boycott factor. If the FSC (or small FSC) or any member of the FSC...) exempt foreign trade income multiplied by the international boycott factor determined under section 999... participation on FSC and small FSC benefits. 1.927(e)-2T Section 1.927(e)-2T Internal Revenue INTERNAL REVENUE...

  8. THE E2/FRB PATHWAY REGULATION OF DNA REPLICATION AND PROTEIN BIOSYNTHESIS

    EPA Science Inventory

    The E2F/Rb pathway plays a pivotal role in the control of cell cycle progression and regulates the expression of genes required for Gl/S transition. Our study examines the genomic response in Drosophila embryos after overexpression and mutation of E2F/Rb pathway molecules. Hierar...

  9. Human ApoE Isoforms Differentially Modulate Glucose and Amyloid Metabolic Pathways in Female Brain: Evidence of the Mechanism of Neuroprotection by ApoE2 and Implications for Alzheimer's Disease Prevention and Early Intervention.

    PubMed

    Keeney, Jeriel Thomas-Richard; Ibrahimi, Shaher; Zhao, Liqin

    2015-01-01

    Three major genetic isoforms of apolipoprotein E (ApoE), ApoE2, ApoE3, and ApoE4, exist in humans and lead to differences in susceptibility to Alzheimer's disease (AD). This study investigated the impact of human ApoE isoforms on brain metabolic pathways involved in glucose utilization and amyloid-β (Aβ) degradation, two major areas that are significantly perturbed in preclinical AD. Hippocampal RNA samples from middle-aged female mice with targeted human ApoE2, ApoE3, and ApoE4 gene replacement were comparatively analyzed with a qRT-PCR custom array for the expression of 85 genes involved in insulin/insulin-like growth factor (Igf) signaling. Consistent with its protective role against AD, ApoE2 brain exhibited the most metabolically robust profile among the three ApoE genotypes. When compared to ApoE2 brain, both ApoE3 and ApoE4 brains exhibited markedly reduced levels of Igf1, insulin receptor substrates (Irs), and facilitated glucose transporter 4 (Glut4), indicating reduced glucose uptake. Additionally, ApoE4 brain exhibited significantly decreased Pparg and insulin-degrading enzyme (Ide), indicating further compromised glucose metabolism and Aβ dysregulation associated with ApoE4. Protein analysis showed significantly decreased Igf1, Irs, and Glut4 in ApoE3 brain, and Igf1, Irs, Glut4, Pparg, and Ide in ApoE4 brain compared to ApoE2 brain. These data provide the first documented evidence that human ApoE isoforms differentially affect brain insulin/Igf signaling and downstream glucose and amyloid metabolic pathways, illustrating a potential mechanism for their differential risk in AD. A therapeutic strategy that enhances brain insulin/Igf1 signaling activity to a more robust ApoE2-like phenotype favoring both energy production and amyloid homeostasis holds promise for AD prevention and early intervention.

  10. Recombinant Swinepox Virus Expressing Glycoprotein E2 of Classical Swine Fever Virus Confers Complete Protection in Pigs upon Viral Challenge.

    PubMed

    Lin, Huixing; Ma, Zhe; Chen, Lei; Fan, Hongjie

    2017-01-01

    Classical swine fever (CSF) is a highly contagious and serious viral disease that affects the pig industry worldwide. The glycoprotein E2 of the classical swine fever virus (CSFV) can induce neutralizing antibodies, and it is widely used for novel vaccine development. To explore the development of a vaccine against CSFV infections, the gene of glycoprotein E2 was inserted into the swinepox virus (SPV) genome by homologous recombination. The culture titers of rSPV-E2 remained at about 4.3 × 10 6 TCID 50 for more than 60 passages in PK15 and swine testis cell lines. The rSPV-E2 could not be replicated in Vero, MDBK or other non-porcine cell lines. After two to three passages, the SPV specific gene of rSPV-E2 could not been detected in the non-porcine cell culture. To evaluate the immunogenicity of rSPV-E2, 20 CSFV seronegative minipigs were immunized with rSPV-E2, a commercial C-strain vaccine, wild-type SPV (wtSPV; negative control), or PBS (a no-challenge control). After challenge with CSFV, pigs in the rSPV-E2-immunized group showed significantly shorter fever duration compared with the wtSPV-treated group ( P  < 0.05). E2-specific antibodies in the rSPV-E2-immunized group increased dramatically after vaccination and increased continuously over time. CSFV genomic copies in the serum of rSPV-E2-immunized pigs were significantly less compared with the wtSPV-treated group at all time points after challenge ( P  < 0.01). Significant reduction in gross lung lesion scores, histopathological liver, spleen, lung, and kidney lesion scores were noted in the rSPV-E2-immunized group compared with the wtSPV-treated group ( P  < 0.01). The results suggested that the recombinant rSPV-E2 provided pigs with significant protection from CSFV infections; thus, rSPV-E2 offers proof of principle for the development of a vaccine for the prevention of CSFV infections in pigs.

  11. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

  12. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 3 2011-01-01 2011-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

  13. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 3 2013-01-01 2013-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

  14. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 3 2014-01-01 2014-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

  15. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 3 2012-01-01 2012-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

  16. Heavy enzymes--experimental and computational insights in enzyme dynamics.

    PubMed

    Swiderek, Katarzyna; Ruiz-Pernía, J Javier; Moliner, Vicent; Tuñón, Iñaki

    2014-08-01

    The role of protein motions in the chemical step of enzyme-catalyzed reactions is the subject of an open debate in the scientific literature. The systematic use of isotopically substituted enzymes has been revealed as a useful tool to quantify the role of these motions. According to the Born-Oppenheimer approximation, changing the mass of the protein does not change the forces acting on the system but alters the frequencies of the protein motions, which in turn can affect the rate constant. Experimental and theoretical studies carried out in this field are presented in this article and discussed in the framework of Transition State Theory. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Exploring diurnal and seasonal characteristics of global carbon cycle with GISS Model E2 GCM

    NASA Astrophysics Data System (ADS)

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

    2017-12-01

    The ability to properly model surface carbon fluxes on the diurnal and seasonal time scale is a necessary requirement for understanding of the global carbon cycle. It is also one of the most challenging tasks faced by modern General Circulation Models (GCMs) due to complexity of the algorithms and variety of relevant spatial and temporal scales. The observational data, though abundant, is difficult to interpret at the global scale, because flux tower observations are very sparse for large impact areas (such as Amazon and African rainforest and most of Siberia) and satellite missions often struggle to produce sufficiently high confidence data over the land and may be missing CO2 amounts near the surface due to the nature of the method. In this work we use the GISS Model E2 GCM to perform a subset of experiments proposed by the Coupled Climate-Carbon Cycle Model Intercomparison Project (C4MIP) and relate the results to available observations.The GISS Model E2 GCM is currently equipped with a complete global carbon cycle algorithm. Its surface carbon fluxes are computed by the Ent Terrestrial Biosphere Model (Ent TBM) over the land with observed leaf area index of the Moderate Resolution Imaging Spectrometer (MODIS) and by the NASA Ocean Biogeochemistry Model (NOBM) over the ocean. The propagation of atmospheric CO2 is performed by a generic Model E2 tracer algorithm, which is based on a quadratic upstream method (Prather 1986). We perform a series spin-up experiments for preindustrial climate conditions and fixed preindustrial atmospheric CO2 concentration. First, we perform separate spin-up simulations each for terrestrial and ocean carbon. We then combine the spun-up states and perform a coupled spin-up simulation until the model reaches a sufficient equilibrium. We then release restrictions on CO2 concentration and allow it evolve freely, driven only by simulated surface fluxes. We then study the results of the unforced run, comparing the amplitude and the phase

  18. Anti-E1E2 antibodies status prior therapy favors direct-acting antiviral treatment efficacy.

    PubMed

    Virlogeux, Victor; Berthillon, Pascale; Bordes, Isabelle; Larrat, Sylvie; Crouy, Stéphanie; Scholtès, Caroline; Pradat, Pierre; Maynard, Marianne; Zoulim, Fabien; Leroy, Vincent; Chemin, Isabelle; Trépo, Christian; Petit, Marie-Anne

    2018-03-15

    Presence of anti-E1E2 antibodies was previously associated with spontaneous cure of hepatitis C virus (HCV) and predictive before treatment of a sustained virological response (SVR) to bi- or tri-therapy in naïve or experienced patients, regardless of HCV genotype. We investigated the impact of anti-E1E2 seroprevalence at baseline on treatment response in patients receiving direct-acting antiviral (DAA) therapy. We screened anti-E1E2 antibodies by ELISA in serum samples collected at treatment initiation for two groups of patients: 59 with SVR at the end of DAA treatment and 44 relapsers after DAA treatment. Nineteen patients received a combination of ribavirin (RBV) or PEG-interferon/ribavirin with sofosbuvir or daclatasvir and others received interferon-free treatment with DAA±RBV. HCV viral load was measured at different time points during treatment in a subgroup of patients. A significant association was observed between presence of anti-E1E2 and HCV viral load<6log10 prior treatment. Among patients with anti-E1E2 at baseline, 70% achieved SVR whereas among patients without anti-E1E2, only 45% achieved SVR. Conversely, 66% of patients experiencing DAA-failure were anti-E1E2 negative at baseline. In the multivariate analysis, presence of anti-E1E2 was significantly associated with SVR after adjustment on potential cofounders such as age, sex, fibrosis stage, prior HCV treatment and alanine aminotransferase (ALT) level. The presence of anti-E1E2 at treatment initiation is a predictive factor of SVR among patients treated with DAA and more likely among patients with low initial HCV viral load (<6log10). Absence of anti-E1E2 at baseline could predict DAA-treatment failure. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  19. Electric Quadrupole E2- Transitions of 170-174 Yb Isotopes

    NASA Astrophysics Data System (ADS)

    Abu El Sheikh, Mohd Kh. M.; Okhunov, Abdurahim A.; Usmanov, Ph. N.; Hassan, Torla HJ

    2017-12-01

    The non-adiabatic effects which is manifested in the electric properties of low-lying states of even-even deformed nuclei are studied. A simple phenomenological model which takes into account the Coriolis mixing of {K}π ={0}n+,{2}n+ and {K}π ={1}ν + state bands. The Calculations for isotopes 170-174 Yb, are carried out. The reduced probability of electric quadrupole transitions from the states {0}ν + and {2}ν + - bands to the ground (gr) state band is calculated and non adiabatic effect is discussed. The ratio of E2- transitions RIK from {0}2+, {0}3+, {2}1+, and {2}2+ bands are calculated and compared with the experimental data.

  20. A Simple Pythagorean Interpretation of E2 = p2c2 + (mc2)2

    NASA Astrophysics Data System (ADS)

    Tobar, J. A.; Vargas, E. L.; Andrianarijaona, V. M.

    2015-03-01

    We are considering the relationship between the relativistic energy, the momentum, and the rest energy, E2 =p2c2 + (mc2)2 , and using geometrical means to analyze each individual portion in a spatial setting. The aforementioned equation suggests that pc and mc2 could be thought of as the two axis of a plane. According to de Broglie's hypothesis λ = h / p therefore suggesting that the pc-axis is connected to the wave properties of a moving object, and subsequently, the mc2-axis is connected to the particle properties. Consequently, these two axis could represent the particle and wave properties of the moving object. An overview of possible models and meaningful interpretations will be presented. Authors wish to give special thanks to Pacific Union College Student Senate in Angwin, California, for their financial support.

  1. A Simple Pythagorean Interpretation of E2 = p2 c2 + (mc2)2

    NASA Astrophysics Data System (ADS)

    Tobar, J. A.; Guillen, C. I.; Vargas, E. L.; Andrianarijaona, V. M.

    2015-04-01

    We are considering the relationship between the relativistic energy, the momentum, and the rest energy, E2 =p2c2 + (mc2)2 , and using geometrical means to analyze each individual portion in a spatial setting. The aforementioned equation suggests that pc and mc2 could be thought of as the two axis of a plane. According to de Broglie's hypothesis λ = h / p therefore suggesting that the pc-axis is connected to the wave properties of a moving object, and subsequently, the mc2-axis is connected to the particle properties such as its moment of inertia. Consequently, these two axes could represent the particle (matter) and wave properties of the moving object. An overview of possible models and meaningful interpretations, which agree with Dirac's prediction of the electron's magnetic moment, will be presented. Authors wish to give special thanks to Pacific Union College Student Senate in Angwin, California, for their financial support.

  2. DFT calculations, spectroscopy and antioxidant activity studies on (E)-2-nitro-4-[(phenylimino)methyl]phenol

    NASA Astrophysics Data System (ADS)

    Temel, Ersin; Alaşalvar, Can; Gökçe, Halil; Güder, Aytaç; Albayrak, Çiğdem; Alpaslan, Yelda Bingöl; Alpaslan, Gökhan; Dilek, Nefise

    2015-02-01

    We have reported synthesis and characterization of (E)-2-nitro-4-[(phenylimino)methyl]phenol by using X-ray crystallographic method, FT-IR and UV-vis spectroscopies and density functional theory (DFT). Optimized geometry and vibrational frequencies of the title compound in the ground state have been computed by using B3LYP with the 6-311G+(d,p) basis set. HOMO-LUMO energy gap, Non-linear optical properties and NBO analysis of the compound are performed at B3LYP/6-311G+(d,p) level. Additionally, as remarkable properties, antioxidant activity of the title compound (CMPD) has been determined by using different antioxidant test methods i.e. ferric reducing antioxidant power (FRAP), hydrogen peroxide scavenging (HPSA), free radical scavenging (FRSA) and ferrous ion chelating activities (FICA). When compared with standards (BHA, BHT, and α-tocopherol), we have concluded that CPMD has effective FRAP, HPSA, FRSA and FICA.

  3. Atomic spectroscopy with twisted photons: Separation of M 1 -E 2 mixed multipoles

    NASA Astrophysics Data System (ADS)

    Afanasev, Andrei; Carlson, Carl E.; Solyanik, Maria

    2018-02-01

    We analyze atomic photoexcitation into the discrete states by twisted photons, or photons carrying extra orbital angular momentum along their direction of propagation. From the angular momentum and parity considerations, we are able to relate twisted-photon photoexcitation amplitudes to their plane-wave analogs, independently of the details of the atomic wave functions. We analyze the photoabsorption cross sections of mixed-multipolarity E 2 -M 1 transitions in ionized atoms and found fundamental differences coming from the photon topology. Our theoretical analysis demonstrates that it is possible to extract the relative transition rates of different multipolar contributions by measuring the photoexcitation rate as a function of the atom's position (or impact parameter) with respect to the optical vortex center. The proposed technique for separation of multipoles can be implemented if the target's atom position is resolved with subwavelength accuracy; for example, with Paul traps. Numerical examples are presented for Boron-like highly charged ions.

  4. Sulfonamide derivatives of styrylheterocycles as a potent inhibitor of COX-2-mediated prostaglandin E2 production.

    PubMed

    Lim, Chaemin; Lee, Minhee; Park, Eun-Jung; Cho, Ran; Park, Hyen-Joo; Lee, Seong Jin; Cho, Heeyeong; Lee, Sang Kook; Kim, Sanghee

    2010-12-01

    The overproduction of prostaglandin E(2) (PGE(2)) plays an important role in a variety of pathophysiological processes including inflammation and carcinogenesis. Therefore, the modulation of PGE(2) production is a promising target in the design of chemotherapeutic agents. In the present study, the inhibitory effects of a series of styrylheterocycles having either a p-SO(2)NH(2) or p-SO(2)Me group on the production of cyclooxygenase-2-mediated PGE(2) were evaluated in lipopolysaccharide-stimulated RAW264.7 murine macrophages. Among the series of styrylheterocycle derivatives, (E)-4-(2-(thiophen-3-yl)vinyl)benzenesulfonamide exhibited a potent inhibitory activity, with an IC(50) value of 0.013 μM. The inhibitory activity against the overproduction of PGE(2) by the active compound was found to be due in part to the suppression of COX-2 mRNA expression. Copyright © 2010 Elsevier Ltd. All rights reserved.

  5. Tromboxane B2 inhibits the pulmonary inactivation of prostaglandin E2 in the dog.

    PubMed Central

    Fitzpatrick, T. M.; Friedman, L. S.; Kot, P. A.; Ramwell, P. W.

    1980-01-01

    1 The systemic vasodepressor response to intravenously administered prostaglandin E2 (PGE2, 0.3, 1.0 and 3.0 micrograms/kg) is potentiated during intravenous infusion of thromboxane B2 (TXB2, 1.0 micrograms kg-1 min-1) in the anaesthetized dog. 2 The augmented haemodynamic response returns toward control values following cessation of the TXB2 infusion. 3 The systemic haemodynamic responses to intra-arterially administered PGE2, PGF2 alpha and PGI2 as well as intravenously administered PGF2 alpha and PGI2 are not altered by TXB2 infusion. 4. This study suggests that TXB2 inhibits the pulmonary inactivation of PGE2. 5 Arachidonic acid metabolites may interact, producing haemodynamic responses differing from their individual effects. PMID:7000216

  6. Energy spectra and E2 transition rates of 124—130Ba

    NASA Astrophysics Data System (ADS)

    Sabri, H.; Seidi, M.

    2016-10-01

    In this paper, we have studied the energy spectra and B(E2) values of 124—130Ba isotopes in the shape phase transition region between the spherical and gamma unstable deformed shapes. We have used a transitional interacting Boson model (IBM), Hamiltonian which is based on affine SU(1,1) Lie algebra in the both IBM-1 and 2 versions and also the Catastrophe theory in combination with a coherent state formalism to generate energy surfaces and determine the exact values of control parameters. Our results for control parameters suggest a combination of U(5) and SO(6) dynamical symmetries in this isotopic chain. Also, the theoretical predictions can be rather well reproduce the experimental counterparts, when the control parameter is approached to the SO(6) limit.

  7. Extreme relativistic electron fluxes at geosynchronous orbit: Analysis of GOES E > 2 MeV electrons

    NASA Astrophysics Data System (ADS)

    Meredith, Nigel P.; Horne, Richard B.; Isles, John D.; Rodriguez, Juan V.

    2015-03-01

    Relativistic electrons (E > 1 MeV) cause internal charging on satellites and are an important space weather hazard. A key requirement in space weather research concerns extreme events and knowledge of the largest flux expected to be encountered over the lifetime of a satellite mission. This is interesting both from scientific and practical points of view since satellite operators, engineers, and the insurance industry need this information to better evaluate the effects of extreme events on their spacecraft. Here we conduct an extreme value analysis of daily averaged E > 2 MeV electron fluxes from the Geostationary Operational Environmental Satellites (GOES) during the 19.5 year period from 1 January 1995 to 30 June 2014. We find that the daily averaged flux measured at GOES West is typically a factor of about 2.5 higher than that measured at GOES East, and we conduct independent analyses for these two locations. The 1 in 10, 1 in 50, and 1 in 100 year daily averaged E > 2 MeV electron fluxes at GOES West are 1.84 ×105, 5.00 ×105, and 7.68 ×105 cm-2 s-1 sr-1, respectively. The corresponding fluxes at GOES East are 6.53 ×104, 1.98 ×105, and 3.25 ×105 cm-2 s-1 sr-1, respectively. The largest fluxes seen during the 19.5 year period on 29 July 2004 were particularly extreme and were seen by satellites at GOES West and GOES East. The extreme value analysis suggests that this event was a 1 in 50 year event.

  8. Comparative study of labour induced by oral prostaglandin E2 and intravenous syntocinon.

    PubMed

    Murray, C P; Clinch, J

    1975-03-22

    The use of prostaglandin E2 for the induction of labor with intact membranes is described and its effectiveness is compared to intravenous syntocinon. 40 primigravida and 60 multigravid patients with previous medical and obstetrical histories were studied. The patients were numbered as they entered the trial, with the odd numbers in each group being given oral prostaglandin and the even numbers intravenous syntocinon. In no case was the pregnancy less than 38 weeks maturity. No patient was in labor prior to being given either drug. Prostaglandin E2 (PGE2) was supplied in ampoules containing 5 milligrams in 0.5 milliliter of ethanol. This was added to 49.5 milliliters of sterile water to produce a concentration of the drug of 0.1 milligrams per ml. The syntocinon infusion was prepared by putting 20 units of syntocinon into 1 liter of 5% dextrose in water to produce a solution concentration of 20 mu/ml. The accepted criteria for diagnosing established labor for both groups of patients was the presence of uterine contractions occurring once every 3 minutes, associated with progressive dilatation of the cervix. For both groups of patients it was decided that cervical dilatation should be at least 6 cm within 18 hours of the infusion starting. Using this criterion there was only 1 failure, occurring in the 1st primigravid patient given PGE2, the labor in this instance being completed with intravenous syntocinon. A further 8 patients failed to complete the trial as they had to be delivered by cesarian section. Syntocin was considerably more efficient than PGE2 in inducing labor in the remaining 91 patients particularly in primigravida. This was the case whether judged by the length of labor or by the induction delivery interval. Toco-dynamometric studies showed that the contractions produced by prostaglandin more closely resembled those of normal labor and were less painful.

  9. A Digital Hydrologic Network Supporting NAWQA MRB SPARROW Modeling--MRB_E2RF1WS

    USGS Publications Warehouse

    Brakebill, J.W.; Terziotti, S.E.

    2011-01-01

    A digital hydrologic network was developed to support SPAtially Referenced Regression on Watershed attributes (SPARROW) models within selected regions of the United States. These regions correspond with the U.S. Geological Survey's National Water Quality Assessment (NAWQA) Program Major River Basin (MRB) study units 2, 3, 4, 5, and 7 (Preston and others, 2009). MRB2, covers the South Atlantic-Gulf and Tennessee River basins. MRB3, covers the Great Lakes, Ohio, Upper Mississippi, and Souris-Red-Rainy River basins. MRB4, covers the Missouri River basins. MRB5, covers the Lower Mississippi, Arkansas-White-Red, and Texas-Gulf River basins. MRB7, covers the Pacific Northwest River basins. The digital hydrologic network described here represents surface-water pathways (MRB_E2RF1) and associated catchments (MRB_E2RF1WS). It serves as the fundamental framework to spatially reference and summarize explanatory information supporting nutrient SPARROW models (Brakebill and others, 2011; Wieczorek and LaMotte, 2011). The principal geospatial dataset used to support this regional effort was based on an enhanced version of a 1:500,000 scale digital stream-reach network (ERF1_2) (Nolan et al., 2002). Enhancements included associating over 3,500 water-quality monitoring sites to the reach network, improving physical locations of stream reaches at or near monitoring locations, and generating drainage catchments based on 100m elevation data. A unique number (MRB_ID) identifies each reach as a single unit. This unique number is also shared by the catchment area drained by the reach, thus spatially linking the hydrologically connected streams and the respective drainage area characteristics. In addition, other relevant physical, environmental, and monitoring information can be associated to the common network and accessed using the unique identification number.

  10. A Digital Hydrologic Network Supporting NAWQA MRB SPARROW Modeling--MRB_E2RF1

    USGS Publications Warehouse

    Brakebill, J.W.; Terziotti, S.E.

    2011-01-01

    A digital hydrologic network was developed to support SPAtially Referenced Regression on Watershed attributes (SPARROW) models within selected regions of the United States. These regions correspond with the U.S. Geological Survey's National Water Quality Assessment (NAWQA) Program Major River Basin (MRB) study units 2, 3, 4, 5, and 7 (Preston and others, 2009). MRB2, covers the South Atlantic-Gulf and Tennessee River basins. MRB3, covers the Great Lakes, Ohio, Upper Mississippi, and Souris-Red-Rainy River basins. MRB4, covers the Missouri River basins. MRB5, covers the Lower Mississippi, Arkansas-White-Red, and Texas-Gulf River basins. MRB7, covers the Pacific Northwest River basins. The digital hydrologic network described here represents surface-water pathways (MRB_E2RF1) and associated catchments (MRB_E2RF1WS). It serves as the fundamental framework to spatially reference and summarize explanatory information supporting nutrient SPARROW models (Brakebill and others, 2011; Wieczorek and LaMotte, 2011). The principal geospatial dataset used to support this regional effort was based on an enhanced version of a 1:500,000 scale digital stream-reach network (ERF1_2) (Nolan et al., 2002). Enhancements included associating over 3,500 water-quality monitoring sites to the reach network, improving physical locations of stream reaches at or near monitoring locations, and generating drainage catchments based on 100m elevation data. A unique number (MRB_ID) identifies each reach as a single unit. This unique number is also shared by the catchment area drained by the reach, thus spatially linking the hydrologically connected streams and the respective drainage area characteristics. In addition, other relevant physical, environmental, and monitoring information can be associated to the common network and accessed using the unique identification number.

  11. The whipworm (Trichuris suis) secretes prostaglandin E2 to suppress proinflammatory properties in human dendritic cells.

    PubMed

    Laan, Lisa C; Williams, Andrew R; Stavenhagen, Kathrin; Giera, Martin; Kooij, Gijs; Vlasakov, Iliyan; Kalay, Hakan; Kringel, Helene; Nejsum, Peter; Thamsborg, Stig M; Wuhrer, Manfred; Dijkstra, Christine D; Cummings, Richard D; van Die, Irma

    2017-02-01

    Clinical trials have shown that administration of the nematode Trichuris suis can be beneficial in treating various immune disorders. To provide insight into the mechanisms by which this worm suppresses inflammatory responses, an active component was purified from T. suis soluble products (TsSPs) that suppress---- TNF and IL-12 secretion from LPS-activated human dendritic cells (DCs). Analysis by liquid chromatography tandem mass spectrometry identified this compound as prostaglandin (PG)E2. The purified compound showed similar properties compared with TsSPs and commercial PGE2 in modulating LPS-induced expression of many cytokines and chemokines and in modulating Rab7B and P2RX7 expression in human DCs. Furthermore, the TsSP-induced reduction of TNF secretion from DCs is reversed by receptor antagonists for EP2 and EP4, indicating PGE2 action. T. suis secretes extremely high amounts of PGE2 (45-90 ng/mg protein) within their excretory/secretory products but few related lipid mediators as established by metabololipidomic analysis. Culture of T. suis with several cyclooxygenase (COX) inhibitors that inhibit mammalian prostaglandin synthesis affected the worm's motility but did not inhibit PGE2 secretion, suggesting that the worms can synthesize PGE2 via a COX-independent pathway. We conclude that T. suis secretes PGE2 to suppress proinflammatory responses in human DCs, thereby modulating the host's immune response.-Laan, L. C., Williams, A. R., Stavenhagen, K., Giera, M., Kooij, G., Vlasakov, I., Kalay, H., Kringel, H., Nejsum, P., Thamsborg, S. M., Wuhrer, M., Dijkstra, C. D., Cummings, R. D., van Die, I. The whipworm (Trichuris suis) secretes prostaglandin E2 to suppress proinflammatory properties in human dendritic cells. © FASEB.

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

    SciTech Connect

    Bu, Yanlong; Tang, Geshi; Cao, Jianfeng

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

  13. Telomere fusion in Drosophila: The role of subtelomeric chromatin

    PubMed Central

    Marzullo, Marta; Gatti, Maurizio

    2015-01-01

    Drosophila telomeres are maintained by transposition to chromosome ends of the HeT-A, TART and TAHRE retrotransposons, collectively designated as HTT. Although all Drosophila telomeres terminate with HTT arrays and are capped by the terminin complex, they differ in the type of subtelomeric chromatin. The HTT sequences of YS, YL, XR, and 4L are juxtaposed to constitutive heterochromatin, while the HTTs of the other telomeres are linked to either the TAS repeat-associated chromatin (XL, 2L, 2R, 3L, 3R) or to the specialized 4R chromatin. We found that mutations in pendolino (peo) cause (telomeric fusions) that preferentially involve the heterochromatin-associated telomeres (Ha-telomeres), a telomeric fusion pattern never observed in the other 10 telomere-capping mutants characterized so far. Peo, is homologous to the E2 variant ubiquitin-conjugating enzymes and is required for DNA replication. Our analyses lead us to hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in Ha-telomeres. These data provide the first demonstration that subtelomeres can affect telomere fusion. PMID:26786804

  14. Mechanism of TRIM25 Catalytic Activation in the Antiviral RIG-I Pathway

    DOE PAGES

    Sanchez, Jacint G.; Chiang, Jessica J.; Sparrer, Konstantin M. J.; ...

    2016-07-14

    Antiviral response pathways induce interferon by higher-order assembly of signaling complexes called signalosomes. Assembly of the RIG-I signalosome is regulated by K63-linked polyubiquitin chains, which are synthesized by the E3 ubiquitin