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Sample records for requirements regulate cortactin

  1. PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin-actin interactions.

    Science.gov (United States)

    Hong, Nan Hyung; Qi, Aidong; Weaver, Alissa M

    2015-08-31

    Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7(+) endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor-induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover.

  2. Histone deacetylase 8 regulates cortactin deacetylation and contraction in smooth muscle tissues.

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    Li, Jia; Chen, Shu; Cleary, Rachel A; Wang, Ruping; Gannon, Olivia J; Seto, Edward; Tang, Dale D

    2014-08-01

    Histone deacetylases (HDACs) are a family of enzymes that mediate nucleosomal histone deacetylation and gene expression. Some members of the HDAC family have also been implicated in nonhistone protein deacetylation, which modulates cell-cycle control, differentiation, and cell migration. However, the role of HDACs in smooth muscle contraction is largely unknown. Here, HDAC8 was localized both in the cytoplasm and the nucleus of mouse and human smooth muscle cells. Knockdown of HDAC8 by lentivirus-encoding HDAC8 shRNA inhibited force development in response to acetylcholine. Treatment of smooth muscle tissues with HDAC8 inhibitor XXIV (OSU-HDAC-44) induced relaxation of precontracted smooth muscle tissues. In addition, cortactin is an actin-regulatory protein that undergoes deacetylation during migration of NIH 3T3 cells. In this study, acetylcholine stimulation induced cortactin deacetylation in mouse and human smooth muscle tissues, as evidenced by immunoblot analysis using antibody against acetylated lysine. Knockdown of HDAC8 by RNAi or treatment with the inhibitor attenuated cortactin deacetylation and actin polymerization without affecting myosin activation. Furthermore, expression of a charge-neutralizing cortactin mutant inhibited contraction and actin dynamics during contractile activation. These results suggest a novel mechanism for the regulation of smooth muscle contraction. In response to contractile stimulation, HDAC8 may mediate cortactin deacetylation, which subsequently promotes actin filament polymerization and smooth muscle contraction.

  3. Src binds cortactin through an SH2 domain cystine-mediated linkage.

    Science.gov (United States)

    Evans, Jason V; Ammer, Amanda G; Jett, John E; Bolcato, Chris A; Breaux, Jason C; Martin, Karen H; Culp, Mark V; Gannett, Peter M; Weed, Scott A

    2012-12-15

    Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

  4. Cortactin affects cell migration by regulating intercellular adhesion and cell spreading

    NARCIS (Netherlands)

    van Rossum, AGSH; Moolenaar, WH; Schuuring, E

    2006-01-01

    Cortactin is an F-actin binding protein that stabilizes F-actin networks and promotes actin polymerization by activating the Arp2/3 complex. Overexpression of cortactin, as observed in several human cancers, stimulates cell migration, invasion, and experimental metastasis; however, the underlying

  5. Roles of Cortactin, an Actin Polymerization Mediator, in Cell Endocytosis

    Institute of Scientific and Technical Information of China (English)

    Li CHEN; Zhi-Wei WANG; Jian-wei ZHU; Xi ZHAN

    2006-01-01

    Cortactin, an actin-binding protein and a substrate of Src, is encoded by the EMS 1 oncogene.Cortactin is known to activate Arp2/3 complex-mediated actin polymerization and interact with dynamin, a large GTPase and proline rich domain-containing protein. Transferrin endocytosis was significantly reduced in cells by knock-down of cortactin expression as well as in vivo introduction of cortactin immunoreagents.Cortactin-dynamin interaction displayed morphologically dynamic co-distribution with a change in the endocytosis level in cells treated with an actin depolymerization reagent, cytochalasin D. In an in vitro beads assay, a branched actin network was recruited onto dynamin-coated beads in a cortactin Src homology domain 3 (SH3)-dependent manner. In addition, cortactin was found to function in the late stage of clathrin coated vesicle formation.Taken together, cortactin is required for optimal clathrin mediated endocytosis in a dynamin directed manner.

  6. Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation

    Institute of Scientific and Technical Information of China (English)

    YU Dan; ZHANG Peijun; ZHAN Xi

    2005-01-01

    Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.

  7. Cell invasion by Neisseria meningitidis requires a functional interplay between the focal adhesion kinase, Src and cortactin.

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    Heiko Slanina

    Full Text Available Entry of Neisseria meningitidis (the meningococcus into human brain microvascular endothelial cells (HBMEC is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK, which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells.

  8. Cortactin Adopts a Globular Conformation and Bundles Actin into Sheets

    Energy Technology Data Exchange (ETDEWEB)

    Cowieson, Nathan P.; King, Gordon; Cookson, David; Ross, Ian; Huber, Thomas; Hume, David A.; Kobe, Bostjan; Martin, Jennifer L. (Queensland); (Aust. Synch.)

    2008-08-21

    Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching.

  9. Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation.

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    Lee, Mi-Sook; Kim, Sudong; Kim, Baek Gil; Won, Cheolhee; Nam, Seo Hee; Kang, Suki; Kim, Hye-Jin; Kang, Minkyung; Ryu, Jihye; Song, Haeng Eun; Lee, Doohyung; Ye, Sang-Kyu; Jeon, Noo Li; Kim, Tai Young; Cho, Nam Hoon; Lee, Jung Weon

    2014-09-01

    Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents.

  10. Cortactin and phagocytosis in isolated Sertoli cells

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    Wolski Katja M

    2005-12-01

    Full Text Available Abstract Background Cortactin, an actin binding protein, has been associated with Sertoli cell ectoplasmic specializations in vivo, based on its immunolocalization around the heads of elongated spermatids, but not previously identified in isolated Sertoli cells. In an in vitro model of Sertoli cell-spermatid binding, cortactin was identified around debris and dead germ cells. Based on this observation, we hypothesized that this actin binding protein may be associated with a non-junction-related physiological function, such as phagocytosis. The purpose of this study was to identify the presence and distribution of cortactin in isolated rat Sertoli cells active in phagocytic activity following the addition of 0.8 μm latex beads. Results Sertoli cell monocultures were incubated with or without follicle stimulating hormone (FSH; 0.1 μg/ml in the presence or absence of cytochalasin D (2 μM, as an actin disrupter. Cortactin was identified by standard immunostaining with anti-cortactin, clone 4F11 (Upstate after incubation times of 15 min, 2 hr, and 24 hr with or without beads. Cells exposed to no hormone and no beads appeared to have a ubiquitous distribution of cortactin throughout the cytoplasm. In the presence of cytochalasin D, cortactin immunostaining was punctate and distributed in a pattern similar to that reported for actin in cells exposed to cytochalasin D. Sertoli cells not exposed to FSH, but activated with beads, did not show cortactin immunostaining around the phagocytized beads at any of the time periods. FSH exposure did not alter the distribution of cortactin within Sertoli cells, even when phagocytic activity was upregulated by the presence of beads. Conclusion Results of this study suggest cortactin is not associated with peripheralized actin at junctional or phagocytic sites. Further studies are necessary to clarify the role of cortactin in Sertoli cells.

  11. The Phosphorylation and Distribution of Cortactin Downstream of Integrin α9β1 Affects Cancer Cell Behaviour

    DEFF Research Database (Denmark)

    Høye, Anette M; Couchman, John R; Wewer, Ulla M;

    2016-01-01

    Integrins, a family of heterodimeric adhesion receptors are implicated in cell migration, development and cancer progression. They can adopt conformations that reflect their activation states and thereby impact adhesion strength and migration. Integrins in an intermediate activation state may...... localises, upon integrin activation. This was commensurate with reduced migration. The localisation and phosphorylation of cortactin Y470 was regulated by Yes kinase and PTEN phosphatase. Cortactin levels influenced fibronectin matrix assembly and active β1 integrin on the cell surface, being inversely...... correlated with migratory behaviour. This study underlines the complex interplay between cortactin and α9β1 integrin that regulates cell-extracellular matrix interactions....

  12. Cortactin overexpression results in sustained epidermal growth factor receptor signaling by preventing ligand-induced receptor degradation in human carcinoma cells

    NARCIS (Netherlands)

    van Rossum, AGSH; Gibcus, J; van der Wal, J; Schuuring, E

    2005-01-01

    The chromosome 11q13 region is frequently amplified in human carcinomas and results in an increased expression of various genes including cortactin, and is also associated with an increased invasive potential. Cortactin acts as an important regulator of the actin cytoskeleton. It is therefore very t

  13. Invadopodia proteins, cortactin, N-WASP and WIP differentially promote local invasiveness in ameloblastoma.

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    Siar, Chong Huat; Rahman, Zainal Ariff Bin Abdul; Tsujigiwa, Hidetsugu; Mohamed Om Alblazi, Kamila; Nagatsuka, Hitoshi; Ng, Kok Han

    2016-09-01

    Cell migration and invasion through interstitial tissues are dependent upon several specialized characteristics of the migratory cell notably generation of proteolytic membranous protrusions or invadopodia. Ameloblastoma is a benign odontogenic epithelial neoplasm with a locally infiltrative behaviour. Cortactin and MMT1-MMP are two invadopodia proteins implicated in its local invasiveness. Other invadopodia regulators, namely N-WASP, WIP and Src kinase remain unclarified. This study addresses their roles in ameloblastoma. Eighty-seven paraffin-embedded ameloblastoma cases (20 unicystic, 47 solid/multicystic, 3 desmoplastic and 17 recurrent) were subjected to immunohistochemistry for expression of cortactin, N-WASP, WIP, Src kinase and F-actin, and findings correlated with clinicopathological parameters. Invadopodia proteins (except Src kinase) and F-actin were widely detected in ameloblastoma (cortactin: n = 73/87, 83.9%; N-WASP: n = 59/87; 67.8%; WIP: n = 77/87; 88.5%; and F-actin: n = 87/87, 100%). Protein localization was mainly cytoplasmic and/or membranous, and occasionally nuclear for F-actin. Cortactin, which functions as an actin-scaffolding protein, demonstrated significantly higher expression levels within ameloblastoma tumoral epithelium than in stroma (P ameloblastoma is dependent upon the migratory potential of its tumour cells as defined by their distribution of cortactin, N-WASP and WIP in correlation with F-actin cytoskeletal dynamics. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Comparative genome analysis of cortactin and HSI : the significance of the F-actin binding repeat domain

    NARCIS (Netherlands)

    van Rossum, AGSH; Schuuring-Scholtes, E; Seggelen, VV; Kluin, PM; Schuuring, E

    2005-01-01

    Background: In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp)2/3 mediated actin polymerization. It shares a high amino acid seque

  15. Comparative genome analysis of cortactin and HS1: the significance of the F-actin binding repeat domain

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    Seggelen Vera

    2005-02-01

    Full Text Available Abstract Background In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp2/3 mediated actin polymerization. It shares a high amino acid sequence and structural similarity to hematopoietic lineage cell-specific protein 1 (HS1 although their functions differ considerable. In this manuscript we describe the genomic organization of these two genes in a variety of species by a combination of cloning and database searches. Based on our analysis, we predict the genesis of the actin-binding repeat domain during evolution. Results Cortactin homologues exist in sponges, worms, shrimps, insects, urochordates, fishes, amphibians, birds and mammalians, whereas HS1 exists in vertebrates only, suggesting that both genes have been derived from an ancestor cortactin gene by duplication. In agreement with this, comparative genome analysis revealed very similar exon-intron structures and sequence homologies, especially over the regions that encode the characteristic highly conserved F-actin-binding repeat domain. Cortactin splice variants affecting this F-actin-binding domain were identified not only in mammalians, but also in amphibians, fishes and birds. In mammalians, cortactin is ubiquitously expressed except in hematopoietic cells, whereas HS1 is mainly expressed in hematopoietic cells. In accordance with their distinct tissue specificity, the putative promoter region of cortactin is different from HS1. Conclusions Comparative analysis of the genomic organization and amino acid sequences of cortactin and HS1 provides inside into their origin and evolution. Our analysis shows that both genes originated from a gene duplication event and subsequently HS1 lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in

  16. SQSTM1/p62 interacts with HDAC6 and regulates deacetylase activity.

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    Jin Yan

    Full Text Available Protein aggregates can form in the cytoplasm of the cell and are accumulated at aggresomes localized to the microtubule organizing center (MTOC where they are subsequently degraded by autophagy. In this process, aggregates are engulfed into autophagosomes which subsequently fuse with lysosomes for protein degradation. A member of the class II histone deacetylase family, histone deacetylase 6(HDAC6 has been shown to be involved in both aggresome formation and the fusion of autophagosomes with lysosomes making it an attractive target to regulate protein aggregation. The scaffolding protein sequestosome 1(SQSTM1/p62 has also been shown to regulate accumulation and autophagic clearance of protein aggregates. Recent studies have revealed colocalization of HDAC6 and p62 to ubiquitinated mitochondria, as well as, ubiquitinated protein aggregates associated with the E3 ubiquitin ligase TRIM50. HDAC6 deacetylase activity is required for aggresome formation and can be regulated by protein interaction with HDAC6. Due to their colocalization at ubiquitinated protein aggregates, we sought to examine if p62 specifically interacted with HDAC6 and if so, if this interaction had any effect on HDAC6 activity and/or the physiological function of cortactin-F-actin assembly. We succeeded in identifying and mapping the direct interaction between HDAC6 and p62. We further show that this interaction regulates HDAC6 deacetylase activity. Data are presented demonstrating that the absence of p62 results in hyperactivation of HDAC6 and deacetylation of α-tubulin and cortactin. Further, upon induction of protein misfolding we show that p62 is required for perinuclear co-localization of cortactin-F-actin assemblies. Thus, our findings indicate that p62 plays a key role in regulating the recruitment of F-actin network assemblies to the MTOC, a critical cellular function that is required for successful autophagic clearance of protein aggregates.

  17. 癌基因CTTN编码蛋白Cortactin与大肠癌细胞内吞作用的关系%A pivotal role of cortactin,a CTTN encoding protein,in endocytosis of human colon cancer

    Institute of Scientific and Technical Information of China (English)

    朱建伟; 马利林; 黄宝玉; 刘培根; 汝凯; 曹翔; 钱飞

    2011-01-01

    目的 分析Cortactin分子与大肠癌细胞内吞作用的关系,了解癌组织高表达Cortactin的内在意义.方法 采用免疫组化和蛋白印迹检测大肠癌组织、癌旁组织及大肠癌细胞系中Cortactin表达水平;采用小干扰RNA抑制癌细胞Cortactin表达,并构建Cortactin不同功能区缺失突变体,检测各种癌细胞Tfn内吞作用的变化;采用免疫荧光化学方法分析Cortactin与癌细胞内吞作用路径的关系.结果 大肠癌组织Cortactin表达高于癌旁组织,表达率分别为77.5%及47.5%(P<0.05).siRNA处理的癌细胞Tfn摄取值为0.61±0.02,对照为1.01±0.16(P<0.05).高表达Cortactin的大肠癌细胞为实现正常的内吞作用,需要足够数量和结构完整的Cortactin蛋白;Cortactin参与内吞物质的胞内运输过程.结论 大肠癌细胞的内吞作用依赖Cortactin分子功能的发挥,高表达Cortactin有利于大肠癌细胞基于内吞作用的信号传导、侵袭和转移.%Objective To explore the role of cortactin in endocytosis of colon cancer cells and clarify the significance of its over-expression in colon cancer tissues.Methods Immunohistochemistry and Western blot were employed to detect the expression of cortactin in benign and malignant tissues and cells.Cell endocytosis was examined in cancer cells after siRNA treatment and DNA transfection with plasmid encoding cortactin wild type and domain deletion mutants.Results Cortactin was over-expressed in colon cancer tissues than in adjacent normal tissues.The expression rate was 77.5% in cancer tissues and 47.5% in normal tissues ( P < 0.05 ).The value of transferrin uptake was 0.61 ± 0.02 in siRNA treated cancer cells and 1.01 ± 0.16 in the control cells (P < 0.05).Intact molecule and sufficient level of cortactin was required for an optimal endocytosis of cancer cells.Cortactin was involved in coated-vesicle transportation in cells.Conclusion Endocytosis in colon cancer cells is dependent on an intact

  18. Persistent Morbillivirus Infection Leads to Altered Cortactin Distribution in Histiocytic Sarcoma Cells with Decreased Cellular Migration Capacity

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    Pfankuche, Vanessa Maria; Sayed-Ahmed, Mohamed; Contioso, Vanessa Bono; Spitzbarth, Ingo; Rohn, Karl; Ulrich, Reiner; Deschl, Ulrich; Kalkuhl, Arno; Baumgärtner, Wolfgang; Puff, Christina

    2016-01-01

    Histiocytic sarcomas represent rare but fatal neoplasms in humans. Based on the absence of a commercially available human histiocytic sarcoma cell line the frequently affected dog displays a suitable translational model. Canine distemper virus, closely related to measles virus, is a highly promising candidate for oncolytic virotherapy. Therapeutic failures in patients are mostly associated with tumour invasion and metastasis often induced by misdirected cytoskeletal protein activities. Thus, the impact of persistent canine distemper virus infection on the cytoskeletal protein cortactin, which is frequently overexpressed in human cancers with poor prognosis, was investigated in vitro in a canine histiocytic sarcoma cell line (DH82). Though phagocytic activity, proliferation and apoptotic rate were unaltered, a significantly reduced migration activity compared to controls (6 hours and 1 day after seeding) accompanied by a decreased number of cortactin mRNA transcripts (1 day) was detected. Furthermore, persistently canine distemper virus infected DH82 cells showed a predominant diffuse intracytoplasmic cortactin distribution at 6 hours and 1 day compared to controls with a prominent membranous expression pattern (p ≤ 0.05). Summarized, persistent canine distemper virus infection induces reduced tumour cell migration associated with an altered intracellular cortactin distribution, indicating cytoskeletal changes as one of the major pathways of virus-associated inhibition of tumour spread. PMID:27911942

  19. Cortactin is associated with perineural invasion in the deep invasive front area of laryngeal carcinomas

    DEFF Research Database (Denmark)

    Ambrosio, Eliane Papa; Rosa, Fabíola Encinas; Domingues, Maria Aparecida Custódio;

    2011-01-01

    . The transcript expression levels were evaluated in a subset of cases. Overexpression of CTTN cytoplasmatic protein (80% of cases in both the deep invasive front and superficial areas) and transcript (30% of samples) was detected in a significant number of cases. In more than 20% of cases, observation verified...... of CTTN in the deep invasive front showed good prognosis parameters, and a second group with moderate to strong expression of CTTN were associated with an unfavorable prognosis, suggesting an association with worse outcome. Taken together, these results suggest that the deep invasive front might...... be considered a grading system in laryngeal carcinomas and that cortactin is a putative marker of worse outcome in the deep invasive front of laryngeal carcinomas....

  20. Expression of DIAPH1 is up-regulated in colorectal cancer and its down-regulation strongly reduces the metastatic capacity of colon carcinoma cells.

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    Lin, Yuan-Na; Izbicki, Jakob R; König, Alexandra; Habermann, Jens K; Blechner, Christine; Lange, Tobias; Schumacher, Udo; Windhorst, Sabine

    2014-04-01

    In most cases, metastatic colorectal cancer is not curable, thus new approaches are necessary to identify novel targets for colorectal cancer therapy. Actin-binding-proteins (ABPs) directly regulate motility of metastasising tumor cells, and for cortactin an association with colon cancer metastasis has been already shown. However, as its depletion only incompletely inhibits metastasis, additional, more suitable cellular targets have to be identified. Here we analyzed expression of the ABPs, DIAPH1, VASP, N-WASP, and fascin in comparison with cortactin and found that, besides cortactin, DIAPH1 was expressed with the highest frequency (63%) in colorectal cancer. As well as cortactin, DIAPH1 was not detectable in normal colon tissue and expression of both proteins was positively correlated with metastasis of colorectal cancer. To analyse the mechanistic role of DIAPH1 for metastasis of colon carcinoma cells in comparison with cortactin, expression of the proteins was stably down-regulated in the human colon carcinoma cell lines HT-29, HROC-24 and HCT-116. Analysis of metastasis of colon carcinoma cells in SCID mice revealed that depletion of DIAPH1 reduced metastasis 60-fold and depletion of cortactin 16-fold as compared with control cells. Most likely the stronger effect of DIAPH1 depletion on colon cancer metastasis is due to the fact that in vitro knock down of DIAPH1 impaired all steps of metastasis; adhesion, invasion and migration while down-regulation of cortactin only reduced adhesion and invasion. This very strong reducing effect of DIAPH1 depletion on colon carcinoma cell metastasis makes the protein a promising therapeutic target for individualized colorectal cancer therapy.

  1. 77 FR 51784 - Federal Acquisition Regulation; Information Collection; Qualification Requirements

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    2012-08-27

    .... Under the qualification requirements, an end item, or a component thereof, may be required to be... Regulation; Information Collection; Qualification Requirements AGENCY: Department of Defense (DOD), General...: Under the provisions of the Paperwork Reduction Act, the Regulatory Secretariat will be submitting...

  2. SDF1 reduces interneuron leading process branching through dual regulation of actin and microtubules.

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    Lysko, Daniel E; Putt, Mary; Golden, Jeffrey A

    2014-04-02

    Normal cerebral cortical function requires a highly ordered balance between projection neurons and interneurons. During development these two neuronal populations migrate from distinct progenitor zones to form the cerebral cortex, with interneurons originating in the more distant ganglionic eminences. Moreover, deficits in interneurons have been linked to a variety of neurodevelopmental disorders underscoring the importance of understanding interneuron development and function. We, and others, have identified SDF1 signaling as one important modulator of interneuron migration speed and leading process branching behavior in mice, although how SDF1 signaling impacts these behaviors remains unknown. We previously found SDF1 inhibited leading process branching while increasing the rate of migration. We have now mechanistically linked SDF1 modulation of leading process branching behavior to a dual regulation of both actin and microtubule organization. We find SDF1 consolidates actin at the leading process tip by de-repressing calpain protease and increasing proteolysis of branched-actin-supporting cortactin. Additionally, SDF1 stabilizes the microtubule array in the leading process through activation of the microtubule-associated protein doublecortin (DCX). DCX stabilizes the microtubule array by bundling microtubules within the leading process, reducing branching. These data provide mechanistic insight into the regulation of interneuron leading process dynamics during neuronal migration in mice and provides insight into how cortactin and DCX, a known human neuronal migration disorder gene, participate in this process.

  3. SDF1 Reduces Interneuron Leading Process Branching through Dual Regulation of Actin and Microtubules

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    Lysko, Daniel E.; Putt, Mary

    2014-01-01

    Normal cerebral cortical function requires a highly ordered balance between projection neurons and interneurons. During development these two neuronal populations migrate from distinct progenitor zones to form the cerebral cortex, with interneurons originating in the more distant ganglionic eminences. Moreover, deficits in interneurons have been linked to a variety of neurodevelopmental disorders underscoring the importance of understanding interneuron development and function. We, and others, have identified SDF1 signaling as one important modulator of interneuron migration speed and leading process branching behavior in mice, although how SDF1 signaling impacts these behaviors remains unknown. We previously found SDF1 inhibited leading process branching while increasing the rate of migration. We have now mechanistically linked SDF1 modulation of leading process branching behavior to a dual regulation of both actin and microtubule organization. We find SDF1 consolidates actin at the leading process tip by de-repressing calpain protease and increasing proteolysis of branched-actin-supporting cortactin. Additionally, SDF1 stabilizes the microtubule array in the leading process through activation of the microtubule-associated protein doublecortin (DCX). DCX stabilizes the microtubule array by bundling microtubules within the leading process, reducing branching. These data provide mechanistic insight into the regulation of interneuron leading process dynamics during neuronal migration in mice and provides insight into how cortactin and DCX, a known human neuronal migration disorder gene, participate in this process. PMID:24695713

  4. The Regulation of Health Requirements for Police and Current Problems

    National Research Council Canada - National Science Library

    Ramazan Akçan; Mahmut Şerif Yıldırım; Aykut Lale; Abdurrahman İsak; Mehmet Cavlak; Aysun Balseven Odabaşı; Ali Rıza Tümer

    2017-01-01

    Introduction: Forensic medicine specialists might prepare reports about health conditions of candidates for Police Organization or assessment of organization's staff based on The Regulation of Health Requirements for Police...

  5. GENERAL CONSIDERATIONS ON REGULATIONS AND SAFETY REQUIREMENTS FOR QUADRICYCLES

    Directory of Open Access Journals (Sweden)

    Ana Pavlovic

    2015-12-01

    Full Text Available In recent years, a new class of compact vehicles has been emerging and wide-spreading all around Europe: the quadricycle. These four-wheeled motor vehicles, originally derived from motorcycles, are a small and fuel-efficient mean of transportation used in rural or urban areas as an alternative to motorbikes or city cars. In some countries, they are also endorsed by local authorities and institutions which support small and environmentally-friendly vehicles. In this paper, several general considerations on quadricycles will be provided including the vehicle classification, evolution of regulations (as homologation, driver licence, emissions, etc, technical characteristics, safety requirements, most relevant investigations, and other additional useful information (e.g. references, links. It represents an important and actual topic of investigation for designers and manufacturers considering that the new EU regulation on the approval and market surveillance of quadricycles will soon enter in force providing conclusive requirements for functional safety environmental protection of these promising vehicles.

  6. Actin is required for IFT regulation in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Avasthi, Prachee; Onishi, Masayuki; Karpiak, Joel; Yamamoto, Ryosuke; Mackinder, Luke; Jonikas, Martin C; Sale, Winfield S; Shoichet, Brian; Pringle, John R; Marshall, Wallace F

    2014-09-01

    Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length.

  7. FTIP1 is an essential regulator required for florigen transport.

    Directory of Open Access Journals (Sweden)

    Lu Liu

    Full Text Available The capacity to respond to day length, photoperiodism, is crucial for flowering plants to adapt to seasonal change. The photoperiodic control of flowering in plants is mediated by a long-distance mobile floral stimulus called florigen that moves from leaves to the shoot apex. Although the proteins encoded by FLOWERING LOCUS T (FT in Arabidopsis and its orthologs in other plants are identified as the long-sought florigen, whether their transport is a simple diffusion process or under regulation remains elusive. Here we show that an endoplasmic reticulum (ER membrane protein, FT-INTERACTING PROTEIN 1 (FTIP1, is an essential regulator required for FT protein transport in Arabidopsis. Loss of function of FTIP1 exhibits late flowering under long days, which is partly due to the compromised FT movement to the shoot apex. FTIP1 and FT share similar mRNA expression patterns and subcellular localization, and they interact specifically in phloem companion cells. FTIP1 is required for FT export from companion cells to sieve elements, thus affecting FT transport through the phloem to the SAM. Our results provide a mechanistic understanding of florigen transport, demonstrating that FT moves in a regulated manner and that FTIP1 mediates FT transport to induce flowering.

  8. FTIP1 is an essential regulator required for florigen transport.

    Science.gov (United States)

    Liu, Lu; Liu, Chang; Hou, Xingliang; Xi, Wanyan; Shen, Lisha; Tao, Zhen; Wang, Yue; Yu, Hao

    2012-01-01

    The capacity to respond to day length, photoperiodism, is crucial for flowering plants to adapt to seasonal change. The photoperiodic control of flowering in plants is mediated by a long-distance mobile floral stimulus called florigen that moves from leaves to the shoot apex. Although the proteins encoded by FLOWERING LOCUS T (FT) in Arabidopsis and its orthologs in other plants are identified as the long-sought florigen, whether their transport is a simple diffusion process or under regulation remains elusive. Here we show that an endoplasmic reticulum (ER) membrane protein, FT-INTERACTING PROTEIN 1 (FTIP1), is an essential regulator required for FT protein transport in Arabidopsis. Loss of function of FTIP1 exhibits late flowering under long days, which is partly due to the compromised FT movement to the shoot apex. FTIP1 and FT share similar mRNA expression patterns and subcellular localization, and they interact specifically in phloem companion cells. FTIP1 is required for FT export from companion cells to sieve elements, thus affecting FT transport through the phloem to the SAM. Our results provide a mechanistic understanding of florigen transport, demonstrating that FT moves in a regulated manner and that FTIP1 mediates FT transport to induce flowering.

  9. 76 FR 50461 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Types of...

    Science.gov (United States)

    2011-08-15

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Types of Contracts AGENCY: Defense Acquisition Regulations System, Department of... information collection requirement. SUMMARY: In compliance with Section 3506(c)(2)(A) of the...

  10. 78 FR 60898 - Regulation on Definition and Requirements for a Nationally Recognized Testing Laboratory...

    Science.gov (United States)

    2013-10-02

    ... Occupational Safety and Health Administration Regulation on Definition and Requirements for a Nationally..., ``definition and requirements for a nationally recognized testing laboratory'' (The Regulation). The Regulation... place all submissions, including any personal information provided, in the public docket...

  11. Rigid performance requirements assure public safety by regulations

    Energy Technology Data Exchange (ETDEWEB)

    Nickell, R.E.; Glass, R.E.

    1987-07-01

    Title 10, Part 71 of the Code of Federal Regulations (10 CFR 71) provides a set of prescriptive performance test requirements for spent nuclear fuel and high-level waste transport packaging containment systems. The hypothetical accident conditions, which involve a sequence of impact, puncture, fire, and water immersion events, are referred to as rigid because of their extremely prescriptive nature. These hypothetical accident events have now been placed within the context of real transportation accidents, at least for conventional austenitic stainless steel/ lead gamma shielded cask designs. The assurance of public safety, including the issue of safety margin for very severe accident events is discussed in this paper for both conventional and innovative cask design concepts. A particular risk assessment approach that follows from work at the Lawrence Livermore National Laboratory is suggested.

  12. Rigid performance requirements assure public safety by regulations

    Energy Technology Data Exchange (ETDEWEB)

    Nickell, R.E.; Glass, R.E.

    1987-01-01

    Title 10, Part 71 of the Code of Federal Regulations (10 CFR 71) provides a set of prescriptive performance test requirements for spent nuclear fuel and high-level waste transport packaging containment systems. The hypothetical accident conditions, which involve a sequence of impact, puncture, fire, and water immersion events, are referred to as rigid because of their extremely prescriptive nature. These hypothetical accident events have now been placed within the context of real transportation accidents, at least for conventional austenitic stainless steel/lead gamma shielded cask designs. The assurance of public safety, including the issue of safety margin for very severe accident events, is discussed in this paper for both conventional and innovative cask design concepts. A particular risk assessment approach that follows from work at the Lawrence Livermore National Laboratory is suggested. 6 refs., 2 figs.

  13. 78 FR 25445 - Federal Acquisition Regulation; Submission for OMB Review; Quality Assurance Requirements

    Science.gov (United States)

    2013-05-01

    ... Regulation; Submission for OMB Review; Quality Assurance Requirements AGENCY: Department of Defense (DOD... information collection requirement concerning quality assurance requirements. A notice was published in the..., Quality Assurance Requirements, by any of the following methods: Regulations.gov : http://www.regulations...

  14. 77 FR 67366 - Federal Acquisition Regulation; Information Collection; Quality Assurance Requirements

    Science.gov (United States)

    2012-11-09

    ... Regulation; Information Collection; Quality Assurance Requirements AGENCY: Department of Defense (DOD... information collection requirement concerning quality assurance requirements. Public comments are particularly... Information Collection 9000- 0077, Quality Assurance Requirements, by any of the following methods...

  15. 77 FR 66464 - Federal Acquisition Regulation; Submission for OMB Review; Value Engineering Requirements

    Science.gov (United States)

    2012-11-05

    ... Regulation; Submission for OMB Review; Value Engineering Requirements AGENCIES: Department of Defense (DOD... approved information collection requirement concerning Value Engineering Requirements. A notice was...: Submit comments identified by Information Collection 9000- 0027, Value Engineering Requirements, by...

  16. 77 FR 43076 - Federal Acquisition Regulation; Information Collection; Value Engineering Requirements

    Science.gov (United States)

    2012-07-23

    ... Regulation; Information Collection; Value Engineering Requirements AGENCIES: Department of Defense (DOD... approved information collection requirement concerning Value Engineering Requirements. Public comments are...: Submit comments identified by Information Collection 9000- 0027, Value Engineering Requirements, by...

  17. Thyroid hormone is required for hypothalamic neurons regulating cardiovascular functions.

    Science.gov (United States)

    Mittag, Jens; Lyons, David J; Sällström, Johan; Vujovic, Milica; Dudazy-Gralla, Susi; Warner, Amy; Wallis, Karin; Alkemade, Anneke; Nordström, Kristina; Monyer, Hannah; Broberger, Christian; Arner, Anders; Vennström, Björn

    2013-01-01

    Thyroid hormone is well known for its profound direct effects on cardiovascular function and metabolism. Recent evidence, however, suggests that the hormone also regulates these systems indirectly through the central nervous system. While some of the molecular mechanisms underlying the hormone's central control of metabolism have been identified, its actions in the central cardiovascular control have remained enigmatic. Here, we describe a previously unknown population of parvalbuminergic neurons in the anterior hypothalamus that requires thyroid hormone receptor signaling for proper development. Specific stereotaxic ablation of these cells in the mouse resulted in hypertension and temperature-dependent tachycardia, indicating a role in the central autonomic control of blood pressure and heart rate. Moreover, the neurons exhibited intrinsic temperature sensitivity in patch-clamping experiments, providing a new connection between cardiovascular function and core temperature. Thus, the data identify what we believe to be a novel hypothalamic cell population potentially important for understanding hypertension and indicate developmental hypothyroidism as an epigenetic risk factor for cardiovascular disorders. Furthermore, the findings may be beneficial for treatment of the recently identified patients that have a mutation in thyroid hormone receptor α1.

  18. Discovery of a Splicing Regulator Required for Cell Cycle Progression

    Energy Technology Data Exchange (ETDEWEB)

    Suvorova, Elena S.; Croken, Matthew; Kratzer, Stella; Ting, Li-Min; Conde de Felipe, Magnolia; Balu, Bharath; Markillie, Lye Meng; Weiss, Louis M.; Kim, Kami; White, Michael W.

    2013-02-01

    In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

  19. Growth Conditions Regulate the Requirements for Caulobacter Chromosome Segregation

    DEFF Research Database (Denmark)

    Shebelut, Conrad W.; Jensen, Rasmus Bugge; Gitai, Zemer

    2009-01-01

    Growth environments are important metabolic and developmental regulators. Here we demonstrate a growth environment-dependent effect on Caulobacter chromosome segregation of a small-molecule inhibitor of the MreB bacterial actin cytoskeleton. Our results also implicate ParAB as important segregation...... determinants, suggesting that multiple distinct mechanisms can mediate Caulobacter chromosome segregation and that their relative contributions can be environmentally regulated....

  20. Growth Conditions Regulate the Requirements for Caulobacter Chromosome Segregation▿ †

    OpenAIRE

    Shebelut, Conrad W.; Jensen, Rasmus B.; Gitai, Zemer

    2008-01-01

    Growth environments are important metabolic and developmental regulators. Here we demonstrate a growth environment-dependent effect on Caulobacter chromosome segregation of a small-molecule inhibitor of the MreB bacterial actin cytoskeleton. Our results also implicate ParAB as important segregation determinants, suggesting that multiple distinct mechanisms can mediate Caulobacter chromosome segregation and that their relative contributions can be environmentally regulated.

  1. Growth conditions regulate the requirements for Caulobacter chromosome segregation.

    Science.gov (United States)

    Shebelut, Conrad W; Jensen, Rasmus B; Gitai, Zemer

    2009-02-01

    Growth environments are important metabolic and developmental regulators. Here we demonstrate a growth environment-dependent effect on Caulobacter chromosome segregation of a small-molecule inhibitor of the MreB bacterial actin cytoskeleton. Our results also implicate ParAB as important segregation determinants, suggesting that multiple distinct mechanisms can mediate Caulobacter chromosome segregation and that their relative contributions can be environmentally regulated.

  2. Thyroid hormone is required for hypothalamic neurons regulating cardiovascular functions

    NARCIS (Netherlands)

    Mittag, J.; Lyons, D.J.; Sällström, J.; Vujoviv, M.; Dudazy-Gralla, S.; Warner, A.; Wallis, K.; Alkemade, A.; Nordström, K.; Monyer, H.; Broberger, C.; Arner, A.; Vennström, B.

    2013-01-01

    Thyroid hormone is well known for its profound direct effects on cardiovascular function and metabolism. Recent evidence, however, suggests that the hormone also regulates these systems indirectly through the central nervous system. While some of the molecular mechanisms underlying the hormone’s

  3. Epigenetic Regulation in Hepatocellular Carcinoma Requires Long Noncoding RNAs

    Directory of Open Access Journals (Sweden)

    Laura Amicone

    2015-01-01

    Full Text Available Recent evidence has proven the relevance of epigenetic changes in the development of hepatocellular carcinoma (HCC, the major adult liver malignancy. Moreover, HCC onset and progression correlate with the deregulation of several long noncoding RNAs (lncRNAs, exhibiting great biological significance. As discussed in this review, many of these transcripts are able to specifically act as tumor suppressors or oncogenes by means of their role as molecular platforms. Indeed, these lncRNAs are able to bind and recruit epigenetic modifiers on specific genomic loci, ultimately resulting in regulation of the gene expression relevant in cancer development. The evidence presented in this review highlights that lncRNAs-mediated epigenetic regulation should be taken into account for potential targeted therapeutic approaches.

  4. Financial Market Regulation in Germany - Capital Requirements of Financial Institutions

    Directory of Open Access Journals (Sweden)

    Daniel Karl Detzer

    2015-03-01

    Full Text Available This paper examines capital adequacy regulation in Germany. The first part reviews capital adequacy regulation from the 1930s up to the financial crisis and identifies two main trends: a gradual softening of the eligibility criteria for equity and increasing reliance on internal risk models. While the first trend has been reversed following the financial crisis, internal risk models still play a central role. Therefore, the second part discusses the problems with the use of internal risk models and discusses the potentials of Basel 2.5 and Basel III to alleviate the identified problems. It is concluded that the relevant problems are not resolved. Therefore, in the final part some suggestions of how the problems could be addressed properly are given.

  5. 75 FR 60263 - Federal Acquisition Regulation; Offering a Construction Requirement-8(a) Program

    Science.gov (United States)

    2010-09-29

    ... Federal Acquisition Regulation; Offering a Construction Requirement--8(a) Program AGENCIES: Department of... Regulations Council (Councils) are issuing a final rule amending the Federal Acquisition Regulation (FAR) to revise FAR subpart 19.8, Contracting with the Small Business Administration (The 8(a) Program),...

  6. 13 CFR 120.1511 - Certification and other reporting and notification requirements for Other Regulated SBLCs.

    Science.gov (United States)

    2010-01-01

    ...) Certification. An SBLC seeking Other Regulated SBLC status must certify to SBA in writing that its lending... or state banking regulator that regulates the lending activities of the SBLC; (2) A statement that... may appeal this decision to the AA/CA. (g) Failure to satisfy requirements. In the event that an...

  7. 78 FR 5452 - Federal Acquisition Regulation; Submission for OMB Review; Qualification Requirements

    Science.gov (United States)

    2013-01-25

    ... award of a contract. Under the qualification requirements, an end item, or a component thereof, may be... offeror before award of a contract. Under the qualification requirements, an end item, or a component... Regulation; Submission for OMB Review; Qualification Requirements AGENCIES: Department of Defense...

  8. 75 FR 14448 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Science.gov (United States)

    2010-03-25

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good Clinical Practices; Public Workshop AGENCY: Food and Drug Administration, HHS. ACTION: Notice of public workshop. SUMMARY: The Food and Drug Administration (FDA) Los...

  9. 26 CFR 55.6001-1 - Notice or regulations requiring records, statements, and special returns.

    Science.gov (United States)

    2010-04-01

    ... INVESTMENT TRUSTS AND REGULATED INVESTMENT COMPANIES Procedure and Administration § 55.6001-1 Notice or regulations requiring records, statements, and special returns. (a) In general. Any person subject to tax... retained so long as the contents thereof may become material in the administration of any internal...

  10. 76 FR 14562 - Federal Acquisition Regulation; Additional Requirements for Market Research

    Science.gov (United States)

    2011-03-16

    ... 52 RIN 9000-AL50 Federal Acquisition Regulation; Additional Requirements for Market Research AGENCY... interim rule amending the Federal Acquisition Regulation (FAR) to implement section 826, Market Research... items engages in market research as necessary before making purchases. DATES: Effective Date: April...

  11. An SH3 binding motif within the nucleocapsid protein of porcine reproductive and respiratory syndrome virus interacts with the host cellular signaling proteins STAMI, TXK, Fyn, Hck, and cortactin.

    Science.gov (United States)

    Kenney, Scott P; Meng, Xiang-Jin

    2015-06-02

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease, and has a complicated virus-host immunomodulation that often leads to a weak Th2 immune response and viral persistence. In this study, we identified a Src homology 3 (SH3) binding motif, PxxPxxP, that is conserved within the N protein of PRRSV strains. Subsequently, we identified five host cellular proteins [signal transducing adaptor molecule (STAM)I, TXK tyrosine kinase (TXK), protein tyrosine kinase fyn (Fyn), hematopoietic cell kinase (Hck), and cortactin] that interact with this SH3 motif. We demonstrated that binding of SH3 proteins with PRRSV N protein depends on at least one intact PxxP motif as disruption of P53 within the motif significantly reduced interaction of each of the 5 proteins. The first PxxP motif appears to be more important for STAMI-N protein interactions whereas the second PxxP motif was more important for Hck interaction. Both STAMI and Hck interactions with PRRSV N protein required an unhindered C-terminal domain as the interaction was only observed with STAMI and Hck proteins with N-terminal but not C-terminal fluorescent tags. We showed that the P56 residue within the SH3 motif is critical for virus lifecycle as mutation resulted in a loss of virus infectivity, however the P50 and P53 mutations did not abolish virus infectivity suggesting that these highly conserved proline residues within the SH3 motif may provide a selective growth advantage through interactions with the host rather than a vital functional element. These results have important implications in understanding PRRSV-host interactions.

  12. Invadopodia Are Required for Cancer Cell Extravasation and Are a Therapeutic Target for Metastasis

    Directory of Open Access Journals (Sweden)

    Hon S. Leong

    2014-09-01

    Full Text Available Tumor cell extravasation is a key step during cancer metastasis, yet the precise mechanisms that regulate this dynamic process are unclear. We utilized a high-resolution time-lapse intravital imaging approach to visualize the dynamics of cancer cell extravasation in vivo. During intravascular migration, cancer cells form protrusive structures identified as invadopodia by their enrichment of MT1-MMP, cortactin, Tks4, and importantly Tks5, which localizes exclusively to invadopodia. Cancer cells extend invadopodia through the endothelium into the extravascular stroma prior to their extravasation at endothelial junctions. Genetic or pharmacological inhibition of invadopodia initiation (cortactin, maturation (Tks5, or function (Tks4 resulted in an abrogation of cancer cell extravasation and metastatic colony formation in an experimental mouse lung metastasis model. This provides direct evidence of a functional role for invadopodia during cancer cell extravasation and distant metastasis and reveals an opportunity for therapeutic intervention in this clinically important process.

  13. 31 CFR 103.120 - Anti-money laundering program requirements for financial institutions regulated by a Federal...

    Science.gov (United States)

    2010-07-01

    ... requirements for financial institutions regulated by a Federal functional regulator or a self-regulatory... financial institutions regulated by a Federal functional regulator or a self-regulatory organization, and... futures commission merchants. A financial institution regulated by a self-regulatory organization shall...

  14. Franchise Values, Regulatory Monitoring, and Capital Requirements in Optimal Bank Regulation

    DEFF Research Database (Denmark)

    Andersen, Thomas Barnebeck; Harr, Thomas

    2008-01-01

    This paper demonstrates that financial deregulation is likely to make standard prudential regulatory instruments less effective in curbing excessive risk-taking incentives among banks. This has interesting implications for optimal bank regulation. When there is an increase in competition......, the optimal capital requirement should increase, whereas regulatory auditing should decrease. In contrast, when there is an increase in gambling yields, auditing should always increase, whereas the optimal capital requirement may increase or decrease....

  15. PR72, a novel regulator of Wnt signaling required for Naked cuticle function

    NARCIS (Netherlands)

    Creyghton, M.P.; Roël, G.; Eichhorn, P.J.A.; Hijmans, E.M.; Maurer, I.; Destrée, O.; Bernards, R.A.

    2005-01-01

    The Wnt signaling cascade is a central regulator of cell fate determination during embryonic development, whose deregulation contributes to oncogenesis. Naked cuticle is the first Wnt-induced antagonist found in this pathway, establishing a negative-feedback loop that limits the Wnt signal required

  16. Capital Regulation, Liquidity Requirements and Taxation in a Dynamic Model of Banking

    NARCIS (Netherlands)

    Di Nicolo, G.; Gamba, A.; Lucchetta, M.

    2011-01-01

    This paper formulates a dynamic model of a bank exposed to both credit and liquidity risk, which can resolve financial distress in three costly forms: fire sales, bond issuance ad equity issuance. We use the model to analyze the impact of capital regulation, liquidity requirements and taxation on ba

  17. Capital Regulation, Liquidity Requirements and Taxation in a Dynamic Model of Banking

    NARCIS (Netherlands)

    Di Nicolo, G.; Gamba, A.; Lucchetta, M.

    2011-01-01

    This paper formulates a dynamic model of a bank exposed to both credit and liquidity risk, which can resolve financial distress in three costly forms: fire sales, bond issuance and equity issuance. We use the model to analyze the impact of capital regulation, liquidity requirements and taxation on b

  18. 77 FR 34315 - National Pollutant Discharge Elimination System-Proposed Regulations to Establish Requirements...

    Science.gov (United States)

    2012-06-11

    .... In other words, the cooling tower was in place but cooling water was used in a single pass mode, with...--Proposed Regulations to Establish Requirements for Cooling Water Intake Structures at Existing Facilities... proposed standards for cooling water intake structures at all existing power generating facilities...

  19. 78 FR 73104 - Federal Travel Regulation (FTR); Agency Requirements for Payment of Expenses Connected With the...

    Science.gov (United States)

    2013-12-05

    ...] RIN 3090-AJ37 Federal Travel Regulation (FTR); Agency Requirements for Payment of Expenses Connected... family member. The employee may elect an alternate destination, but it must be approved by your agency...) The new CONUS location if in transit; or (c) An alternate destination as approved by the agency....

  20. 50 CFR 25.23 - What are the general regulations and information collection requirements?

    Science.gov (United States)

    2010-10-01

    ... collect information to assist us in administering our programs in accordance with statutory authorities... information collection requirements? 25.23 Section 25.23 Wildlife and Fisheries UNITED STATES FISH AND... PROVISIONS Administrative Provisions § 25.23 What are the general regulations and information...

  1. PR72, a novel regulator of Wnt signaling required for Naked cuticle function

    NARCIS (Netherlands)

    Creyghton, M.P.; Roël, G.; Eichhorn, P.J.A.; Hijmans, E.M.; Maurer, I.; Destrée, O.; Bernards, R.A.

    2005-01-01

    The Wnt signaling cascade is a central regulator of cell fate determination during embryonic development, whose deregulation contributes to oncogenesis. Naked cuticle is the first Wnt-induced antagonist found in this pathway, establishing a negative-feedback loop that limits the Wnt signal required

  2. 75 FR 51824 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Science.gov (United States)

    2010-08-23

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good Clinical Practice; Public Workshop AGENCY: Food and Drug Administration, HHS. ACTION: Notice of public workshop. ] SUMMARY: The Food and Drug Administration (FDA) New Jersey...

  3. Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

    DEFF Research Database (Denmark)

    Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K

    2013-01-01

    ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together...

  4. Hydrogen Monitoring Requirements in the Global Technical Regulation on Hydrogen and Fuel Cell Vehicles: Preprint

    Energy Technology Data Exchange (ETDEWEB)

    Buttner, William; Rivkin, Carl; Burgess, Robert; Hartmann, Kevin; Bubar, Max; Post, Matthew; Boon-Brett, Lois; Weidner, Eveline; Moretto, Pietro

    2016-07-01

    The United Nations Global Technical Regulation (GTR) Number 13 (Global Technical Regulation on Hydrogen and Fuel Cell Vehicles) is the defining document regulating safety requirements in hydrogen vehicles, and in particular fuel cell electric vehicles (FCEV). GTR Number 13 has been formally implemented and will serve as the basis for the national regulatory standards for FCEV safety in North America (Canada, United States), Japan, Korea, and the European Union. The GTR defines safety requirement for these vehicles, including specifications on the allowable hydrogen levels in vehicle enclosures during in-use and post-crash conditions and on the allowable hydrogen emissions levels in vehicle exhaust during certain modes of normal operation. However, in order to be incorporated into national regulations, that is, in order to be binding, methods to verify compliance to the specific requirements must exist. In a collaborative program, the Sensor Laboratories at the National Renewable Energy Laboratory in the United States and the Joint Research Centre, Institute for Energy and Transport in the Netherlands have been evaluating and developing analytical methods that can be used to verify compliance to the hydrogen release requirement as specified in the GTR.

  5. Oogenesis requires germ cell-specific transcriptional regulators Sohlh1 and Lhx8

    Science.gov (United States)

    Pangas, Stephanie A.; Choi, Youngsok; Ballow, Daniel J.; Zhao, Yangu; Westphal, Heiner; Matzuk, Martin M.; Rajkovic, Aleksandar

    2006-01-01

    Mammalian oogenesis requires oocyte-specific transcriptional regulators. The full complement of oocyte-specific transcription factors is unknown. Here, we describe the finding that Sohlh1, a spermatogenesis and oogenesis basic helix–loop–helix transcription factor in females, is preferentially expressed in oocytes and required for oogenesis. Sohlh1 disruption perturbs follicular formation in part by causing down-regulation of two genes that are known to disrupt folliculogenesis: newborn ovary homeobox gene (Nobox) and factor in the germ-line alpha (Figla). In addition, we show that Lhx8 is downstream of Sohlh1 and critical in fertility. Thus, Sohlh1 and Lhx8 are two germ cell-specific, critical regulators of oogenesis. PMID:16690745

  6. Additional requirements for charitable hospitals; community health needs assessments for charitable hospitals; requirement of a section 4959 excise tax return and time for filing the return. Final regulations and removal of temporary regulations.

    Science.gov (United States)

    2014-12-31

    This document contains final regulations that provide guidance regarding the requirements for charitable hospital organizations added by the Patient Protection and Affordable Care Act of 2010. The regulations will affect charitable hospital organizations

  7. Endocytosis-dependent coordination of multiple actin regulators is required for wound healing.

    Science.gov (United States)

    Matsubayashi, Yutaka; Coulson-Gilmer, Camilla; Millard, Tom H

    2015-08-01

    The ability to heal wounds efficiently is essential for life. After wounding of an epithelium, the cells bordering the wound form dynamic actin protrusions and/or a contractile actomyosin cable, and these actin structures drive wound closure. Despite their importance in wound healing, the molecular mechanisms that regulate the assembly of these actin structures at wound edges are not well understood. In this paper, using Drosophila melanogaster embryos, we demonstrate that Diaphanous, SCAR, and WASp play distinct but overlapping roles in regulating actin assembly during wound healing. Moreover, we show that endocytosis is essential for wound edge actin assembly and wound closure. We identify adherens junctions (AJs) as a key target of endocytosis during wound healing and propose that endocytic remodeling of AJs is required to form "signaling centers" along the wound edge that control actin assembly. We conclude that coordination of actin assembly, AJ remodeling, and membrane traffic is required for the construction of a motile leading edge during wound healing.

  8. RpoS is required for natural transformation of Vibrio cholerae through regulation of chitinases.

    Science.gov (United States)

    Dalia, Ankur B

    2016-11-01

    Vibrio species naturally reside in the aquatic environment and a major metabolite in this habitat is the chitinous exoskeletons of crustacean zooplankton. In addition to serving as a nutrient, chitin-derived oligosaccharides also induce natural genetic competence in many Vibrio spp., a physiological state in which bacteria take up DNA from the extracellular environment and can integrate it into their chromosome by homologous recombination. Another inducing cue required for competence are quorum-sensing autoinducers. The alternative sigma factor RpoS is critical for natural transformation in Vibrio cholerae, and it was previously presumed to exert this effect through regulation of quorum sensing. Here, we show that RpoS does not affect quorum sensing-dependent regulation of competence. Instead, we show that an rpoS mutant has reduced chitinase activity, which is required to liberate the soluble chitin oligosaccharides that serve as an inducing cue for competence. Consistent with this, we demonstrate that RpoS is required for growth of V. cholerae on insoluble chitin. RpoS also regulates the mucosal escape response in pathogenic strains of V. cholerae. Thus, in addition to promoting egress from its human host, RpoS may also prime this pathogen for successful reentry into the aquatic environment. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  9. REQUIREMENTS ECO-DESIGN VACUUMS BY COMMISSION REGULATION (EU NO 666/2013

    Directory of Open Access Journals (Sweden)

    Przemysław Kurczewski

    2014-10-01

    Full Text Available For several years, the executive acts related to the Directive of the European Parliament and of the Council 2009/125/EC of 21 October 2009 establishing a framework for the setting of ecodesign requirements for energy-related products are systematically adopted as a part of European Union legislation. Especially the Regulation 666/2013 concerning the eco-design requirements for vacuum cleaners introduced in July 2013 is strongly discussed. This article presents the background to the Directive 2009/125/EC and explains the concept that underlies the formulation of ecodesign requirements for different powered energy products, with special focus on the role of environmental life cycle assessment (LCA.

  10. Cost-optimal levels of minimum energy performance requirements in the Danish Building Regulations

    Energy Technology Data Exchange (ETDEWEB)

    Aggerholm, S.

    2013-09-15

    The purpose of the report is to analyse the cost optimality of the energy requirements in the Danish Building Regulations 2010, BR10 to new building and to existing buildings undergoing major renovation. The energy requirements in the Danish Building Regulations have by tradition always been based on the cost and benefits related to the private economical or financial perspective. Macro economical calculations have in the past only been made in addition. The cost optimum used in this report is thus based on the financial perspective. Due to the high energy taxes in Denmark there is a significant difference between the consumer price and the macro economical for energy. Energy taxes are also paid by commercial consumers when the energy is used for building operation e.g. heating, lighting, ventilation etc. In relation to the new housing examples the present minimum energy requirements in BR 10 all shows gaps that are negative with a deviation of up till 16 % from the point of cost optimality. With the planned tightening of the requirements to new houses in 2015 and in 2020, the energy requirements can be expected to be tighter than the cost optimal point, if the costs for the needed improvements don't decrease correspondingly. In relation to the new office building there is a gap of 31 % to the point of cost optimality in relation to the 2010 requirement. In relation to the 2015 and 2020 requirements there are negative gaps to the point of cost optimality based on today's prices. If the gaps for all the new buildings are weighted to an average based on mix of building types and heat supply for new buildings in Denmark there is a gap of 3 % in average for the new building. The excessive tightness with today's prices is 34 % in relation to the 2015 requirement and 49 % in relation to the 2020 requirement. The component requirement to elements in the building envelope and to installations in existing buildings adds up to significant energy efficiency

  11. Exploring the structural requirements for jasmonates and related compounds as novel plant growth regulators

    Science.gov (United States)

    Chen, Ke-Xian

    2009-01-01

    Jasmonates and related compounds have been highlighted recently in the field of plant physiology and plant molecular biology due to their significant regulatory roles in the signaling pathway for the diverse aspects of plant development and survival. Though a considerable amount of studies concerning their biological effects in different plants have been widely reported, the molecular details of the signaling mechanism are still poorly understood. This review sheds new light on the structural requirements for the bioactivity/property of jasmonic acid derivatives in current computational perspective, which differs from previous research that mainly focus on their biological evaluation, gene and metabolic regulation and the enzymes in their biosynthesis. The computational results may contribute to further understanding the mechanism of drug-receptor interactions in their signaling pathway and designing novel plant growth regulators as high effective ecological pesticides. PMID:20009552

  12. Interpretation of Section 52.21(i) (3) of the Regulations for the PSD Requirement

    Science.gov (United States)

    This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

  13. Agreement that the PSD Regulations Require a Source to Commence Construction

    Science.gov (United States)

    This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

  14. Iron-regulated biofilm formation in Staphylococcus aureus Newman requires ica and the secreted protein Emp.

    Science.gov (United States)

    Johnson, Miranda; Cockayne, Alan; Morrissey, Julie A

    2008-04-01

    Staphylococcus aureus biofilm formation is induced in iron-restricted growth conditions in vitro. In this study, we showed that Emp and Eap play important roles in low-iron-induced biofilm formation of S. aureus Newman. Eap and Emp are secreted proteins which are non-covalently attached to the S. aureus cell surface and have previously been implicated in a number of aspects of S. aureus pathogenesis. We showed here that the transcription of these important virulence factors is induced by growth in low-iron medium, reflective of the in vivo environment. Our results show that iron regulation of Eap and Emp is Fur independent. However, Fur is required for full induction of eap and emp expression in low-iron conditions. In this study, we demonstrated that in addition to Fur, low-iron-induced biofilm formation requires Sae, Agr, and SarA. In iron-restricted growth conditions, Sae and Agr are essential for Emp and Eap expression and hence for biofilm formation, whereas SarA appears to have a less-significant role. We also showed that expression of the ica operon is required for biofilm formation in iron-restricted growth conditions. We demonstrated that in fact, ica is required for the expression of the important multifunctional virulence determinants eap and emp.

  15. Fis is required for proper regulation of ssaG expression in Salmonella enterica serovar Typhimurium.

    Science.gov (United States)

    Lim, Sangyong; Kim, Byeongkwan; Choi, Hyun-Sook; Lee, Younghoon; Ryu, Sangryeol

    2006-07-01

    Salmonella pathogenicity island 2 (SPI2) encodes a type III secretion system (TTSS) necessary for bacterial survival and replication in intracellular environment of host cells. SPI2 genes are transcribed preferentially after Salmonella enters the host cells. Transcriptional regulation of ssaG encoding the component of SPI2-TTSS apparatus was studied in vivo and in vitro. Fis, one of the major components of bacterial nucleoid, activated the stationary phase-specific expression of ssaG when Salmonella was grown in LB media. Gel-shift and footprinting analysis showed Fis bound to four distinct sites of the ssaG promoter region with different affinities. All four Fis-binding sites were required for timely transcription activation of ssaG after Salmonella entered macrophage cells. Gentamicin protection experiments using bacteria grown to stationary phase prior to infection showed that the ability of the fis mutant strain to replicate within the RAW264.7 macrophage cells was lower than the wild type. These observations confirm that Fis plays an important role in regulations of SPI2 as well as SPI1 for an efficient regulation of the virulence genes.

  16. Medical regulation and health outcomes: the effect of the physician examination requirement.

    Science.gov (United States)

    Cotet, Anca M; Benjamin, Daniel K

    2013-04-01

    This article investigates the effect on health outcomes of the regulation prohibiting physicians from prescribing drugs without a prior physical examination. This requirement could improve health by reducing illegal access to prescription drugs. However, it reduces access to health care by making it more difficult for patients and physicians to use many forms of telemedicine. Thus, this regulation generates a trade-off between access and safety. Using matching techniques, we find that the physician examination requirement leads to an increase of 1% in mortality rates from disease, the equivalent of 8.5 more deaths per 100,000 people, and a decrease of 6.7% in injury mortality, the equivalent of 2.5 deaths per 100,000 people. The magnitude of these effects is larger in rural areas and in areas with low physician density and is accompanied by an 18% increase in the number of days lost each month to illness. Copyright © 2012 John Wiley & Sons, Ltd.

  17. Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

    2006-05-25

    Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

  18. UHRF1 regulation of Dnmt1 is required for pre-gastrula zebrafish development.

    Science.gov (United States)

    Kent, Brandon; Magnani, Elena; Walsh, Martin J; Sadler, Kirsten C

    2016-04-01

    Landmark epigenetic events underlie early embryonic development, yet how epigenetic modifiers are regulated to achieve rapid epigenome re-patterning is not known. Uhrf1 and DNA methyltransferase 1 (Dnmt1) are known to largely mediate maintenance DNA methylation and Uhrf1 is also required for both Dnmt1 localization and stability. Here, we investigate how these two key epigenetic modifiers regulate early zebrafish development and characterize the developmental consequences of disrupting their homeostatic relationship. Unlike Uhrf1 knockdown, which causes developmental arrest and death prior to gastrulation, overexpression of human UHRF1 (WT-UHRF1) caused asymmetric epiboly, inefficient gastrulation and multi-systemic defects. UHRF1 phosphorylation was previously demonstrated as essential for zebrafish embryogenesis, and we found that penetrance of the asymmetric epiboly phenotype was significantly increased in embryos injected with mRNA encoding non-phosphorylatable UHRF1 (UHRF1(S661A)). Surprisingly, both WT-UHRF1 and UHRF1(S661A) overexpression caused DNA hypomethylation. However, since other approaches that caused an equivalent degree of DNA hypomethylation did not cause the asymmetric epiboly phenotype, we conclude that bulk DNA methylation is not the primary mechanism. Instead, UHRF1(S661A) overexpression resulted in accumulation of Dnmt1 protein and the overexpression of both WT and a catalytically inactive Dnmt1 phenocopied the assymetric epiboly phenotype. Dnmt1 knockdown suppressed the phenotype caused by UHRF1(S661A) overexpression, and Uhrf1 knockdown suppressed the effect of Dnmt1 overexpression. Therefore, we conclude that the interaction between these two proteins is the mechanism underlying the gastrulation defects. This indicates that Dnmt1 stability requires UHRF1 phosphorylation and that crosstalk between the proteins is essential for the function of these two important epigenetic regulators during gastrulation.

  19. Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis.

    Science.gov (United States)

    Schweizer, Nina; Ferrás, Cristina; Kern, David M; Logarinho, Elsa; Cheeseman, Iain M; Maiato, Helder

    2013-12-23

    Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. Here, we show that Tpr is required for normal SAC response by stabilizing Mad1 and Mad2 before mitosis. Tpr coimmunoprecipitated with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1–c-Mad2 recruitment to NPCs. Interestingly, Tpr was normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1–c-Mad2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-Mad2 restored normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, we provide evidence that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response.

  20. Meeting the International Health Regulations (2005) surveillance core capacity requirements at the subnational level in Europe

    DEFF Research Database (Denmark)

    Ziemann, Alexandra; Rosenkötter, Nicole; Riesgo, Luis Garcia-Castrillo;

    2015-01-01

    public health emergencies of international concern: (i) can syndromic surveillance support countries, especially the subnational level, to meet the International Health Regulations (2005) core surveillance capacity requirements, (ii) are European syndromic surveillance systems comparable to enable cross......-border surveillance, and (iii) at which administrative level should syndromic surveillance best be applied? DISCUSSION: Despite the ongoing criticism on the usefulness of syndromic surveillance which is related to its clinically nonspecific output, we demonstrate that it was a suitable supplement for timely...... assessment of the impact of three different public health emergencies affecting Europe. Subnational syndromic surveillance analysis in some cases proved to be of advantage for detecting an event earlier compared to national level analysis. However, in many cases, syndromic surveillance did not detect local...

  1. Spindly/CCDC99 is required for efficient chromosome congression and mitotic checkpoint regulation.

    Science.gov (United States)

    Barisic, Marin; Sohm, Bénédicte; Mikolcevic, Petra; Wandke, Cornelia; Rauch, Veronika; Ringer, Thomas; Hess, Michael; Bonn, Günther; Geley, Stephan

    2010-06-15

    Spindly recruits a fraction of cytoplasmic dynein to kinetochores for poleward movement of chromosomes and control of mitotic checkpoint signaling. Here we show that human Spindly is a cell cycle-regulated mitotic phosphoprotein that interacts with the Rod/ZW10/Zwilch (RZZ) complex. The kinetochore levels of Spindly are regulated by microtubule attachment and biorientation induced tension. Deletion mutants lacking the N-terminal half of the protein (NDelta253), or the conserved Spindly box (DeltaSB), strongly localized to kinetochores and failed to respond to attachment or tension. In addition, these mutants prevented the removal of the RZZ complex and that of MAD2 from bioriented chromosomes and caused cells to arrest at metaphase, showing that RZZ-Spindly has to be removed from kinetochores to terminate mitotic checkpoint signaling. Depletion of Spindly by RNAi, however, caused cells to arrest in prometaphase because of a delay in microtubule attachment. Surprisingly, this defect was alleviated by codepletion of ZW10. Thus, Spindly is not only required for kinetochore localization of dynein but is a functional component of a mechanism that couples dynein-dependent poleward movement of chromosomes to their efficient attachment to microtubules.

  2. Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis.

    Science.gov (United States)

    Kachaner, David; Pinson, Xavier; El Kadhi, Khaled Ben; Normandin, Karine; Talje, Lama; Lavoie, Hugo; Lépine, Guillaume; Carréno, Sébastien; Kwok, Benjamin H; Hickson, Gilles R; Archambault, Vincent

    2014-10-27

    Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks.

  3. Learned stressor resistance requires extracellular signal-regulated kinase in the prefrontal cortex

    Directory of Open Access Journals (Sweden)

    John Paul Christianson

    2014-10-01

    Full Text Available Behaviorally controllable stressors confer protection from the neurochemical and behavioral consequences of future uncontrollable stressors, a phenomenon termed behavioral immunization. Recent data implicate neuroplasticity within the ventromedial prefrontal cortex (mPFC as critical to behavioral immunization. Adult, male Sprague-Dawley rats were exposed to a series of controllable tailshocks and one week later to uncontrollable tailshocks, followed 24h later by social exploration and shuttlebox escape tests. To test the involvement of N-methyl-D-aspartate receptors (NMDAR and the extracellular signal-regulated kinase (ERK cascade in behavioral immunization, either D-AP5 or the MEK inhibitor U0126 was injected to the prelimbic (PL or infralimbic (IL mPFC prior to controllable stress exposure. Phosphorylated ERK and P70S6K, regulators of transcription and translation, were quantified by Western blot or immunohistochemistry after controllable or uncontrollable tailshocks. Prior controllable stress prevented the social exploration and shuttlebox performance deficits caused by the later uncontrollable stressor, and this effect was blocked by injections of D-AP5 into mPFC. A significant increase in phosphorylated ERK1 and ERK2, but not P70S6K, occurred within the PL and IL in rats exposed to controllable stress, but not to uncontrollable stress. However, U0126 only prevented behavioral immunization when injected to the PL. We provide evidence that NMDAR and ERK dependent plasticity within the PL region is required for behavioral immunization, a learned form of stressor resistance.

  4. Th9 cell development requires a BATF-regulated transcriptional network.

    Science.gov (United States)

    Jabeen, Rukhsana; Goswami, Ritobrata; Awe, Olufolakemi; Kulkarni, Aishwarya; Nguyen, Evelyn T; Attenasio, Andrea; Walsh, Daniel; Olson, Matthew R; Kim, Myung H; Tepper, Robert S; Sun, Jie; Kim, Chang H; Taparowsky, Elizabeth J; Zhou, Baohua; Kaplan, Mark H

    2013-11-01

    T helper 9 (Th9) cells are specialized for the production of IL-9, promote allergic inflammation in mice, and are associated with allergic disease in humans. It has not been determined whether Th9 cells express a characteristic transcriptional signature. In this study, we performed microarray analysis to identify genes enriched in Th9 cells compared with other Th subsets. This analysis defined a transcriptional regulatory network required for the expression of a subset of Th9-enriched genes. The activator protein 1 (AP1) family transcription factor BATF (B cell, activating transcription factor–like) was among the genes enriched in Th9 cells and was required for the expression of IL-9 and other Th9-associated genes in both human and mouse T cells. The expression of BATF was increased in Th9 cultures derived from atopic infants compared with Th9 cultures from control infants. T cells deficient in BATF expression had a diminished capacity to promote allergic inflammation compared with wild-type controls. Moreover, mouse Th9 cells ectopically expressing BATF were more efficient at promoting allergic inflammation than control transduced cells. These data indicate that BATF is a central regulator of the Th9 phenotype and contributes to the development of allergic inflammation.

  5. Revocation of General Safety Test Regulations That Are Duplicative of Requirements in Biologics License Applications. Final rule.

    Science.gov (United States)

    2015-07-02

    The Food and Drug Administration (FDA) is amending the biologics regulations by removing the general safety test (GST) requirements for biological products. FDA is finalizing this action because the existing codified GST regulations are duplicative of requirements that are also specified in biologics license applications (BLAs), or are no longer necessary or appropriate to help ensure the safety, purity, and potency of licensed biological products. FDA is taking this action as part of its retrospective review of its regulations to promote improvement and innovation, in response to the Executive order.

  6. A monomer is the minimum functional unit required for channel and ATPase activity of the cystic fibrosis transmembrane conductance regulator.

    Science.gov (United States)

    Ramjeesingh, M; Li, C; Kogan, I; Wang, Y; Huan, L J; Bear, C E

    2001-09-04

    The cystic fibrosis transmembrane conductance regulator (CFTR) normally functions as a phosphorylation-regulated chloride channel on the apical surface of epithelial cells, and lack of this function is the primary cause for the fatal disease cystic fibrosis (CF). Previous studies showed that purified, reconstituted CFTR can function as a chloride channel and, further, that its intrinsic ATPase activity is required to regulate opening and closing of the channel gate. However, these previous studies did not identify the quaternary structure required to mediate conduction and catalysis. Our present studies show that CFTR molecules may self-associate in CHO and Sf9 membranes, as complexes close to the predicted size of CFTR dimers can be captured by chemical cross-linking reagents and detected using nondissociative PAGE. However, CFTR function does not require a multimeric complex for function as we determined that purified, reconstituted CFTR monomers are sufficient to mediate regulated chloride conduction and ATPase activity.

  7. Profiles of Motivated Self-Regulation in College Computer Science Courses: Differences in Major versus Required Non-Major Courses

    Science.gov (United States)

    Shell, Duane F.; Soh, Leen-Kiat

    2013-01-01

    The goal of the present study was to utilize a profiling approach to understand differences in motivation and strategic self-regulation among post-secondary STEM students in major versus required non-major computer science courses. Participants were 233 students from required introductory computer science courses (194 men; 35 women; 4 unknown) at…

  8. BDNF activates mTOR to regulate GluR1 expression required for memory formation.

    Directory of Open Access Journals (Sweden)

    Leandro Slipczuk

    Full Text Available BACKGROUND: The mammalian target of Rapamycin (mTOR kinase plays a key role in translational control of a subset of mRNAs through regulation of its initiation step. In neurons, mTOR is present at the synaptic region, where it modulates the activity-dependent expression of locally-translated proteins independently of mRNA synthesis. Indeed, mTOR is necessary for different forms of synaptic plasticity and long-term memory (LTM formation. However, little is known about the time course of mTOR activation and the extracellular signals governing this process or the identity of the proteins whose translation is regulated by this kinase, during mnemonic processing. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that consolidation of inhibitory avoidance (IA LTM entails mTOR activation in the dorsal hippocampus at the moment of and 3 h after training and is associated with a rapid and rapamycin-sensitive increase in AMPA receptor GluR1 subunit expression, which was also blocked by intra-hippocampal delivery of GluR1 antisense oligonucleotides (ASO. In addition, we found that pre- or post-training administration of function-blocking anti-BDNF antibodies into dorsal CA1 hampered IA LTM retention, abolished the learning-induced biphasic activation of mTOR and its readout, p70S6K and blocked GluR1 expression, indicating that BDNF is an upstream factor controlling mTOR signaling during fear-memory consolidation. Interestingly, BDNF ASO hindered LTM retention only when given into dorsal CA1 1 h after but not 2 h before training, suggesting that BDNF controls the biphasic requirement of mTOR during LTM consolidation through different mechanisms: an early one involving BDNF already available at the moment of training, and a late one, happening around 3 h post-training that needs de novo synthesis of this neurotrophin. CONCLUSIONS/SIGNIFICANCE: IN CONCLUSION, OUR FINDINGS DEMONSTRATE THAT: 1 mTOR-mediated mRNA translation is required for memory consolidation during

  9. Experiences in certification of packages for transportation of fresh nuclear fuel in the context of new safety requirements established by IAEA regulations (IAEA-96 regulations, ST-1) for air transportation of nuclear materials (requirements to C-type packages)

    Energy Technology Data Exchange (ETDEWEB)

    Dudai, V.I.; Kovtun, A.D.; Matveev, V.Z.; Morenko, A.I.; Nilulin, V.M.; Shapovalov, V.I.; Yakushev, V.A.; Bobrovsky, V.S.; Rozhkov, V.V.; Agapov, A.M.; Kolesnikov, A.S. [Russian Federal Nuclear Centre - All-Russian Research Inst. of Experimental Physics, Sarov (Russian Federation)]|[JSC ' ' MSZ' ' , Electrostal (Russian Federation)]|[JSC ' ' NPCC' ' , Novosibirsk (Russian Federation)]|[Minatom of Russia, Moscow (Russian Federation)]|[Gosatomnadzor of Russia, Moscow (Russian Federation)

    2004-07-01

    Every year in Russia, a large amount of domestic and international transportation of fresh nuclear fuel (FNF) used in Russian and foreign energy and research atomic reactors and referred to fissile materials based on IAEA Regulations is performed. Here, bulk transportation is performed by air, and it concerns international transportation in particular. According to national ''Main Regulations for Safe Transport and physical Protection of Nuclear Materials (OPBZ- 83)'' and ''Regulations for the Safe Transport of Radioactive Materials'' of the International Atomic Energy Agency (IAEA Regulations), nuclear and radiation security under normal (accident free) and accident conditions of transport must be completely provided by the package design. In this context, high requirements to fissile packages exposed to heat and mechanical loads in transport accidents are imposed. A long-standing experience in accident free transportation of FM has shown that such approach to provide nuclear and radiation security pays for itself completely. Nevertheless, once in 10 years the International Atomic Energy Agency on every revision of the ''Regulations for the Safe Transport of Radioactive Materials'' places more stringent requirements upon the FM and transportation thereof, resulting from the objectively increasing risk associated with constant rise in volume and density of transportation, and also strained social and economical situation in a number of regions in the world. In the new edition of the IAEA Regulations (ST-1), published in 1996 and brought into force in 2001 (IAEA-96 Regulations), the requirements to FM packages conveyed by aircraft were radically changed. These requirements are completely presented in new Russian ''Regulations for the Safe Transport of Radioactive Materials'' (PBTRM- 2004) which will be brought into force in the time ahead.

  10. Dentate Gyrus Development Requires ERK Activity to Maintain Progenitor Population and MAPK Pathway Feedback Regulation.

    Science.gov (United States)

    Vithayathil, Joseph; Pucilowska, Joanna; Goodnough, L Henry; Atit, Radhika P; Landreth, Gary E

    2015-04-29

    The ERK/MAPK pathway is an important developmental signaling pathway. Mutations in upstream elements of this pathway result in neuro-cardio-facial cutaneous (NCFC) syndromes, which are typified by impaired neurocognitive abilities that are reliant upon hippocampal function. The role of ERK signaling during hippocampal development has not been examined and may provide critical insight into the cause of hippocampal dysfunction in NCFC syndromes. In this study, we have generated ERK1 and conditional ERK2 compound knock-out mice to determine the role of ERK signaling during development of the hippocampal dentate gyrus. We found that loss of both ERK1 and ERK2 resulted in 60% fewer granule cells and near complete absence of neural progenitor pools in the postnatal dentate gyrus. Loss of ERK1/2 impaired maintenance of neural progenitors as they migrate from the dentate ventricular zone to the dentate gyrus proper, resulting in premature depletion of neural progenitor cells beginning at E16.5, which prevented generation of granule cells later in development. Finally, loss of ERK2 alone does not impair development of the dentate gyrus as animals expressing only ERK1 developed a normal hippocampus. These findings establish that ERK signaling regulates maintenance of progenitor cells required for development of the dentate gyrus. Copyright © 2015 the authors 0270-6474/15/356836-13$15.00/0.

  11. CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

    Science.gov (United States)

    Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

    2016-11-16

    For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

  12. Reciprocal regulation of Wnt and Gpr177/mouse Wntless is required for embryonic axis formation.

    Science.gov (United States)

    Fu, Jiang; Jiang, Ming; Mirando, Anthony J; Yu, Hsiao-Man Ivy; Hsu, Wei

    2009-11-03

    Members of the Wnt family are secreted glycoproteins that trigger cellular signals essential for proper development of organisms. Cellular signaling induced by Wnt proteins is involved in diverse developmental processes and human diseases. Previous studies have generated an enormous wealth of knowledge on the events in signal-receiving cells. However, relatively little is known about the making of Wnt in signal-producing cells. Here, we describe that Gpr177, the mouse orthologue of Drosophila Wls, is expressed during formation of embryonic axes. Embryos with deficient Gpr177 exhibit defects in establishment of the body axis, a phenotype highly reminiscent to the loss of Wnt3. Although many different mammalian Wnt proteins are required for a wide range of developmental processes, the Wnt3 ablation exhibits the earliest developmental abnormality. This suggests that the Gpr177-mediated Wnt production cannot be substituted. As a direct target of Wnt, Gpr177 is activated by beta-catenin and LEF/TCF-dependent transcription. This activation alters the cellular distributions of Gpr177 which binds to Wnt proteins and assists their sorting and secretion in a feedback regulatory mechanism. Our findings demonstrate that the loss of Gpr177 affects Wnt production in the signal-producing cells, leading to alterations of Wnt signaling in the signal-receiving cells. A reciprocal regulation of Wnt and Gpr177 is essential for the patterning of the anterior-posterior axis during mammalian development.

  13. Fgf19 regulated by Hh signaling is required for zebrafish forebrain development.

    Science.gov (United States)

    Miyake, Ayumi; Nakayama, Yoshiaki; Konishi, Morichika; Itoh, Nobuyuki

    2005-12-01

    Fibroblast growth factor (Fgf) signaling plays important roles in brain development. Fgf3 and Fgf8 are crucial for the formation of the forebrain and hindbrain. Fgf8 is also required for the midbrain to form. Here, we identified zebrafish Fgf19 and examined its roles in brain development by knocking down Fgf19 function. We found that Fgf19 expressed in the forebrain, midbrain and hindbrain was involved in cell proliferation and cell survival during embryonic brain development. Fgf19 was also essential for development of the ventral telencephalon and diencephalon. Regional specification is linked to cell type specification. Fgf19 was also essential for the specification of gamma-aminobutyric acid (GABA)ergic interneurons and oligodendrocytes generated in the ventral telencephalon and diencephalon. The cross talk between Fgf and Hh signaling is critical for brain development. In the forebrain, Fgf19 expression was down-regulated on inhibition of Hh but not of Fgf3/Fgf8, and overexpression of Fgf19 rescued partially the phenotype on inhibition of Hh. The present findings indicate that Fgf19 signaling is crucial for forebrain development by interacting with Hh and provide new insights into the roles of Fgf signaling in brain development.

  14. ADAM10 regulates transcription factor expression required for plasma cell function.

    Directory of Open Access Journals (Sweden)

    Natalia S Chaimowitz

    Full Text Available A disintegrin and metalloprotease 10 (ADAM10 is a key regulator of cellular processes by shedding extracellular domains of transmembrane proteins. We have previously demonstrated that deletion of B cell expressed ADAM10 results in changes in lymphoid tissue architecture and impaired germinal center (GC formation. In this study, mice were generated in which ADAM10 is deleted in B cells following class switch recombination (ADAM10(Δ/ΔIgG1-cre(+/- mice. Despite normal GC formation, antibody responses were impaired in ADAM10(Δ/ΔIgG1-cre(+/- mice, implicating ADAM10 in post-GC and extrafollicular B cell terminal differentiation. Surprisingly, plasma cell (PC numbers were normal in ADAM10(Δ/ΔIgG1-cre(+/- mice when compared to controls. However, PCs isolated from ADAM10(Δ/ΔIgG1-cre(+/- mice exhibited decreased expression of transcription factors important for PC function: Prdm1, Xbp1 and Irf4. Bcl6 is a GC transcriptional repressor that inhibits the PC transcriptional program and thus must be downregulated for PC differentiation to occur. Bcl6 expression was increased in PCs isolated from ADAM10(Δ/ΔIgG1-cre(+/- mice at both the mRNA and protein level. These results demonstrate that ADAM10 is required for proper transcription factor expression in PCs and thus, for normal PC function.

  15. 75 FR 20825 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DoD Pilot...

    Science.gov (United States)

    2010-04-21

    ... Regulation Supplement; DoD Pilot Mentor-Protege Program AGENCY: Defense Acquisition Regulations System... Acquisition Regulation Supplement (DFARS) Appendix I, DoD Pilot Mentor-Protege Program; OMB Control Number... Mentor-Protege Program have been met. The purposes of the Program are to (1) provide incentives to...

  16. The European Union's REACH regulation: a review of its history and requirements.

    Science.gov (United States)

    Williams, E Spencer; Panko, Julie; Paustenbach, Dennis J

    2009-01-01

    In 2006, the European Union (EU) promulgated a monumental regulatory initiative for the Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH). To date, several thousand pages of text have been needed to describe the expectations of this regulation. There were numerous reasons for the promulgation of REACH, but, by and large, it is an extension of the global desire to produce fewer industrial chemicals, to understand the possible human and ecological hazards of those that are produced, and to insure that any major threat is anticipated, as well as prevented. Most industry-related groups consider it the most wide-ranging and costly regulatory initiatives related to health risk assessment ever to be promulgated. This review presents a description of REACH that should inform scientists, managers, and others about its objectives and the means to satisfy them. Registration is required for all chemicals manufactured or imported into the EU, unless specifically exempted. Registration is expected to be a collaborative process among companies, which will generate a dossier containing data on physicochemical characteristics, as well as toxicological and ecotoxicological properties. Though the magnitude of the gaps in the data required for registration is uncertain at this point, it is clear that basic toxicology testing will have to be conducted for many chemical substances that have not undergone formal review up to this point. For many chemicals, an examination of hazards and risks arising from the use of these substances will also be required in the form of a chemical safety report (CSR). Beginning with the dual processes of dossier and substance evaluation, the European Chemicals Agency (ECHA), the Member States of the EU, and the European Commission will identify chemicals that may pose unacceptable hazards to human health and/or the environment, and will curtail or restrict their usage. The implementation of REACH will expand and deepen the

  17. Novel insights into iron regulation and requirement in marine medaka Oryzias melastigma

    Science.gov (United States)

    Wang, Jian; Wang, Wen-Xiong

    2016-05-01

    Iron (Fe) is an essential trace element for marine fish. However, our knowledge of Fe requirements at different development stages of marine fish is still limited. Here, we reported the efficient Fe absorption strategies adopted by larval fish under different dietary Fe supplementary levels (i.e., 0-640 mg/kg). Biokinetically, the larval fish controlled their dietary Fe assimilation efficiency (AE, 1.6-18.5%), and enhanced their waterborne Fe uptake (ca. 2.5 fold change of uptake rate constant) once the dietary Fe was deficient (i.e., 27.4 mg Fe/kg feed). Transcriptionally, the expression of hepcidin1 (hep1; Fe regulator; i.e., 2.3-15.7 fold change) in larval fish was positively correlated with the Fe supplementary levels. Comparatively, the female adult fish were poor in assimilating the added Fe source (i.e., ferric form) with similar life-sustainable levels of Fe (i.e., 0.046-0.12 μg/g/d assimilated for Fe supplementary levels of 27.4, 162 and 657 mg Fe/kg feed). The overall feeding experiments suggested that dietary net Fe flux sufficient for the normal growth of larval medaka was 0.71-1.75 μg/g/d (i.e., 83.9 mg Fe/kg feed), consistent with the modeled value (i.e., 1.09-2.16 μg/g/d). In female adults, the estimated essential net Fe flux was 0.88-0.90 μg/g/d.

  18. 25 CFR 518.7 - If a tribe holds a certificate of self-regulation, is it required to report information to the...

    Science.gov (United States)

    2010-04-01

    ... 25 Indians 2 2010-04-01 2010-04-01 false If a tribe holds a certificate of self-regulation, is it... REGULATION OF CLASS II GAMING § 518.7 If a tribe holds a certificate of self-regulation, is it required to... certificate of self-regulation shall be required to submit a self-regulation report annually to the Commission...

  19. 78 FR 30898 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Rights in...

    Science.gov (United States)

    2013-05-23

    ... Technical Data and Computer Software--Small Business Innovation Research (SBIR) Program. In accordance with... Regulation Supplement; Rights in Technical Data and Computer Software AGENCY: Defense Acquisition Regulations... in Technical Data, and Subpart 227.72, Rights in Computer Software and Computer Software...

  20. Melanocortin 4 receptor is not required for estrogenic regulations on energy homeostasis and reproduction

    Science.gov (United States)

    Brain estrogen receptor-a (ERa) is essential for estrogenic regulation of energy homeostasis and reproduction. We previously showed that ERa expressed by pro-opiomelanocortin (POMC) neurons mediates estrogen's effects on food intake, body weight, negative regulation of hypothalamic–pituitary–gonadal...

  1. 77 FR 53870 - Availability of a Legal Entity Identifier Meeting the Requirements of the Regulations of the...

    Science.gov (United States)

    2012-09-04

    ... in recordkeeping and swap data reporting. Section 45.6(f)(1) provides that: When a legal entity... COMMISSION Availability of a Legal Entity Identifier Meeting the Requirements of the Regulations of the Commodity Futures Trading Commission and Designation of Provider of Legal Entity Identifiers To Be Used...

  2. 76 FR 4555 - Authority To Require Supervision and Regulation of Certain Nonbank Financial Companies

    Science.gov (United States)

    2011-01-26

    ... those activities most prone to systemic risk through contagion. To measure interconnectedness, commenters suggested evaluating, among other things, liquidity profile, contagion risk, counterparty credit... prudential regulators or the Office of Financial Research to make its determinations. 11. Should the degree...

  3. 76 FR 78126 - Disclosure Requirements for Depository Institutions Lacking Federal Deposit Insurance (Regulation I)

    Science.gov (United States)

    2011-12-16

    ... INFORMATION CONTACT: Krista Ayoub or Jane Gao, Office of Regulations, at (202) 435-7700. SUPPLEMENTARY... accepts deposits or opens accounts (excluding automated teller machines or point of sale terminals), and...

  4. 78 FR 4788 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Science.gov (United States)

    2013-01-23

    ... under section 4(a) of the Administrative Procedure Act (APA) (5 U.S.C. 553(b)). This provision... follows: PART 165--REGULATED NAVIGATION AREAS AND LIMITED ACCESS AREAS 0 1. The authority citation...

  5. 76 FR 51040 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Science.gov (United States)

    2011-08-17

    ..., in co-sponsorship with the Society of Clinical Research Associates (SoCRA) is announcing a public... Clinical Trial; (2) Adverse Event Reporting--Science, Regulation, Error, and Safety; (3) Part 11 Compliance...

  6. 77 FR 8886 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Science.gov (United States)

    2012-02-15

    ...-sponsorship with the Society of Clinical Research Associates (SoCRA) is announcing a public workshop. The... Pharmaceutical Clinical Trial; (2) Adverse Event Reporting--Science, Regulation, Error, and Safety; (3) Part 11...

  7. Histone Methylation Regulator PTIP Is Required for PPARγ and C/EBPα Expression and Adipogenesis

    OpenAIRE

    Cho, Young-Wook; Hong, SunHwa; Jin, Qihuang; Wang, Lifeng; Lee, Ji-Eun; Gavrilova, Oksana; Ge, Kai

    2009-01-01

    PPARγ and C/EBPα cooperate to control preadipocyte differentiation (adipogenesis). However, the factors that regulate PPARγ and C/EBPα expression during adipogenesis remain largely unclear. Here we show PTIP, a protein that associates with histone H3K4 methyltransferases, regulates PPARγ and C/EBPα expression in mouse embryonic fibroblasts (MEFs) and during preadipocyte differentiation. PTIP deletion in MEFs leads to marked decreases of PPARγ expression and PPARγ-stimulated C/EBPα expression....

  8. A mechanism regulating G protein-coupled receptor signaling that requires cycles of protein palmitoylation and depalmitoylation.

    Science.gov (United States)

    Jia, Lixia; Chisari, Mariangela; Maktabi, Mohammad H; Sobieski, Courtney; Zhou, Hao; Konopko, Aaron M; Martin, Brent R; Mennerick, Steven J; Blumer, Kendall J

    2014-02-28

    Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K(+) (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.

  9. Down-regulation of stathmin expression is required for megakaryocyte maturation and platelet production.

    Science.gov (United States)

    Iancu-Rubin, Camelia; Gajzer, David; Tripodi, Joseph; Najfeld, Vesna; Gordon, Ronald E; Hoffman, Ronald; Atweh, George F

    2011-04-28

    The final stages of of megakaryocyte (MK) maturation involve a series of steps, including polyploidization and proplatelet formation. Although these processes are highly dependent on dynamic changes in the microtubule (MT) cytoskeleton, the mechanisms responsible for regulation of MTs in MKs remain poorly defined. Stathmin is a highly conserved MT-regulatory protein that has been suggested to play a role in MK differentiation of human leukemic cell lines. However, previous studies defining this relationship have reached contradictory conclusions. In this study, we addressed this controversy and investigated the role of stathmin in primary human MKs. To explore the importance of stathmin down-regulation during megakaryocytopoiesis, we used a lentiviral-mediated gene delivery system to prevent physiologic down-regulation of stathmin in primary MKs. We demonstrated that sustained expression of constitutively active stathmin delayed cytoplasmic maturation (ie, glycoprotein GPIb and platelet factor 4 expression) and reduced the ability of MKs to achieve high levels of ploidy. Moreover, platelet production was impaired in MKs in which down-regulation of stathmin expression was prevented. These studies indicate that suppression of stathmin is biologically important for MK maturation and platelet production and support the importance of MT regulation during the final stages of thrombopoiesis.

  10. Profiles of Motivated Self-Regulation in College Computer Science Courses: Differences in Major versus Required Non-Major Courses

    Science.gov (United States)

    Shell, Duane F.; Soh, Leen-Kiat

    2013-12-01

    The goal of the present study was to utilize a profiling approach to understand differences in motivation and strategic self-regulation among post-secondary STEM students in major versus required non-major computer science courses. Participants were 233 students from required introductory computer science courses (194 men; 35 women; 4 unknown) at a large Midwestern state university. Cluster analysis identified five profiles: (1) a strategic profile of a highly motivated by-any-means good strategy user; (2) a knowledge-building profile of an intrinsically motivated autonomous, mastery-oriented student; (3) a surface learning profile of a utility motivated minimally engaged student; (4) an apathetic profile of an amotivational disengaged student; and (5) a learned helpless profile of a motivated but unable to effectively self-regulate student. Among CS majors and students in courses in their major field, the strategic and knowledge-building profiles were the most prevalent. Among non-CS majors and students in required non-major courses, the learned helpless, surface learning, and apathetic profiles were the most prevalent. Students in the strategic and knowledge-building profiles had significantly higher retention of computational thinking knowledge than students in other profiles. Students in the apathetic and surface learning profiles saw little instrumentality of the course for their future academic and career objectives. Findings show that students in STEM fields taking required computer science courses exhibit the same constellation of motivated strategic self-regulation profiles found in other post-secondary and K-12 settings.

  11. 76 FR 71707 - Revising Underground Storage Tank Regulations-Revisions to Existing Requirements and New...

    Science.gov (United States)

    2011-11-18

    ..., classroom training must be signed by the trainer; computer based training does not require a signature but... Containment and Operator Training; Proposed Rule #0;#0;Federal Register / Vol. 76 , No. 223 / Friday, November... Requirements for Secondary Containment and Operator Training AGENCY: Environmental Protection Agency...

  12. 75 FR 34277 - Federal Acquisition Regulation; FAR Case 2008-007, Additional Requirements for Market Research

    Science.gov (United States)

    2010-06-16

    ... executive agencies. Specifically, the head of the agency must conduct market research before issuing an... million for the procurement of items other than commercial items is required to conduct market research... 10 U.S.C. 2377(c), ``Preliminary Market Research'', to require the head of an agency to...

  13. 76 FR 14548 - Federal Acquisition Regulation; Requirements for Acquisitions Pursuant to Multiple-Award Contracts

    Science.gov (United States)

    2011-03-16

    ...- going competition, when deciding how many BPAs are appropriate and document the decision in the... GSA Advantage! on-line shopping service, by reviewing the catalogs or pricelists of at least three... requirement(s); (B) The benefits of on-going competition and the need to periodically compare multiple...

  14. 76 FR 68027 - Federal Acquisition Regulation; Certification Requirement and Procurement Prohibition Relating to...

    Science.gov (United States)

    2011-11-02

    .... Applicability to Construction Comment: One respondent was concerned that the prescription at FAR 25.1103, which... be interpreted to exclude construction. The respondent suggested changing the prescription to require... require use of the provision in ``all solicitations.'' B. Commercial Database of Persons Doing...

  15. 78 FR 72620 - Federal Acquisition Regulation; Higher-Level Contract Quality Requirements

    Science.gov (United States)

    2013-12-03

    ... posted without change to http://www.regulations.gov , including any personal and/or business confidential... counterfeit electronic parts. However, because of the globalization of the marketplace, the problem of... and industry. Globalization in the marketplace increased the risk of counterfeit items in...

  16. Regulation of Hepatic Triacylglycerol Metabolism by CGI-58 Does Not Require ATGL Co-activation

    Directory of Open Access Journals (Sweden)

    Caleb C. Lord

    2016-07-01

    Full Text Available Adipose triglyceride lipase (ATGL and comparative gene identification 58 (CGI-58 are critical regulators of triacylglycerol (TAG turnover. CGI-58 is thought to regulate TAG mobilization by stimulating the enzymatic activity of ATGL. However, it is not known whether this coactivation function of CGI-58 occurs in vivo. Moreover, the phenotype of human CGI-58 mutations suggests ATGL-independent functions. Through direct comparison of mice with single or double deficiency of CGI-58 and ATGL, we show here that CGI-58 knockdown causes hepatic steatosis in both the presence and absence of ATGL. CGI-58 also regulates hepatic diacylglycerol (DAG and inflammation in an ATGL-independent manner. Interestingly, ATGL deficiency, but not CGI-58 deficiency, results in suppression of the hepatic and adipose de novo lipogenic program. Collectively, these findings show that CGI-58 regulates hepatic neutral lipid storage and inflammation in the genetic absence of ATGL, demonstrating that mechanisms driving TAG lipolysis in hepatocytes differ significantly from those in adipocytes.

  17. 78 FR 19467 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Science.gov (United States)

    2013-04-01

    ... Number: Defense Federal Acquisition Regulation Supplement (DFARS), Appendix I, DoD Pilot Mentor-Protege... purposes of the DoD Pilot Mentor-Protege Program have been met. The purposes of the Program are to (1) provide incentives to major DoD contractors to assist protege firms in enhancing their capabilities...

  18. RovA, a global regulator of Yersinia pestis, specifically required for bubonic plague.

    Science.gov (United States)

    Cathelyn, Jason S; Crosby, Seth D; Lathem, Wyndham W; Goldman, William E; Miller, Virginia L

    2006-09-05

    The pathogenic species of Yersinia contain the transcriptional regulator RovA. In Yersinia pseudotuberculosis and Yersinia enterocolitica, RovA regulates expression of the invasion factor invasin (inv), which mediates translocation across the intestinal epithelium. A Y. enterocolitica rovA mutant has a significant decrease in virulence by LD(50) analysis and an altered rate of dissemination compared with either wild type or an inv mutant, suggesting that RovA regulates multiple virulence factors. Here, we show the involvement of RovA in the virulence of Yersinia pestis, which naturally lacks a functional inv gene. A Y. pestis DeltarovA mutant is attenuated approximately 80-fold by LD(50) and is defective in dissemination/colonization of spleens and lungs after s.c. inoculation. However, the DeltarovA mutant is only slightly attenuated when given via an intranasal or i.p. route, indicating a more important role for RovA in bubonic plague than pneumonic plague or systemic infection. Microarray analysis was used to define the RovA regulon. The psa locus was among the most highly down-regulated loci in the DeltarovA mutant. A DeltapsaA mutant had a significant dissemination defect after s.c. infection but only slight attenuation by the pneumonic-disease model, closely mimicking the virulence defect seen with the DeltarovA mutant. DNA-binding studies revealed that RovA specifically interacts with the psaE and psaA promoter regions, indicating a direct role for RovA in regulating this locus. Thus, RovA appears to be a global transcription factor in Y. pestis and, through its regulatory influence on genes such as psaEFABC, contributes to the virulence of Y. pestis.

  19. Good for your health? An analysis of the requirements for scientific substantiation in European health claims regulation

    Directory of Open Access Journals (Sweden)

    Oliver Todt

    2016-05-01

    Full Text Available Objective. To identify the various types of evidence, as well as their relative importance in European health claims regulation, in order to analyze the consequences for consumer protection of the requirements for scientific substantiation in this regulation. Materials and methods. Qualitative analysis of various documents relevant to the regulatory process, particularly as to the implications of the standards of proof for the functional food market, as well as consumer behavior. Results. European regulation defines a hierarchy of evidence that turns randomized controlled trials into a necessary and sufficient condition for health claim autho- rizations. Conclusions. Consumer protection can be interpreted in different manners. High standards of proof protect consumers from false information about the health outcomes of functional foods, while lower standards lead to more, albeit less accurate information about such outcomes being available to consumers.

  20. glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans.

    Science.gov (United States)

    Austin, J; Kimble, J

    1987-11-20

    In the wild-type C. elegans germ line there are both mitotic and meiotic germ cells. Mutations in glp-1 cause germ cells that would normally divide mitotically to enter meiosis. This mutant phenotype mimics the effect of killing the distal tip cell, a somatic cell that interacts with the germ line to regulate the mitotic/meiotic decision. In addition, wild-type glp-1 product is required maternally for embryogenesis. Temperature-shift experiments indicate that the temporal requirement for glp-1 activity in the germ line is the same as that for distal tip cell regulation. Mosaic analyses suggest that glp-1 is produced in the germ line. We propose that glp-1 acts as part of the receiving mechanism in the interaction between the distal tip cell and germ line.

  1. Development of technology-neutral safety requirements for the regulation of future nuclear power reactors: Back to basics

    Energy Technology Data Exchange (ETDEWEB)

    Tronea, Madalina, E-mail: madalina.tronea@gmail.co [Faculty of Physics, University of Bucharest (Romania)

    2011-03-15

    This paper explores the current trends as regards the development of technology-neutral safety requirements to be used in the regulation of future nuclear power reactors and the role of the quantitative safety goals in the design of reactor safety systems. The use of the recommendations of the International Commission on Radiological Protection (ICRP) on protection against potential exposure could form the basis of a technology-neutral framework for safety requirements on new reactor designs and could contribute to international harmonisation of nuclear safety assessment practices as part of the licensing processes for future nuclear power plants.

  2. Feedback regulation of NEUROG2 activity by MTGR1 is required for progression of neurogenesis.

    Science.gov (United States)

    Aaker, Joshua D; Patineau, Andrea L; Yang, Hyun-Jin; Ewart, David T; Gong, Wuming; Li, Tongbin; Nakagawa, Yasushi; McLoon, Steven C; Koyano-Nakagawa, Naoko

    2009-12-01

    The sequential steps of neurogenesis are characterized by highly choreographed changes in transcription factor activity. In contrast to the well-studied mechanisms of transcription factor activation during neurogenesis, much less is understood regarding how such activity is terminated. We previously showed that MTGR1, a member of the MTG family of transcriptional repressors, is strongly induced by a proneural basic helix-loop-helix transcription factor, NEUROG2 in developing nervous system. In this study, we describe a novel feedback regulation of NEUROG2 activity by MTGR1. We show that MTGR1 physically interacts with NEUROG2 and represses transcriptional activity of NEUROG2. MTGR1 also prevents DNA binding of the NEUROG2/E47 complex. In addition, we provide evidence that proper termination of NEUROG2 activity by MTGR1 is necessary for normal progression of neurogenesis in the developing spinal cord. These results highlight the importance of feedback regulation of proneural gene activity in neurodevelopment.

  3. Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis

    DEFF Research Database (Denmark)

    Confalonieri, S; Salcini, A E; Puri, C;

    2000-01-01

    for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function...... of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular...... determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis....

  4. Netrin-1 up-regulation in inflammatory bowel diseases is required for colorectal cancer progression.

    Science.gov (United States)

    Paradisi, Andrea; Maisse, Carine; Coissieux, Marie-May; Gadot, Nicolas; Lépinasse, Florian; Delloye-Bourgeois, Céline; Delcros, Jean-Guy; Svrcek, Magali; Neufert, Clemens; Fléjou, Jean-François; Scoazec, Jean-Yves; Mehlen, Patrick

    2009-10-06

    Chronic inflammation and cancer are intimately associated. This is particularly true for inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease, which show a major increased risk for colorectal cancer. While the understanding of the molecular pathogenesis of IBD has recently improved, the mechanisms that link these chronic inflammatory states to colorectal cancer development are in large part unknown. One of these mechanisms is NF-kappaB pathway activation which in turn may contribute to tumor formation by providing anti-apoptotic survival signals to the epithelial cells. Based on the observation that netrin-1, the anti-apoptotic ligand for the dependence receptors DCC and UNC5H is up-regulated in colonic crypts in response to NF-kappaB, we show here that colorectal cancers from inflammatory bowel diseases patients have selected up-regulation of netrin-1. Moreover, we demonstrate that this inflammation-driven netrin-1 up-regulation is causal for colorectal cancer development as interference with netrin-1 autocrine loop in a mouse model for ulcerative colitis-associated colorectal cancer, while showing no effect on inflammation, inhibits colorectal cancer progression.

  5. Multinational Firm’s Production Decisions under Overlapping Free Trade Agreements: Rule of Origin Requirements and Environmental Regulation

    Directory of Open Access Journals (Sweden)

    Sung Hee Lee

    2016-12-01

    Full Text Available In this paper, we study the impact of the rule of origin (ROO requirements accompanied by free trade agreements (FTAs, which specify the minimum portion of supplies that should satisfy the origin requirement, on a multinational firm’s production decisions. We consider a multinational firm who exports its product to multiple countries and analyze its production decision in the presence of multiple free trading agreements (FTAs. The ROO requirements in FTAs not only refer to the origin of supplies but are also involved with an environmental regulation of a country of the supplies. As such, meeting multiple ROO requirements can be costly since it may be involved with an adjustment of production facility and suppliers according to different environmental standards. We investigate a multinational firm’s choice of the ROO level in its production decision under multiple FTAs. It is well known that, in the presence of overlapping FTAs, the firm may strategically choose not to comply with the minimum ROO requirements in the FTAs. Instead, the firm may choose to comply with an ROO level that is higher than required, or pay the tariff instead without enjoying tariff exemption by the FTA in the new country. Such unintended outcomes in the FTAs are called the Spaghetti Bowl Effect. We characterize and quantify two types of such Spaghetti Bowl Effects with the optimal production decisions of a multinational firm under multiple ROO requirements and derive policy implications.

  6. Leptin is required for hypothalamic regulation of miRNAs targeting POMC 3’UTR

    Directory of Open Access Journals (Sweden)

    Adel eDerghal

    2015-05-01

    Full Text Available The central nervous system monitors modifications in metabolic parameters or hormone levels and elicits adaptive responses such as food intake regulation. Particularly, within the hypothalamus, leptin modulates the activity of pro-opiomelanocortin (POMC neurons which are critical regulators of energy balance. Consistent with a pivotal role of the melanocortin system in the control of energy homeostasis, disruption of the POMC gene causes hyperphagia and obesity. MicroRNAs (miRNAs are short noncoding RNA molecules that post-transcriptionally repress the expression of genes by binding to 3'-untranslated regions (3'UTR of the target mRNAs. However, little is known regarding the role of miRNAs that target POMC 3’UTR in the central control energy homeostasis. Particularly, their interaction with the leptin signaling pathway remain unclear. First, we used common prediction programs to search for potential miRNAs target sites on 3'UTR of POMC mRNA. This screening identified a set of conserved miRNAs seed sequences for mir-383, mir-384-3p and mir-488. We observed that mir-383, mir-384-3p and mir-488 are up-regulated in the hypothalamus of leptin deficient ob/ob mice. In accordance with these observations, we also showed that mir-383, mir-384-3p and mir-488 were increased in db/db mice that exhibit a non-functional leptin receptor. The intraperitoneal injection of leptin down-regulated the expression of these miRNAs of interest in the hypothalamus of ob/ob mice showing the involvement of leptin in the expression of mir-383, mir-384-3p and mir-488. Finally, the evaluation of responsivity to intracerebroventricular administration of leptin exhibited that a chronic treatment with leptin decreased mir-488 expression in hypothalamus of C57BL/6 mice. In summary, these results suggest that leptin modulates the expression of miRNAs that target POMC mRNA in hypothalamus.

  7. Leptin is required for hypothalamic regulation of miRNAs targeting POMC 3′UTR

    Science.gov (United States)

    Derghal, Adel; Djelloul, Mehdi; Airault, Coraline; Pierre, Clément; Dallaporta, Michel; Troadec, Jean-Denis; Tillement, Vanessa; Tardivel, Catherine; Bariohay, Bruno; Trouslard, Jérôme; Mounien, Lourdes

    2015-01-01

    The central nervous system (CNS) monitors modifications in metabolic parameters or hormone levels and elicits adaptive responses such as food intake regulation. Particularly, within the hypothalamus, leptin modulates the activity of pro-opiomelanocortin (POMC) neurons which are critical regulators of energy balance. Consistent with a pivotal role of the melanocortin system in the control of energy homeostasis, disruption of the POMC gene causes hyperphagia and obesity. MicroRNAs (miRNAs) are short noncoding RNA molecules that post-transcriptionally repress the expression of genes by binding to 3′-untranslated regions (3′UTR) of the target mRNAs. However, little is known regarding the role of miRNAs that target POMC 3′UTR in the central control energy homeostasis. Particularly, their interaction with the leptin signaling pathway remain unclear. First, we used common prediction programs to search for potential miRNAs target sites on 3′UTR of POMC mRNA. This screening identified a set of conserved miRNAs seed sequences for mir-383, mir-384-3p, and mir-488. We observed that mir-383, mir-384-3p, and mir-488 are up-regulated in the hypothalamus of leptin deficient ob/ob mice. In accordance with these observations, we also showed that mir-383, mir-384-3p, and mir-488 were increased in db/db mice that exhibit a non-functional leptin receptor. The intraperitoneal injection of leptin down-regulated the expression of these miRNAs of interest in the hypothalamus of ob/ob mice showing the involvement of leptin in the expression of mir-383, mir-384-3p, and mir-488. Finally, the evaluation of responsivity to intracerebroventricular administration of leptin exhibited that a chronic treatment with leptin decreased mir-488 expression in hypothalamus of C57BL/6 mice. In summary, these results suggest that leptin modulates the expression of miRNAs that target POMC mRNA in hypothalamus. PMID:25999818

  8. Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling.

    OpenAIRE

    Stähli, Alexandra Beatrice; Bosshardt, Dieter; Sculean, Anton; Gruber, Reinhard

    2014-01-01

    Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB43...

  9. Emdogain-Regulated Gene Expression in Palatal Fibroblasts Requires TGF-βRI Kinase Signaling

    OpenAIRE

    Alexandra Stähli; Dieter Bosshardt; Anton Sculean; Reinhard Gruber

    2014-01-01

    Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB43...

  10. Determining the requirements for e-selection in a small recruitment company - using the regulative cycle

    NARCIS (Netherlands)

    Bondarouk, T.V.; Ruel, H.J.M.; Timmermans, P.; Buragga, K.A.; Zaman, N.

    2013-01-01

    The requirements for e-selection technology to be of practical use for Company T have been investigated. Company T’s main business is in identifying and seconding personnel, especially in the technical sector, and it had been anticipating a shortage in the supply of candidates. This served as the in

  11. 78 FR 69267 - Defense Federal Acquisition Regulation Supplement: Requirements Relating to Supply Chain Risk...

    Science.gov (United States)

    2013-11-18

    ... supply chain risk. This rule implements section 806's three supply-chain risk-management approaches as... to improve supply chain risk management (SCRM). In doing so, the DoDI requires, among other things... consider the impact of supply chain risk in specified types of procurements related to national...

  12. 78 FR 54403 - Removal of Transferred OTS Regulations Regarding Recordkeeping and Confirmation Requirements for...

    Science.gov (United States)

    2013-09-04

    ... minor technical nuances,\\23\\ the OTS's rule was otherwise substantively similar to the Agencies... descriptive charts. Additionally, the FDIC recognizes that some State savings associations availed themselves.... Regulatory Analysis and Procedure A. The Paperwork Reduction Act In accordance with the requirements of the...

  13. 77 FR 59139 - Prompt Corrective Action, Requirements for Insurance, and Promulgation of NCUA Rules and Regulations

    Science.gov (United States)

    2012-09-26

    ...'' credit union for determining whether risk-based net worth requirements apply, and 12 CFR 741.3(b)(5)(i... risk to the National Credit Union Share Insurance Fund (NCUSIF). The Board believes the $10 million... the NCUSIF based on recent trends. (iii) Credit Union Complexity and NCUSIF Risk The Board...

  14. 78 FR 63461 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Foreign...

    Science.gov (United States)

    2013-10-24

    ... Canada--Submission with Offer, and 252.225-7004, Report of Intended Performance Outside the United States and Canada-- Submission after Award, as prescribed in 225.7204(a) and (b) respectively, require... list any qualifying country, NAFTA country, or other foreign end product that it intends to furnish...

  15. Arabidopsis MSI1 Is Required for Negative Regulation of the Response to Drought Stress

    Institute of Scientific and Technical Information of China (English)

    Cristina Alexandre; Yvonne M(o)ller-Steinbach; Nicole Sch(o)nrock; Wilhelm Gruissem; Lars Hennig

    2009-01-01

    Arabidopsis MSI1 has fundamental functions in plant development.MSI1 is a subunit of Polycomb group protein complexes and Chromatin assembly factor 1,and it interacts with the Retinoblastoma-related protein 1.Altered levels of MSI1 result in pleiotropic phenotypes,reflecting the complexity of MSI1 protein functions.In order to uncover additional functions of MSI1,we performed transcriptional profiling of wild-type and plants with highly reduced MSI1 levels (msil-cs).Surprisingly,the known functions of MSI1 could only account for a minor part of the transcriptional changes in msi1-cs plants.One of the most striking unexpected observations was the up-regulation of a subset of ABA-responsive genes eliciting the response to drought and salt stress.We report that MSI1 can bind to the chromatin of the drought-inducible downstream target RD20 and suggest a new role for MSI1 in the negative regulation of the Arabidopsis drought-stress response.

  16. Downregulating EMS1/cortactin inhibits migration and endocytosis of MHCC-97H%下调EMS1/cortactin对人高转移潜能肝癌细胞株迁移和内吞的影响

    Institute of Scientific and Technical Information of China (English)

    周家名; 陈莉; 叶蕴瑶; 孙树刚; 王桂兰; 吴圆圆

    2012-01-01

    目的 观察下调人高转移潜能肝癌细胞株(MHCC-97H)中EMS1/cortactin对细胞迁移和内吞的影响,探讨EMS1/cortactin在细胞内可能存在的功能.方法 构建靶向EMS1的shRNA真核表达质粒(EMS1-PSilencer4.1-GFP-shRNA,EMS1-shRNA);通过Lipofectamine 2000处理后将EMS1-shRNA转染MHCC-97H细胞,免疫荧光显微镜下观察转染效率;采用RT-qPCR和Westen blot分别检测EMS1 mRNA和cortactin蛋白表达水平;利用划痕、Transwell小室实验、转铁蛋白(transferrin,Tfn)内吞实验分别研究细胞迁移和内吞作用.结果 成功构建EMS1-shRNA.免疫荧光显微镜下见转染细胞中明显的绿色荧光蛋白(green fluorescence protein,GFP),荧光强度在48 h达到峰值,其转染效率达84.76%.与未处理组相比,转染EMS1-shRNA细胞中EMS1 mRNA水平下降至19.00%,cortactin蛋白水平下降至36.74%、细胞相对迁移距离下降至55.52%、相对穿膜细胞数下降至27.79%、细胞内吞荧光标记Tfn水平显著减弱(P均<0.05).结论 下调EMS1/cortactin后,MHCC-97H细胞迁移、内吞能力明显下降,提示EMS1/cortactin与原发性肝癌(primary human heptatocellular,HCC)转移的生物学行为有关.%Purpose To observe its effect on migration and endocytosis of MHCC-97H by downregulating EMSl/cortactin, and to explore the possible function of EMSl/xontactin within cell. Methods ShRNA eukaryotic expression plasmid targeting EMSl( EMS1-PSilencer4. 1-GFP-shrank, EMSl-shRNA ) was constructed and transfected into MHCC-97H after treatment with Lipofectamine 2000 . Transfection efficiency was evaluated under immunofluorescence microscopy. The expression EMS1 mRNA and protein were detected u-sing real-time quantitative RT-qPCR and western blotting. The migration and endocytosis were tested through wound-healing assay, tr-answell invasion and transferrin endocytosis assay, respectively. Results EMSl-shRNA was constructed successfully. GFP within the transfected cell was obviously observed under

  17. The Ustilago maydis Nit2 homolog regulates nitrogen utilization and is required for efficient induction of filamentous growth.

    Science.gov (United States)

    Horst, Robin J; Zeh, Christine; Saur, Alexandra; Sonnewald, Sophia; Sonnewald, Uwe; Voll, Lars M

    2012-03-01

    Nitrogen catabolite repression (NCR) is a regulatory strategy found in microorganisms that restricts the utilization of complex and unfavored nitrogen sources in the presence of favored nitrogen sources. In fungi, this concept has been best studied in yeasts and filamentous ascomycetes, where the GATA transcription factors Gln3p and Gat1p (in yeasts) and Nit2/AreA (in ascomycetes) constitute the main positive regulators of NCR. The reason why functional Nit2 homologs of some phytopathogenic fungi are required for full virulence in their hosts has remained elusive. We have identified the Nit2 homolog in the basidiomycetous phytopathogen Ustilago maydis and show that it is a major, but not the exclusive, positive regulator of nitrogen utilization. By transcriptome analysis of sporidia grown on artificial media devoid of favored nitrogen sources, we show that only a subset of nitrogen-responsive genes are regulated by Nit2, including the Gal4-like transcription factor Ton1 (a target of Nit2). Ustilagic acid biosynthesis is not under the control of Nit2, while nitrogen starvation-induced filamentous growth is largely dependent on functional Nit2. nit2 deletion mutants show the delayed initiation of filamentous growth on maize leaves and exhibit strongly compromised virulence, demonstrating that Nit2 is required to efficiently initiate the pathogenicity program of U. maydis.

  18. Wdr18 is required for Kupffer's vesicle formation and regulation of body asymmetry in zebrafish.

    Directory of Open Access Journals (Sweden)

    Wei Gao

    Full Text Available Correct specification of the left-right (L-R axis is important for organ morphogenesis. Conserved mechanisms involving cilia rotation inside node-like structures and asymmetric Nodal signaling in the lateral plate mesoderm (LPM, which are important symmetry-breaking events, have been intensively studied. In zebrafish, the clustering and migration of dorsal forerunner cells (DFCs is critical for the formation of the Kuppfer's vesicle (KV. However, molecular events underlying DFC clustering and migration are less understood. The WD-repeat proteins function in a variety of biological processes, including cytoskeleton assembly, intracellular trafficking, mRNA splicing, transcriptional regulation and cell migration. However, little is known about the function of WD-repeat proteins in L-R asymmetry determination. Here, we report the identification and functional analyses of zebrafish wdr18, a novel gene that encodes a WD-repeat protein that is highly conserved among vertebrate species. wdr18 was identified from a Tol2 transposon-mediated enhancer trap screen. Follow-up analysis of wdr18 mRNA expression showed that it was detected in DFCs or the KV progenitor cells and later in the KV at early somitogenesis stages. Morpholino knockdown of wdr18 resulted in laterality defects in the visceral organs, which were preceded by the mis-expression of Nodal-related genes, including spaw and pitx2. Examination of morphants at earlier stages revealed that the KV had fewer and shorter cilia which are immotile and a smaller cavity. We further investigated the organization of DFCs in wdr18 morphant embryos using ntl and sox17 as specific markers and found that the clustering and migration of DFC was altered, leading to a disorganized KV. Finally, through a combination of wdr18 and itgb1b morpholino injections, we provided evidence that wdr18 and itgb1b genetically interact in the laterality determination process. Thus, we reveal a new and essential role for WD

  19. DNMT1 is a Required Genomic Regulator for Murine Liver Histogenesis and Regeneration

    DEFF Research Database (Denmark)

    Kaji, Kosuke; Factor, Valentina M; Andersen, Jesper B

    2016-01-01

    conditional knockout mice (Dnmt1(Δalb) ) by crossing Dnmt1(fl/fl) with Albumin-Cre (Alb-Cre) transgenic mice. Serum, liver tissues and primary hepatocytes were collected from 1-20 week old mice. The Dnmt1(Δalb) phenotype was assessed by histology, confocal and electron microscopy, biochemistry as well...... hepatocytes caused global hypomethylation, enhanced DNA damage response and initiated a senescence state causing a progressive inability to maintain tissue homeostasis and proliferate in response to injury. The liver regenerated via activation and repopulation from progenitors due to lineage...... hepatocytes did not affect liver homeostasis. CONCLUSION: These results establish the indispensable role of DNMT1-mediated epigenetic regulation in postnatal liver growth and regeneration. The Dnmt1(Δalb) mice provide a unique experimental model to study the role of senescence and contribution of progenitor...

  20. Regulation of extracellular matrix genes by arecoline in primary gingival fibroblasts requires epithelial factors.

    Science.gov (United States)

    Thangjam, G S; Agarwal, P; Balapure, A K; Rao, S Girish; Kondaiah, P

    2009-12-01

    Oral submucous fibrosis, a disease of collagen disorder, has been attributed to arecoline present in the saliva of betel quid chewers. However, the molecular basis of the action of arecoline in the pathogenesis of oral submucous fibrosis is poorly understood. The basic aim of our study was to elucidate the mechanism underlying the action of arecoline on the expression of genes in oral fibroblasts. Human keratinocytes (HaCaT cells) and primary human gingival fibroblasts were treated with arecoline in combination with various pathway inhibitors, and the expression of transforming growth factor-beta isoform genes and of collagen isoforms was assessed using reverse transcription-polymerase chain reaction analysis. We observed the induction of transforming growth factor-beta2 by arecoline in HaCaT cells and this induction was found to be caused by activation of the M-3 muscarinic acid receptor via the induction of calcium and the protein kinase C pathway. Most importantly, we showed that transforming growth factor-beta2 was significantly overexpressed in oral submucous fibrosis tissues (p = 0.008), with a median of 2.13 (n = 21) compared with 0.75 (n = 18) in normal buccal mucosal tissues. Furthermore, arecoline down-regulated the expression of collagens 1A1 and 3A1 in human primary gingival fibroblasts; however these collagens were induced by arecoline in the presence of spent medium of cultured human keratinocytes. Treatment with a transforming growth factor-beta blocker, transforming growth factor-beta1 latency-associated peptide, reversed this up-regulation of collagen, suggesting a role for profibrotic cytokines, such as transforming growth factor-beta, in the induction of collagens. Taken together, our data highlight the importance of arecolineinduced epithelial changes in the pathogenesis of oral submucous fibrosis.

  1. The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation

    Directory of Open Access Journals (Sweden)

    Bertin Stéphane

    2011-10-01

    Full Text Available Abstract Background Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. Results In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. Conclusions These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

  2. Phytochromes Regulate SA and JA Signaling Pathways in Rice and Are Required for Developmentally Controlled Resistance to Magnaporthe grisea

    Institute of Scientific and Technical Information of China (English)

    Xian-Zhi Xie; Yan-Jiu Xue; Jin-Jun Zhoua; Bin Zhang; Hong Chang; Makoto Takano

    2011-01-01

    Old leaves of wild-type rice plants (Oryza sativa L. Cv. Nipponbare)are more resistant to blast fungus (Mag-naporthe grisea)than new leaves. In contrast, both old and new leaves of the rice phytochrome triple mutant (phyAphyB-phyC)are susceptible to blast fungus. We demonstrate that pathogenesis-related class 1 (PR1)proteins are rapidly and strongly induced during M. Grisea infection and following exogenous jasmonate (JA)or salicylic acid (SA)exposure in the old leaves, but not in the new leaves of the wild-type. In contrast, the accumulation of PR1 proteins was significantly attenuated in old and new leaves of the phyAphyBphyC mutant. These results suggest that phytochromes are required for the induction of PR1 proteins in rice. Basal transcription levels of Prla and PRlb were substantially higher in the wild-type as compared to the phyAphyBphyC mutant, suggesting that phytochromes also are required for basal expression of PR1 genes. Moreover, the transcript levels of genes known to function in SA-or JA-dependent defense pathways were regulated by leaf age and functional phytochromes. Taken together, our findings demonstrate that phytochromes are required in rice for age-related resistance to M, grisea and may indirectly increase PR1 gene expression by regulating SA-and JA-dependent defense pathways.

  3. Differential developmental requirement and peripheral regulation for dermal Vγ4 and Vγ6T17 cells in health and inflammation.

    Science.gov (United States)

    Cai, Yihua; Xue, Feng; Fleming, Chris; Yang, Jie; Ding, Chuanlin; Ma, Yunfeng; Liu, Min; Zhang, Huang-ge; Zheng, Jie; Xiong, Na; Yan, Jun

    2014-06-09

    Dermal IL-17-producing γδT cells have a critical role in skin inflammation. However, their development and peripheral regulation have not been fully elucidated. Here we demonstrate that dermal γδT cells develop from the embryonic thymus and undergo homeostatic proliferation after birth with diversified TCR repertoire. Vγ6T cells are bona fide resident, but precursors of dermal Vγ4T cells may require extrathymic environment for imprinting skin-homing properties. Thymic Vγ6T cells are more competitive than Vγ4 for dermal γδT cell reconstitution and TCRδ(-/-) mice reconstituted with Vγ6 develop psoriasis-like inflammation after IMQ-application. Although both IL-23 and IL-1β promote Vγ4 and Vγ6 proliferation, Vγ4 are the main source of IL-17 production that requires IL-1 signalling. Mice with deficiency of IL-1RI signalling have significantly decreased skin inflammation. These studies reveal a differential developmental requirement and peripheral regulation for dermal Vγ6 and Vγ4 γδT cells, implying a new mechanism that may be involved in skin inflammation.

  4. Exploring the structural requirements for jasmonates and related compounds as novel plant growth regulators: a current computational perspective.

    Science.gov (United States)

    Chen, Ke-Xian; Li, Zu-Guang

    2009-11-01

    Jasmonates and related compounds have been highlighted recently in the field of plant physiology and plant molecular biology due to their significant regulatory roles in the signaling pathway for the diverse aspects of plant development and survival. Though a considerable amount of studies concerning their biological effects in different plants have been widely reported, the molecular details of the signaling mechanism are still poorly understood. This review sheds new light on the structural requirements for the bioactivity/property of jasmonic acid derivatives in current computational perspective, which differs from previous research that mainly focus on their biological evaluation, gene and metabolic regulation and the enzymes in their biosynthesis. The computational results may contribute to further understanding the mechanism of drug-receptor interactions in their signaling pathway and designing novel plant growth regulators as high effective ecological pesticides.

  5. ChREBP, but not LXRs, is required for the induction of glucose-regulated genes in mouse liver

    Science.gov (United States)

    Denechaud, Pierre-Damien; Bossard, Pascale; Lobaccaro, Jean-Marc A.; Millatt, Lesley; Staels, Bart; Girard, Jean; Postic, Catherine

    2008-01-01

    The transcription factor carbohydrate-responsive element–binding protein (ChREBP) has emerged as a central regulator of lipid synthesis in liver because it is required for glucose-induced expression of the glycolytic enzyme liver–pyruvate kinase (L-PK) and acts in synergy with SREBP to induce lipogenic genes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Liver X receptors (LXRs) are also important regulators of the lipogenic pathway, and the recent finding that ChREBP is a direct target of LXRs and that glucose itself can bind and activate LXRs prompted us to study the role of LXRs in the induction of glucose-regulated genes in liver. Using an LXR agonist in wild-type mice, we found that LXR stimulation did not promote ChREBP phosphorylation or nuclear localization in the absence of an increased intrahepatic glucose flux. Furthermore, the induction of ChREBP, L-PK, and ACC by glucose or high-carbohydrate diet was similar in LXRα/β knockout compared with wild-type mice, suggesting that the activation of these genes by glucose occurs by an LXR-independent mechanism. We used fluorescence resonance energy transfer analysis to demonstrate that glucose failed to promote the interaction of LXRα/β with specific cofactors. Finally, siRNA silencing of ChREBP in LXRα/β knockout hepatocytes abrogated glucose-induced expression of L-PK and ACC, further demonstrating the central role of ChREBP in glucose signaling. Taken together, our results demonstrate that glucose is required for ChREBP functional activity and that LXRs are not necessary for the induction of glucose-regulated genes in liver. PMID:18292813

  6. Regulation of the ERK pathway in the dentate gyrus by in vivo dopamine D1 receptor stimulation requires glutamatergic transmission.

    Science.gov (United States)

    Gangarossa, Giuseppe; Valjent, Emmanuel

    2012-11-01

    Acute systemic administration of the dopamine D1/D5 receptors (D1Rs) agonist, SKF81297, activates the extracellular signal-regulated protein kinases (ERK) pathway selectively in the granule cells of the dentate gyrus. In this study, we examined the mechanisms involved in this regulation and investigated the molecular components that could promote ERK-dependent transcription and translation. SKF81297 induced phosphorylation of ERK and histone H3 required intact glutamatergic transmission. Blockade of glutamate release achieved by the mGluR2/3 agonist, LY354740 or the selective adenosine A1R agonist, CCPA as well as neurotoxic lesions of lateral entorhinal cortex reduced the ability of SKF81297 to induce ERK activation in the dentate gyrus. This activation required the combined stimulation of NR2B-containing NMDARs, mGluR1 and mGluR5. SKF81297 evoked phosphorylation of the ribosomal protein S6 (rpS6) selectively at the Ser235/236 site while the Ser240/244 site remains unchanged. The SKF81297 induced increased phosphorylation of rpS6 was dependent on PKC and ERK/p90RSK activation. Surprisingly, administration of D1Rs agonist suppressed mTORC1/p70S6K pathway suggesting an mTOR-independent regulation of rpS6 phosphorylation. Taken together, our results show that intact glutamatergic transmission plays a major role in the regulation of ERK-dependent phosphorylation of histone H3 and rpS6 observed in the mouse dentate gyrus after systemic administration of SKF81297.

  7. Vangl2-regulated polarisation of second heart field-derived cells is required for outflow tract lengthening during cardiac development.

    Directory of Open Access Journals (Sweden)

    Simon A Ramsbottom

    2014-12-01

    Full Text Available Planar cell polarity (PCP is the mechanism by which cells orient themselves in the plane of an epithelium or during directed cell migration, and is regulated by a highly conserved signalling pathway. Mutations in the PCP gene Vangl2, as well as in other key components of the pathway, cause a spectrum of cardiac outflow tract defects. However, it is unclear why cells within the mesodermal heart tissue require PCP signalling. Using a new conditionally floxed allele we show that Vangl2 is required solely within the second heart field (SHF to direct normal outflow tract lengthening, a process that is required for septation and normal alignment of the aorta and pulmonary trunk with the ventricular chambers. Analysis of a range of markers of polarised epithelial tissues showed that in the normal heart, undifferentiated SHF cells move from the dorsal pericardial wall into the distal outflow tract where they acquire an epithelial phenotype, before moving proximally where they differentiate into cardiomyocytes. Thus there is a transition zone in the distal outflow tract where SHF cells become more polarised, turn off progenitor markers and start to differentiate to cardiomyocytes. Membrane-bound Vangl2 marks the proximal extent of this transition zone and in the absence of Vangl2, the SHF-derived cells are abnormally polarised and disorganised. The consequent thickening, rather than lengthening, of the outflow wall leads to a shortened outflow tract. Premature down regulation of the SHF-progenitor marker Isl1 in the mutants, and accompanied premature differentiation to cardiomyocytes, suggests that the organisation of the cells within the transition zone is important for maintaining the undifferentiated phenotype. Thus, Vangl2-regulated polarisation and subsequent acquisition of an epithelial phenotype is essential to lengthen the tubular outflow vessel, a process that is essential for on-going cardiac morphogenesis.

  8. Vangl2-regulated polarisation of second heart field-derived cells is required for outflow tract lengthening during cardiac development.

    Science.gov (United States)

    Ramsbottom, Simon A; Sharma, Vipul; Rhee, Hong Jun; Eley, Lorraine; Phillips, Helen M; Rigby, Hannah F; Dean, Charlotte; Chaudhry, Bill; Henderson, Deborah J

    2014-12-01

    Planar cell polarity (PCP) is the mechanism by which cells orient themselves in the plane of an epithelium or during directed cell migration, and is regulated by a highly conserved signalling pathway. Mutations in the PCP gene Vangl2, as well as in other key components of the pathway, cause a spectrum of cardiac outflow tract defects. However, it is unclear why cells within the mesodermal heart tissue require PCP signalling. Using a new conditionally floxed allele we show that Vangl2 is required solely within the second heart field (SHF) to direct normal outflow tract lengthening, a process that is required for septation and normal alignment of the aorta and pulmonary trunk with the ventricular chambers. Analysis of a range of markers of polarised epithelial tissues showed that in the normal heart, undifferentiated SHF cells move from the dorsal pericardial wall into the distal outflow tract where they acquire an epithelial phenotype, before moving proximally where they differentiate into cardiomyocytes. Thus there is a transition zone in the distal outflow tract where SHF cells become more polarised, turn off progenitor markers and start to differentiate to cardiomyocytes. Membrane-bound Vangl2 marks the proximal extent of this transition zone and in the absence of Vangl2, the SHF-derived cells are abnormally polarised and disorganised. The consequent thickening, rather than lengthening, of the outflow wall leads to a shortened outflow tract. Premature down regulation of the SHF-progenitor marker Isl1 in the mutants, and accompanied premature differentiation to cardiomyocytes, suggests that the organisation of the cells within the transition zone is important for maintaining the undifferentiated phenotype. Thus, Vangl2-regulated polarisation and subsequent acquisition of an epithelial phenotype is essential to lengthen the tubular outflow vessel, a process that is essential for on-going cardiac morphogenesis.

  9. cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

    Directory of Open Access Journals (Sweden)

    Ghania Ramdani

    2015-05-01

    Full Text Available Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites.

  10. Down-regulation of Stathmin Is Required for the Phenotypic Changes and Classical Activation of Macrophages.

    Science.gov (United States)

    Xu, Kewei; Harrison, Rene E

    2015-07-31

    Macrophages are important cells of innate immunity with specialized capacity for recognition and elimination of pathogens and presentation of antigens to lymphocytes for adaptive immunity. Macrophages become activated upon exposure to pro-inflammatory cytokines and pathogenic stimuli. Classical activation of macrophages with interferon-γ (IFNγ) and lipopolysaccharide (LPS) triggers a wide range of signaling events and morphological changes to induce the immune response. Our previous microtubule (MT) proteomic work revealed that the stathmin association with MTs is considerably reduced in activated macrophages, which contain significantly more stabilized MTs. Here, we show that there is a global decrease in stathmin levels, an MT catastrophe protein, in activated macrophages using both immunoblotting and immunofluorescent microscopy. This is an LPS-specific response that induces proteasome-mediated degradation of stathmin. We explored the functions of stathmin down-regulation in activated macrophages by generating a stable cell line overexpressing stathmin-GFP. We show that stathmin-GFP overexpression impacts MT stability, impairs cell spreading, and reduces activation-associated phenotypes. Furthermore, overexpressing stathmin reduces complement receptor 3-mediated phagocytosis and cellular activation, implicating a pivotal inhibitory role for stathmin in classically activated macrophages.

  11. Redox regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress.

    Science.gov (United States)

    Stankovic-Valentin, Nicolas; Drzewicka, Katarzyna; König, Cornelia; Schiebel, Elmar; Melchior, Frauke

    2016-06-15

    To sense and defend against oxidative stress, cells depend on signal transduction cascades involving redox-sensitive proteins. We previously identified SUMO (small ubiquitin-related modifier) enzymes as downstream effectors of reactive oxygen species (ROS). Hydrogen peroxide transiently inactivates SUMO E1 and E2 enzymes by inducing a disulfide bond between their catalytic cysteines. How important their oxidation is in light of many other redox-regulated proteins has however been unclear. To selectively disrupt this redox switch, we identified a catalytically fully active SUMO E2 enzyme variant (Ubc9 D100A) with strongly reduced propensity to maintain a disulfide with the E1 enzyme in vitro and in cells. Replacement of Ubc9 by this variant impairs cell survival both under acute and mild chronic oxidative stresses. Intriguingly, Ubc9 D100A cells fail to maintain activity of the ATM-Chk2 DNA damage response pathway that is induced by hydrogen peroxide. In line with this, these cells are also more sensitive to the ROS-producing chemotherapeutic drugs etoposide/Vp16 and Ara-C. These findings reveal that SUMO E1~E2 oxidation is an essential redox switch in oxidative stress.

  12. Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling.

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    Alexandra Stähli

    Full Text Available Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p10-fold. Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro.

  13. Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling.

    Science.gov (United States)

    Stähli, Alexandra; Bosshardt, Dieter; Sculean, Anton; Gruber, Reinhard

    2014-01-01

    Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p10-fold). Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro.

  14. Mitotic destruction of the cell cycle regulated NIMA protein kinase of Aspergillus nidulans is required for mitotic exit.

    OpenAIRE

    Pu, R T; Osmani, S A

    1995-01-01

    NIMA is a cell cycle regulated protein kinase required, in addition to p34cdc2/cyclin B, for initiation of mitosis in Aspergillus nidulans. Like cyclin B, NIMA accumulates when cells are arrested in G2 and is degraded as cells traverse mitosis. However, it is stable in cells arrested in mitosis. NIMA, and related kinases, have an N-terminal kinase domain and a C-terminal extension. Deletion of the C-terminus does not completely inactivate NIMA kinase activity but does prevent functional compl...

  15. High Silicon Accumulation in the Shoot is Required for Down-Regulating the Expression of Si Transporter Genes in Rice.

    Science.gov (United States)

    Mitani-Ueno, Namiki; Yamaji, Naoki; Ma, Jian Feng

    2016-12-01

    Rice requires high silicon (Si) for its high and sustainable yield. The efficient uptake of Si in rice is mediated by two transporters OsLsi1 and OsLsi2, which function as influx and efflux transporters, respectively. Our previous studies showed that the mRNA expression levels of these transporter genes were down-regulated by Si. Herein we investigated the mechanism underlying regulation of OsLsi1 and OsLsi2 expression. There was a negative correlation between the expression level of OsLsi1 and OsLsi2 and shoot Si accumulation when the rice seedlings were exposed to different Si supply conditions. A split root experiment showed that the expression of both OsLsi1 and OsLsi2 was also down-regulated in half the roots without direct Si exposure when the other half of the roots were exposed to Si. Analysis with transgenic rice carrying different lengths of OsLsi1 promoter regions fused with green fluorescent protein (GFP) as a reporter gene revealed that the region responsible for the Si response of OsLsi1 expression is present between -327 to -292 in the promoter. However, this region was not associated with the tissue and cellular localization of OsLsi1. In conclusion, the Si-induced down-regulation of Si transporter genes is controlled by shoot Si, not root Si, and the region between -327 and -292 in the OsLsi1 promoter is involved in this regulation of OsLsi1 expression in rice.

  16. Using reporting requirements to improve employer wellness incentives and their regulation.

    Science.gov (United States)

    Madison, Kristin; Schmidt, Harald; Volpp, Kevin G

    2014-10-01

    Employer interest in offering financial incentives for healthy behaviors has been increasing. Some employers have begun to tie health plan-based rewards or penalties to standards involving tobacco use or biometric measures such as body mass index. The Patient Protection and Affordable Care Act attempts to strike a balance between the potential benefits and risks of wellness incentive programs by permitting these incentives but simultaneously limiting their use. Evidence about the implications of the newest generation of incentive programs for health, health costs, and burdens on individual employees will be critical for informing both private and public decision makers. After describing the many pieces of information that would be valuable for assessing these programs, this article proposes more narrowly targeted reporting requirements that could facilitate incentive program development, evaluation, and oversight. Copyright © 2014 by Duke University Press.

  17. Basic Requirements Of The Basel Committee On Regulating Capital Adequacy And Liquidity Of Commercial Banks

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    Bahriddin Berdiyarov

    2012-03-01

    Full Text Available The current paper highlights theBaselI, Basel II & Basel III requirements on capital adequacy and liquidity of commercial banks.  In the paper, Basel II structure, methods of loan risk assessment, coefficients of loan risk assessment, credit risk measurement for counterparty banks are discussed.  Moreover, assessments of Basel III on bank chances against crisis driven from financial and economic crunches, risk management, performance quality and bank transparency improvement measures are presented.  At the end, the author gives his conclusions on the essence and necessity of new regulatory standards of the Basel Committee on bank’s supervision in the structure of the supervision of credit institutions.

  18. Heterochromatic genome stability requires regulators of histone H3 K9 methylation.

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    Jamy C Peng

    2009-03-01

    Full Text Available Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance. Here, we show that Drosophila that lack the Su(var3-9 H3K9 methyltransferase display significantly elevated frequencies of spontaneous DNA damage in heterochromatin, in both somatic and germ-line cells. Accumulated DNA damage in these mutants correlates with chromosomal defects, such as translocations and loss of heterozygosity. DNA repair and mitotic checkpoints are also activated in mutant animals and are required for their viability. Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2. These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability.

  19. Heterochromatic genome stability requires regulators of histone H3 K9 methylation.

    Science.gov (United States)

    Peng, Jamy C; Karpen, Gary H

    2009-03-01

    Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance. Here, we show that Drosophila that lack the Su(var)3-9 H3K9 methyltransferase display significantly elevated frequencies of spontaneous DNA damage in heterochromatin, in both somatic and germ-line cells. Accumulated DNA damage in these mutants correlates with chromosomal defects, such as translocations and loss of heterozygosity. DNA repair and mitotic checkpoints are also activated in mutant animals and are required for their viability. Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2. These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability.

  20. Survival of Listeria monocytogenes in Soil Requires AgrA-Mediated Regulation.

    Science.gov (United States)

    Vivant, Anne-Laure; Garmyn, Dominique; Gal, Laurent; Hartmann, Alain; Piveteau, Pascal

    2015-08-01

    In a recent paper, we demonstrated that inactivation of the Agr system affects the patterns of survival of Listeria monocytogenes (A.-L. Vivant, D. Garmyn, L. Gal, and P. Piveteau, Front Cell Infect Microbiol 4:160, http://dx.doi.org/10.3389/fcimb.2014.00160). In this study, we investigated whether the Agr-mediated response is triggered during adaptation in soil, and we compared survival patterns in a set of 10 soils. The fate of the parental strain L. monocytogenes L9 (a rifampin-resistant mutant of L. monocytogenes EGD-e) and that of a ΔagrA deletion mutant were compared in a collection of 10 soil microcosms. The ΔagrA mutant displayed significantly reduced survival in these biotic soil microcosms, and differential transcriptome analyses showed large alterations of the transcriptome when AgrA was not functional, while the variations in the transcriptomes between the wild type and the ΔagrA deletion mutant were modest under abiotic conditions. Indeed, in biotic soil environments, 578 protein-coding genes and an extensive repertoire of noncoding RNAs (ncRNAs) were differentially transcribed. The transcription of genes coding for proteins involved in cell envelope and cellular processes, including the phosphotransferase system and ABC transporters, and proteins involved in resistance to antimicrobial peptides was affected. Under sterilized soil conditions, the differences were limited to 86 genes and 29 ncRNAs. These results suggest that the response regulator AgrA of the Agr communication system plays important roles during the saprophytic life of L. monocytogenes in soil.

  1. N-cadherin regulates signaling mechanisms required for lens fiber cell elongation and lens morphogenesis.

    Science.gov (United States)

    Logan, Caitlin M; Rajakaruna, Suren; Bowen, Caitlin; Radice, Glenn L; Robinson, Michael L; Menko, A Sue

    2017-08-01

    Tissue development and regeneration involve high-ordered morphogenetic processes that are governed by elements of the cytoskeleton in conjunction with cell adhesion molecules. Such processes are particularly important in the lens whose structure dictates its function. Studies of our lens-specific N-cadherin conditional knockout mouse (N-cadcKO) revealed an essential role for N-cadherin in the migration of the apical tips of differentiating lens fiber cells along the apical surfaces of the epithelium, a region termed the Epithelial Fiber Interface (EFI), that is necessary for normal fiber cell elongation and the morphogenesis. Studies of the N-cadcKO lens suggest that N-cadherin function in fiber cell morphogenesis is linked to the activation of Rac1 and myosin II, both signaling pathways central to the regulation of cell motility including determining the directionality of cellular movement. The absence of N-cadherin did not disrupt lateral contacts between fiber cells during development, and the maintenance of Aquaporin-0 and increased expression of EphA2 at cell-cell interfaces suggests that these molecules may function in this role. E-cadherin was maintained in newly differentiating fiber cells without interfering with expression of lens-specific differentiation proteins but was not able to replace N-cadherin function in these cells. The dependence of migration of the fiber cell apical domains along the EFI for lens morphogenesis on N-cadherin provides new insight into the process of tissue development. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence.

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    Bidyottam Mittra

    2016-01-01

    Full Text Available Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast and mitoferrin-1 (human that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1 showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes.

  3. The Drosophila cell adhesion molecule klingon is required for long-term memory formation and is regulated by Notch.

    Science.gov (United States)

    Matsuno, Motomi; Horiuchi, Junjiro; Tully, Tim; Saitoe, Minoru

    2009-01-06

    The ruslan (rus) mutant was previously identified in a behavioral screen for mutants defective in long-lasting memory, which consists of two consolidated memory types, anesthesia-resistant memory, and protein synthesis-dependent long-term memory (LTM). We demonstrate here that rus is a new allele of klingon (klg), which encodes a homophilic cell adhesion molecule. Klg is acutely required for LTM but not anesthesia-resistant memory formation, and Klg expression increases upon LTM induction. LTM formation also requires activity of the Notch cell-surface receptor. Although defects in Notch have been implicated in memory loss because of Alzheimer's disease, downstream signaling linking Notch to memory have not been determined. Strikingly, we found that Notch activity increases upon LTM induction and regulates Klg expression. Furthermore, Notch-induced enhancement of LTM is disrupted by a klg mutation. We propose that Klg is a downstream effector of Notch signaling that links Notch activity to memory.

  4. Fyn tyrosine kinase regulates oligodendroglial cell development but is not required for morphological differentiation of oligodendrocytes.

    Science.gov (United States)

    Sperber, B R; McMorris, F A

    2001-02-15

    The non-receptor protein tyrosine kinase Fyn, which is a member of the Src family of kinases, has been shown to be essential for normal myelination and has been suggested to play a role in oligodendrocyte development. However, oligodendrocyte development has not been studied directly in cells lacking Fyn. Additionally, because Fyn is expressed in neurons as well as oligodendrocytes, it is possible that normal myelination requires Fyn expression in neurons but not in oligodendrocytes. To address these issues, we analyzed the development of oligodendrocytes in neuron-free glial cell cultures from fyn(-/-) mice that express no Fyn protein. We observed that oligodendrocytes develop to the stage where they elaborate an extensive network of membranous processes and express the antigenic components of mature oligodendrocytes in the complete absence of Fyn. However, as compared with fyn(+/+) controls, fewer oligodendroglia developed in fyn(-/-) cell cultures, and a smaller proportion of them matured to the stage characterized by a high degree of morphological complexity. In addition, we found that insulin-like growth factor-I, a potent stimulator of oligodendrocyte development, failed to stimulate morphological maturation of fyn(-/-) oligodendroglia. The pyrazolopyrimidine PP2, believed to be a selective inhibitor of Fyn, did not prevent the development of morphologically complex oligodendrocytes. Unexpectedly, however, it was toxic to both fyn(+/+) and fyn(-/-) glial cells, indicating that this class of inhibitors can have significant effects that are independent of Fyn.

  5. Distinct Akt phosphorylation states are required for insulin regulated Glut4 and Glut1-mediated glucose uptake.

    Science.gov (United States)

    Beg, Muheeb; Abdullah, Nazish; Thowfeik, Fathima Shazna; Altorki, Nasser K; McGraw, Timothy E

    2017-06-07

    Insulin, downstream of Akt activation, promotes glucose uptake into fat and muscle cells to lower postprandial blood glucose, an enforced change in cellular metabolism to maintain glucose homeostasis. This effect is mediated by the Glut4 glucose transporter. Growth factors also enhance glucose uptake to fuel an anabolic metabolism required for tissue growth and repair. This activity is predominantly mediated by the Glut1. Akt is activated by phosphorylation of its kinase and hydrophobic motif (HM) domains. We show that insulin-stimulated Glut4-mediated glucose uptake requires PDPK1 phosphorylation of the kinase domain but not mTORC2 phosphorylation of the HM domain. Nonetheless, an intact HM domain is required for Glut4-mediated glucose uptake. Whereas, Glut1-mediated glucose uptake also requires mTORC2 phosphorylation of the HM domain, demonstrating both phosphorylation-dependent and independent roles of the HM domain in regulating glucose uptake. Thus, mTORC2 links Akt to the distinct physiologic programs related to Glut4 and Glut1-mediated glucose uptake.

  6. Surface swarming motility by Pectobacterium atrosepticum is a latent phenotype that requires O antigen and is regulated by quorum sensing.

    Science.gov (United States)

    Bowden, Steven D; Hale, Nicola; Chung, Jade C S; Hodgkinson, James T; Spring, David R; Welch, Martin

    2013-11-01

    We describe a previously cryptic phenotype associated with the opportunistic phytopathogen Pectobacterium atrosepticum (Pca): surface swarming. We found that when Pca was spotted onto plates containing <0.5% (w/v) agar, the culture produced copious amounts of extracellular matrix material containing highly motile cells. Once produced, this 'slime layer' spread rapidly across the plate either as an advancing front or as tendrils. Transposon mutagenesis was used to identify mutants that were affected in swarming. Hypo-swarmer mutants mostly carried insertions in a horizontally acquired island (HAI5), which encodes a cluster of genes involved in O antigen biosynthesis. Hyper-swarmer mutants mostly carried insertions in hexY, a known antagonist of the class I flagellar master regulator, FlhD4C2. In addition, we found that the nucleoid protein, histone-like nuclear structuring protein 2 (H-NS2), also regulated swarming behaviour. A mutant in which hns2 was overexpressed displayed a hyper-swarming phenotype, whereas a mutant in which the hns2 ORF was inactivated had a hypo-swarming phenotype. Swarming was also regulated by quorum sensing (QS) and by the carbon source being utilized. We show, using a range of epistasis experiments, that optimal swarming requires both motility and O antigen biosynthesis, and that H-NS2 and QS both promote swarming through their effects on motility.

  7. Differential requirement for the CD45 splicing regulator hnRNPLL for accumulation of NKT and conventional T cells.

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    Mehmet Yabas

    Full Text Available Natural killer T (NKT cells represent an important regulatory T cell subset that develops in the thymus and contains immature (NK1.1(lo and mature (NK1.1(hi cell subsets. Here we show in mice that an inherited mutation in heterogeneous ribonucleoprotein L-like protein (hnRNPLL(thunder, that shortens the survival of conventional T cells, has no discernible effect on NKT cell development, homeostasis or effector function. Thus, Hnrpll deficiency effectively increases the NKT∶T cell ratio in the periphery. However, Hnrpll mutation disrupts CD45RA, RB and RC exon silencing of the Ptprc mRNA in both NKT and conventional T cells, and leads to a comparably dramatic shift to high molecular weight CD45 isoforms. In addition, Hnrpll mutation has a cell intrinsic effect on the expression of the developmentally regulated cell surface marker NK1.1 on NKT cells in the thymus and periphery but does not affect cell numbers. Therefore our results highlight both overlapping and divergent roles for hnRNPLL between conventional T cells and NKT cells. In both cell subsets it is required as a trans-acting factor to regulate alternative splicing of the Ptprc mRNA, but it is only required for survival of conventional T cells.

  8. Ezrin is required for the functional regulation of the epithelial sodium proton exchanger, NHE3.

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    Hisayoshi Hayashi

    Full Text Available The sodium hydrogen exchanger isoform 3 (NHE3 mediates absorption of sodium, bicarbonate and water from renal and intestinal lumina. This activity is fundamental to the maintenance of a physiological plasma pH and blood pressure. To perform this function NHE3 must be present in the apical membrane of renal tubular and intestinal epithelia. The molecular determinants of this localization have not been conclusively determined, although linkage to the apical actin cytoskeleton through ezrin has been proposed. We set out to evaluate this hypothesis. Functional studies of NHE3 activity were performed on ezrin knockdown mice (Vil2(kd/kd and NHE3 activity similar to wild-type animals detected. Interpretation of this finding was difficult as other ERM (ezrin/radixin/moesin proteins were present. We therefore generated an epithelial cell culture model where ezrin was the only detectable ERM. After knockdown of ezrin expression with siRNA, radixin and moesin expression remained undetectable. Consistent with the animal ultrastructural data, cells lacking ezrin retained an epithelial phenotype but had shortened and thicker microvilli. NHE3 localization was identical to cells transfected with non-targeting siRNA. The attachment of NHE3 to the apical cytoskeleton was unaltered as assessed by fluorescent recovery after photobleaching (FRAP and the solubility of NHE3 in Triton X-100. Baseline NHE3 activity was unaltered, however, cAMP-dependent inhibition of NHE3 was largely lost even though NHE3 was phosphorylated at serines 552 and 605. Thus, ezrin is not necessary for the apical localization, attachment to the cytoskeleton, baseline activity or cAMP induced phosphrylation of NHE3, but instead is required for cAMP mediated inhibition.

  9. CovR and VicRK regulate cell surface biogenesis genes required for biofilm formation in Streptococcus mutans.

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    Rafael N Stipp

    Full Text Available The two-component system VicRK and the orphan regulator CovR of Streptococcus mutans co-regulate a group of virulence genes associated with the synthesis of and interaction with extracellular polysaccharides of the biofilm matrix. Knockout mutants of vicK and covR display abnormal cell division and morphology phenotypes, although the gene function defects involved are as yet unknown. Using transcriptomic comparisons between parent strain UA159 with vicK (UAvic or covR (UAcov deletion mutants together with electrophoretic motility shift assays (EMSA, we identified genes directly regulated by both VicR and CovR with putative functions in cell wall/surface biogenesis, including gbpB, wapE, smaA, SMU.2146c, and lysM. Deletion mutants of genes regulated by VicR and CovR (wapE, lysM, smaA, or regulated only by VicR (SMU.2146c or CovR (epsC promoted significant alterations in biofilm initiation, including increased fragility, defects in microcolony formation, and atypical cell morphology and/or chaining. Significant reductions in mureinolytic activity and/or increases in DNA release during growth were observed in knockout mutants of smaA, wapE, lysM, SMU.2146c and epsC, implying roles in cell wall biogenesis. WapE and lysM mutations also affected cell hydrophobicity and sensitivity to osmotic or oxidative stress. Finally, vicR, covR and VicRK/CovR-targets (gbpB, wapE, smaA, SMU.2146c, lysM, epsC are up-regulated in UA159 during biofilm initiation, in a sucrose-dependent manner. These data support a model in which VicRK and CovR coordinate cell division and surface biogenesis with the extracellular synthesis of polysaccharides, a process apparently required for formation of structurally stable biofilms in the presence of sucrose.

  10. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa.

    Science.gov (United States)

    Whitney, John C; Whitfield, Gregory B; Marmont, Lindsey S; Yip, Patrick; Neculai, A Mirela; Lobsanov, Yuri D; Robinson, Howard; Ohman, Dennis E; Howell, P Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

  11. Notification: Hotline Complaint – Region 7 Compliance with Bid, Payment, and Performance Bond Requirements Outlined in Federal Acquisition Regulation (FAR) Part 28

    Science.gov (United States)

    June 1, 2012. In response to an anonymous hotline complaint, the OIG plans to conduct a review of Region 7’s compliance with bid, payment, and performance bond requirements outlined in Federal Acquisition Regulation (FAR) Part 28.

  12. Protein kinase A regulation of P2X(4) receptors: requirement for a specific motif in the C-terminus.

    Science.gov (United States)

    Brown, David A; Yule, David I

    2010-02-01

    The P2X purinergic receptor sub-family of ligand-gated ion channels are subject to protein kinase modulation. We have previously demonstrated that P2X(4)R signaling can be positively regulated by increasing intracellular cAMP levels. The molecular mechanism underlying this effect was, however, unknown. The present study initially addressed whether protein kinase A (PKA) activation was required. Subsequently a mutational approach was utilized to determine which region of the receptor was required for this potentiation. In both DT-40 3KO and HEK-293 cells transiently expressing P2X(4)R, forskolin treatment enhanced ATP-mediated signaling. Specific PKA inhibitors prevented the forskolin-induced enhancement of ATP-mediated inward currents in P2X(4)R expressing HEK-293 cells. To define which region of the P2X(4)R was required for the potentiation, mutations were generated in the cytoplasmic C-terminal tail. It was determined that a limited region of the C-terminus, consisting of a non-canonical tyrosine based sorting motif, was required for the effects of PKA. Of note, this region does not harbor any recognizable PKA phosphorylation motifs, and no direct phosphorylation of P2X(4)R was detected, suggesting that PKA phosphorylation of an accessory protein interacts with the endocytosis motif in the C-terminus of the P2X(4)R. In support of this notion, using Total Internal Reflection Fluorescence Microscopy (TIRF)\\ P2X(4)-EGFP was shown to accumulate at/near the plasma membrane following forskolin treatment. In addition, disrupting the endocytosis machinery using a dominant-negative dynamin construct also prevented the PKA-mediated enhancement of ATP-stimulated Ca(2+) signals. Our results are consistent with a novel mechanism of P2XR regulation, whereby PKA activity, without directly phosphorylating P2X(4)R, markedly enhances ATP-stimulated P2X(4)R currents and hence cytosolic Ca(2+) signals. This may occur at least in part, by altering the trafficking of a population of

  13. The ciliogenic transcription factor RFX3 regulates early midline distribution of guidepost neurons required for corpus callosum development.

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    Carine Benadiba

    Full Text Available The corpus callosum (CC is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3 is a transcription factor involved in the control of ciliogenesis, and Rfx3-deficient mice show several hallmarks of ciliopathies including left-right asymmetry defects and hydrocephalus. Here we show that Rfx3-deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8 at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3 repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.

  14. Fragile X protein FMRP is required for homeostatic plasticity and regulation of synaptic strength by retinoic acid.

    Science.gov (United States)

    Soden, Marta E; Chen, Lu

    2010-12-15

    Homeostatic synaptic plasticity adjusts the strength of synapses during global changes in neural activity, thereby stabilizing the overall activity of neural networks. Suppression of synaptic activity increases synaptic strength by inducing synthesis of retinoic acid (RA), which activates postsynaptic synthesis of AMPA-type glutamate receptors (AMPARs) in dendrites and promotes synaptic insertion of newly synthesized AMPARs. Here, we show that fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates dendritic protein synthesis, is essential for increases in synaptic strength induced by RA or by blockade of neural activity in the mouse hippocampus. Although activity-dependent RA synthesis is maintained in Fmr1 knock-out neurons, RA-dependent dendritic translation of GluR1-type AMPA receptors is impaired. Intriguingly, FMRP is only required for the form of homeostatic plasticity that is dependent on both RA signaling and local protein synthesis. Postsynaptic expression of wild-type or mutant FMRP(I304N) in knock-out neurons reduced the total, surface, and synaptic levels of AMPARs, implying a role for FMRP in regulating AMPAR abundance. Expression of FMRP lacking the RGG box RNA-binding domain had no effect on AMPAR levels. Importantly, postsynaptic expression of wild-type FMRP, but not FMRP(I304N) or FMRPΔRGG, restored synaptic scaling when expressed in knock-out neurons. Together, these findings identify an unanticipated role for FMRP in regulating homeostatic synaptic plasticity downstream of RA. Our results raise the possibility that at least some of the symptoms of fragile X syndrome reflect impaired homeostatic plasticity and impaired RA signaling.

  15. Mycobacterium tuberculosis universal stress protein Rv2623 regulates bacillary growth by ATP-Binding: requirement for establishing chronic persistent infection.

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    Joshua E Drumm

    2009-05-01

    Full Text Available Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  16. ST2 negatively regulates TLR2 signaling, but is not required for bacterial lipoprotein-induced tolerance.

    LENUS (Irish Health Repository)

    Liu, Jinghua

    2010-05-15

    Activation of TLR signaling is critical for host innate immunity against bacterial infection. Previous studies reported that the ST2 receptor, a member of the Toll\\/IL-1 receptor superfamily, functions as a negative regulator of TLR4 signaling and maintains LPS tolerance. However, it is undetermined whether ST2 negatively regulates TLR2 signaling and furthermore, whether a TLR2 agonist, bacterial lipoprotein (BLP)-induced tolerance is dependent on ST2. In this study, we show that BLP stimulation-induced production of proinflammatory cytokines and immunocomplex formation of TLR2-MyD88 and MyD88-IL-1R-associated kinase (IRAK) were significantly enhanced in ST2-deficient macrophages compared with those in wild-type controls. Furthermore, overexpression of ST2 dose-dependently attenuated BLP-induced NF-kappaB activation, suggesting a negative regulatory role of ST2 in TLR2 signaling. A moderate but significantly attenuated production of TNF-alpha and IL-6 on a second BLP stimulation was observed in BLP-pretreated, ST2-deficient macrophages, which is associated with substantially reduced IRAK-1 protein expression and downregulated TLR2-MyD88 and MyD88-IRAK immunocomplex formation. ST2-deficient mice, when pretreated with a nonlethal dose of BLP, benefitted from an improved survival against a subsequent lethal BLP challenge, indicating BLP tolerance develops in the absence of the ST2 receptor. Taken together, our results demonstrate that ST2 acts as a negative regulator of TLR2 signaling, but is not required for BLP-induced tolerance.

  17. Nfil3 is a glucocorticoid-regulated gene required for glucocorticoid-induced apoptosis in male murine T cells.

    Science.gov (United States)

    Carey, Kirstyn T; Tan, Kheng H; Ng, Judy; Liddicoat, Douglas R; Godfrey, Dale I; Cole, Timothy J

    2013-04-01

    Glucocorticoids (GCs) have essential roles in the regulation of development, integrated metabolism, and immune and neurological responses, and act primarily via the glucocorticoid receptor (GR). In most cells, GC treatment results in down-regulation of GR mRNA and protein levels via negative feedback mechanisms. However, in GC-treated thymocytes, GR protein levels are maintained at a high level, increasing sensitivity of thymocytes to GCs, resulting in apoptosis termed glucocorticoid-induced cell death (GICD). CD4(+)CD8(+) double-positive thymocytes and thymic natural killer T cells in particular are highly sensitive to GICD. Although GICD is exploited via the use of synthetic GC analogues in the treatment of hematopoietic malignancies, the intracellular molecular pathway of GICD is not well understood. To explore GICD in thymocytes, the authors performed whole genome expression microarray analysis in mouse GR exon 2 null vs wild-type thymus RNA 3 hours after dexamethasone treatment. Identified and validated direct GR targets included P21 and Bim, in addition to an important transcriptional regulator Nfil3, which previously has been associated with GICD and is essential for natural killer cell development in vivo. Immunostaining of NFIL3 in whole thymus localized NFIL3 primarily to the medullary region, and double labeling colocalized NFIL3 to apoptotic cells. In silico analysis revealed a putative GC response element 5 kb upstream of the Nfil3 promoter that is strongly conserved in the rat genome and was confirmed to bind GR by chromatin immunoprecipitation. The knockdown of Nfil3 mRNA levels to 20% of normal using specific small interfering RNAs abrogated GICD, indicating that NFIL3 is required for normal GICD in CTLL-2 T cells.

  18. Transcriptome Reprogramming by Plasmid-Encoded Transcriptional Regulators Is Required for Host Niche Adaption of a Macrophage Pathogen

    Science.gov (United States)

    Coulson, Garry B.; Miranda-CasoLuengo, Aleksandra A.; Miranda-CasoLuengo, Raúl; Wang, Xiaoguang; Oliver, Jenna; Willingham-Lane, Jennifer M.

    2015-01-01

    Rhodococcus equi is a facultative intracellular pathogen of macrophages, relying on the presence of a conjugative virulence plasmid harboring a 21-kb pathogenicity island (PAI) for growth in host macrophages. The PAI encodes a family of 6 virulence-associated proteins (Vaps) in addition to 20 other proteins. The contribution of these to virulence has remained unclear. We show that the presence of only 3 virulence plasmid genes (of 73 in total) is required and sufficient for intracellular growth. These include a single vap family member, vapA, and two PAI-located transcriptional regulators, virR and virS. Both transcriptional regulators are essential for wild-type-level expression of vapA, yet vapA expression alone is not sufficient to allow intracellular growth. A whole-genome microarray analysis revealed that VirR and VirS substantially integrate themselves into the chromosomal regulatory network, significantly altering the transcription of 18% of all chromosomal genes. This pathoadaptation involved significant enrichment of select gene ontologies, in particular, enrichment of genes involved in transport processes, energy production, and cellular metabolism, suggesting a major change in cell physiology allowing the bacterium to grow in the hostile environment of the host cell. The results suggest that following the acquisition of the virulence plasmid by an avirulent ancestor of R. equi, coevolution between the plasmid and the chromosome took place, allowing VirR and VirS to regulate the transcription of chromosomal genes in a process that ultimately promoted intracellular growth. Our findings suggest a mechanism for cooption of existing chromosomal traits during the evolution of a pathogenic bacterium from an avirulent saprophyte. PMID:26015480

  19. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

    Energy Technology Data Exchange (ETDEWEB)

    Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  20. Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity.

    Science.gov (United States)

    Schewe, Bettina; Blenau, Wolfgang; Walz, Bernd

    2012-04-15

    Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H(+)-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na(+)-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na(+)-dependent glutamate transporter; (2) the maintenance of resting pH(i) is Na(+), Cl(-), concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na(+) sensitive and requires V-ATPase activity; (4) the Na(+)/H(+) antiporter is not involved in pH(i) recovery after a NH(4)Cl prepulse; and (5) at least one Na(+)-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na(+)-dependent transporter maintain normal pH(i) values of pH 7.5. We have also detected the presence of a Na(+)-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.

  1. S1P-Yap1 signaling regulates endoderm formation required for cardiac precursor cell migration in zebrafish.

    Science.gov (United States)

    Fukui, Hajime; Terai, Kenta; Nakajima, Hiroyuki; Chiba, Ayano; Fukuhara, Shigetomo; Mochizuki, Naoki

    2014-10-13

    To form the primary heart tube in zebrafish, bilateral cardiac precursor cells (CPCs) migrate toward the midline beneath the endoderm. Mutants lacking endoderm and fish with defective sphingosine 1-phosphate (S1P) signaling exhibit cardia bifida. Endoderm defects lead to the lack of foothold for the CPCs, whereas the cause of cardia bifida in S1P signaling mutants remains unclear. Here we show that S1P signaling regulates CPC migration through Yes-associated protein 1 (Yap1)-dependent endoderm survival. Cardia bifida seen in spns2 (S1P transporter) morphants and s1pr2 (S1P receptor-2) morphants could be rescued by endodermal expression of nuclear localized form of yap1. yap1 morphants had decreased expression of the Yap1/Tead target connective tissue growth factor a (Ctgfa) and consequently increased endodermal cell apoptosis. Consistently, ctgfa morphants showed defects of the endodermal sheet and cardia bifida. Collectively, we show that S1pr2/Yap1-regulated ctgfa expression is essential for the proper endoderm formation required for CPC migration.

  2. Regulation of the New Arabidopsis Imprinted Gene AtBMI1C Requires the Interplay of Different Epigenetic Mechanisms

    Institute of Scientific and Technical Information of China (English)

    Fabian Bratzel; ChaoYang; Alexandra Ancelova; Gema López-Torrejón; Marcus Koch; Juan Carlos del Pozo; Myriam Calonje

    2012-01-01

    Recently,it has been shown that plants contain homologs to the animal Polycomb repressive complex 1 (PRC1)components BMI1 and RING1A/B.In Arabidopsis,there are three BMI1-like genes,two of which,AtBMI1A and B,are required during post-embryonic plant growth to repress embryonic traits and allow cell differentiation.However,little is known about the third BMI1-like gene,AtBMI1C.In this work,we show that AtBMI1C is only expressed during endosperm and stamen development.AtBMI1C is an imprinted gene expressed from the maternal allele in the endosperm but biallelically expressed in stamen.We found that the characteristic expression pattern of AtBMI1C is the result of a complex epigenetic regulation that involves CG DNA methylation,RNA-directed non-CG DNA methylation (RdDM),and PcG activity.Our results show the orchestrated interplay of different epigenetic mechanisms in regulating gene expression throughout development,shedding light on the current hypotheses for the origin and mechanism of imprinting in plant endosperm.

  3. Phytophthora capsici homologue of the cell cycle regulator SDA1 is required for sporangial morphology, mycelial growth and plant infection.

    Science.gov (United States)

    Zhu, Chunyuan; Yang, Xiaoyan; Lv, Rongfei; Li, Zhuang; Ding, Xiaomeng; Tyler, Brett M; Zhang, Xiuguo

    2016-04-01

    SDA1 encodes a highly conserved protein that is widely distributed in eukaryotic organisms. SDA1 is essential for cell cycle progression and organization of the actin cytoskeleton in yeasts and humans. In this study, we identified a Phytophthora capsici orthologue of yeast SDA1, named PcSDA1. In P. capsici, PcSDA1 is strongly expressed in three asexual developmental states (mycelium, sporangia and germinating cysts), as well as late in infection. Silencing or overexpression of PcSDA1 in P. capsici transformants affected the growth of hyphae and sporangiophores, sporangial development, cyst germination and zoospore release. Phalloidin staining confirmed that PcSDA1 is required for organization of the actin cytoskeleton. Moreover, 4',6-diamidino-2-phenylindole (DAPI) staining and PcSDA1-green fluorescent protein (GFP) fusions revealed that PcSDA1 is involved in the regulation of nuclear distribution in hyphae and sporangia. Both silenced and overexpression transformants showed severely diminished virulence. Thus, our results suggest that PcSDA1 plays a similar role in the regulation of the actin cytoskeleton and nuclear division in this filamentous organism as in non-filamentous yeasts and human cells.

  4. HDAC1 negatively regulates Bdnf and Pvalb required for parvalbumin interneuron maturation in an experience-dependent manner.

    Science.gov (United States)

    Koh, Dawn X P; Sng, Judy C G

    2016-11-01

    During early postnatal development, neuronal circuits are sculpted by sensory experience provided by the external environment. This experience-dependent regulation of circuitry development consolidates the balance of excitatory-inhibitory (E/I) neurons in the brain. The cortical barrel-column that innervates a single principal whisker is used to provide a clear reference frame for studying the consolidation of E/I circuitry. Sensory deprivation of S1 at birth disrupts the consolidation of excitatory-inhibitory balance by decreasing inhibitory transmission of parvalbumin interneurons. The molecular mechanisms underlying this decrease in inhibition are not completely understood. Our findings show that epigenetic mechanisms, in particular histone deacetylation by histone deacetylases, negatively regulate the expression of brain-derived neurotrophic factor (Bdnf) and parvalbumin (Pvalb) genes during development, which are required for the maturation of parvalbumin interneurons. After whisker deprivation, increased histone deacetylase 1 expression and activity led to increased histone deacetylase 1 binding and decreased histone acetylation at Bdnf promoters I-IV and Pvalb promoter, resulting in the repression of Bdnf and Pvalb gene transcription. The decrease in Bdnf expression further affected parvalbumin interneuron maturation at layer II/III in S1, demonstrated by decreased parvalbumin expression, a marker for parvalbumin interneuron maturation. Knockdown of HDAC1 recovered Bdnf and Pvalb gene transcription and also prevented the decrease of inhibitory synapses accompanying whisker deprivation.

  5. FgFlbD regulates hyphal differentiation required for sexual and asexual reproduction in the ascomycete fungus Fusarium graminearum.

    Science.gov (United States)

    Son, Hokyoung; Kim, Myung-Gu; Chae, Suhn-Kee; Lee, Yin-Won

    2014-11-01

    Fusarium graminearum is a filamentous fungal plant pathogen that infects major cereal crops. The fungus produces both sexual and asexual spores in order to endure unfavorable environmental conditions and increase their numbers and distribution across plants. In a model filamentous fungus, Aspergillus nidulans, early induction of conidiogenesis is orchestrated by the fluffy genes. The objectives of this study were to characterize fluffy gene homologs involved in conidiogenesis and their mechanism of action in F. graminearum. We characterized five fluffy gene homologs in F. graminearum and found that FlbD is the only conserved regulator for conidiogenesis in A. nidulans and F. graminearum. Deletion of fgflbD prevented hyphal differentiation and the formation of perithecia. Successful interspecies complementation using A. nidulans flbD demonstrated that the molecular mechanisms responsible for FlbD functions are conserved in F. graminearum. Moreover, abaA-wetA pathway is positively regulated by FgFlbD during conidiogenesis in F. graminearum. Deleting fgflbD abolished morphological effects of abaA overexpression, which suggests that additional factors for FgFlbD or an AbaA-independent pathway for conidiogenesis are required for F. graminearum conidiation. Importantly, this study led to the construction of a genetic pathway of F. graminearum conidiogenesis and provides new insights into the genetics of conidiogenesis in fungi.

  6. The bifunctional abiotic stress signalling regulator and endogenous RNA silencing suppressor FIERY1 is required for lateral root formation

    KAUST Repository

    Chen, Hao

    2010-09-28

    The Arabidopsis FIERY1 (FRY1) locus was originally identified as a negative regulator of stress-responsive gene expression and later shown to be required for suppression of RNA silencing. In this study we discovered that the FRY1 locus also regulates lateral root formation. Compared with the wild type, fry1 mutant seedlings generated significantly fewer lateral roots under normal growth conditions and also exhibited a dramatically reduced sensitivity to auxin in inducing lateral root initiation. Using transgenic plants that overexpress a yeast homolog of FRY1 that possesses only the 3\\', 5\\'-bisphosphate nucleotidase activity but not the inositol 1-phosphatase activity, we demonstrated that the lateral root phenotypes in fry1 result from loss of the nucleotidase activity. Furthermore, a T-DNA insertion mutant of another RNA silencing suppressor, XRN4 (but not XRN2 or XRN3), which is an exoribonuclease that is inhibited by the substrate of the FRY1 3\\', 5\\'-bisphosphate nucleotidase, exhibits similar lateral root defects. Although fry1 and xrn4 exhibited reduced sensitivity to ethylene, our experiments demonstrated that restoration of ethylene sensitivity in the fry1 mutant is not sufficient to rescue the lateral root phenotypes of fry1. Our results indicate that RNA silencing modulated by FRY1 and XRN4 plays an important role in shaping root architecture. © 2010 Blackwell Publishing Ltd.

  7. Greater-than-Class C low-level radioactive waste transportation regulations and requirements study. National Low-Level Waste Management Program

    Energy Technology Data Exchange (ETDEWEB)

    Tyacke, M.; Schmitt, R.

    1993-07-01

    The purpose of this report is to identify the regulations and requirements for transporting greater-than-Class C (GTCC) low-level radioactive waste (LLW) and to identify planning activities that need to be accomplished in preparation for transporting GTCC LLW. The regulations and requirements for transporting hazardous materials, of which GTCC LLW is included, are complex and include several Federal agencies, state and local governments, and Indian tribes. This report is divided into five sections and three appendices. Section 1 introduces the report. Section 2 identifies and discusses the transportation regulations and requirements. The regulations and requirements are divided into Federal, state, local government, and Indian tribes subsections. This report does not identify the regulations or requirements of specific state, local government, and Indian tribes, since the storage, treatment, and disposal facility locations and transportation routes have not been specifically identified. Section 3 identifies the planning needed to ensure that all transportation activities are in compliance with the regulations and requirements. It is divided into (a) transportation packaging; (b) transportation operations; (c) system safety and risk analysis, (d) route selection; (e) emergency preparedness and response; and (f) safeguards and security. This section does not provide actual planning since the details of the Department of Energy (DOE) GTCC LLW Program have not been finalized, e.g., waste characterization and quantity, storage, treatment and disposal facility locations, and acceptance criteria. Sections 4 and 5 provide conclusions and referenced documents, respectively.

  8. Regulation of carotenoid and bacteriochlorophyll biosynthesis genes and identification of an evolutionarily conserved gene required for bacteriochlorophyll accumulation.

    Science.gov (United States)

    Armstrong, G A; Cook, D N; Ma, D; Alberti, M; Burke, D H; Hearst, J E

    1993-05-01

    The temporal expression of ten clustered genes required for carotenoid (crt) and bacteriochlorophyll (bch) biosynthesis was examined during the transition from aerobic respiration to anaerobiosis requisite for the development of the photosynthetic membrane in the bacterium Rhodobacter capsulatus. Accumulation of crtA, crtC, crtD, crtE, crtF, crtK, bchC and bchD mRNAs increased transiently and coordinately, up to 12-fold following removal of oxygen from the growth medium, paralleling increases in mRNAs encoding pigment-binding polypeptides of the photosynthetic apparatus. The crtB and crtI genes, in contrast, were expressed similarly in the presence or absence of oxygen. The regulation patterns of promoters for the crtA and crtI genes and the bchCXYZ operon were characterized using lacZ transcriptional fusion and qualitatively reflected the corresponding mRNA accumulation patterns. We also report that the bchI gene product, encoded by a DNA sequence previously considered to be a portion of crtA, shares 49% sequence identity with the nuclear-encoded Arabidopsis thaliana Cs chloroplast protein required for normal pigmentation in plants.

  9. Up-regulation of insulin-like growth factor 2 by ketamine requires glycogen synthase kinase-3 inhibition.

    Science.gov (United States)

    Grieco, Steven F; Cheng, Yuyan; Eldar-Finkelman, Hagit; Jope, Richard S; Beurel, Eléonore

    2017-01-04

    An antidepressant dose of the rapidly-acting ketamine inhibits glycogen synthase kinase-3 (GSK3) in mouse hippocampus, and this inhibition is required for the antidepressant effect of ketamine in learned helplessness depression-like behavior. Here we report that treatment with an antidepressant dose of ketamine (10mg/kg) increased expression of insulin-like growth factor 2 (IGF2) in mouse hippocampus, an effect that required ketamine-induced inhibition of GSK3. Ketamine also inhibited hippocampal GSK3 and increased expression of hippocampal IGF2 in mice when administered after the induction of learned helplessness. Treatment with the specific GSK3 inhibitor L803-mts was sufficient to up-regulate hippocampal IGF2 expression. Administration of IGF2 siRNA reduced ketamine's antidepressant effect in the learned helplessness paradigm. Mice subjected to the learned helplessness paradigm were separated into two groups, those that were resilient (non-depressed) and those that were susceptible (depressed). Non-depressed resilient mice displayed higher expression of IGF2 than susceptible mice. These results indicate that IGF2 contributes to ketamine's antidepressant effect and that IGF2 may confer resilience to depression-like behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM

    Science.gov (United States)

    Georgiev, Alexander; Sullivan, David P.; Kersting, Michael C.; Dittman, Jeremy S.; Beh, Christopher T.; Menon, Anant K.

    2011-01-01

    Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP-dependent, non-vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologues of the mammalian oxysterol-binding protein (Osh1–7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM-derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔ osh4-1ts) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse-chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM-enriched fractions as well by extracting sterols from intact cells with methyl-β-cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh-deficient cells, the rate at which it moves within the PM to equilibrate with the methyl-β-cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in nonvesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM. PMID:21689253

  11. Half Pint/Puf68 is required for negative regulation of splicing by the SR factor Transformer2

    Science.gov (United States)

    Wang, Shanzhi; Wagner, Eric J; Mattox, William

    2013-01-01

    The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion. PMID:23880637

  12. Half pint/Puf68 is required for negative regulation of splicing by the SR splicing factor Transformer2.

    Science.gov (United States)

    Wang, Shanzhi; Wagner, Eric J; Mattox, William

    2013-08-01

    The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion.

  13. From the Accounting Treatment Required by the Accounting Regulations Corresponding with European Directives to the Accounting Treatment Required by IFRS - Legislative Steps Taken by Romanian Credit Institutions

    OpenAIRE

    Doroº Alexandra

    2013-01-01

    Legislative stages in accordance with IFRS accounting treatment began to OMF no. 907/2005- approving categories of entities applying accounting rules in accordance with International Financial Reporting Standards, accounting regulations that comply with European directives, as amended, continued with NBR Order no. 13/2008, NBR Order no. 15/2009, NBR Order no. 9/2010 and ends with NBR Order No. 27/16.12.2010 - for approval of Accounting Regulations in accordance with International Financial Re...

  14. CXCR3 Requirement for the Interleukin-13-Mediated Up-Regulation of Interleukin-13Rα2 in Pulmonary Fibroblasts.

    Science.gov (United States)

    Barnes, Jennifer C; Lumsden, Robert V; Worrell, Julie; Counihan, Ian P; O'Beirne, Sarah L; Belperio, John A; Fabre, Aurelie; Donnelly, Seamas C; Boylan, Denise; Kane, Rosemary; Keane, Michael P

    2015-08-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by fibrosis and abnormal vascularity. IL-13, a profibrotic cytokine that plays a role in IPF, functions through the Jak/STAT pathway after binding to the IL-13 receptor α1 (IL-13Rα1)/IL-4Rα complex. IL-13 also binds to IL-13Rα2, which has been thought to function as a nonsignaling decoy receptor, although possible signaling roles of this receptor have been proposed. CXCR3 and its IFN-inducible ligands-CXCL9, CXCL10, and CXCL11-have been implicated in vascular remodeling and fibroblast motility during the development of IPF. In this study, CXCR3 expression was demonstrated in cultured pulmonary fibroblasts from wild-type BALB/c mice and was found to be necessary for the IL-13-mediated gene and protein up-regulation of IL-13Rα2. In fibroblasts from CXCR3-deficient mice, STAT6 activation was prolonged. This study is the first to demonstrate the expression of CXCR3 in fibroblasts and its association with the expression of IL-13Rα2. Taken together, the results from this study point strongly to a requirement for CXCR3 for IL-13-mediated IL-13Rα2 gene expression. Understanding the function of CXCR3 in IL-13-mediated lung injury may lead to novel approaches to combat the development of pulmonary fibrosis, whether by limiting the effects of IL-13 or by manipulation of angiostatic pathways. The elucidation of the complex relationship between these antifibrotic receptors and manipulation of the CXCR3-mediated regulation of IL-13Rα2 may represent a novel therapeutic modality in cases of acute lung injury or chronic inflammation that may progress to fibrosis.

  15. Fyn requires HnRNPA2B1 and Sam68 to synergistically regulate apoptosis in pancreatic cancer.

    Science.gov (United States)

    Chen, Zhi-Yu; Cai, Lei; Zhu, Jin; Chen, Min; Chen, Jian; Li, Zhi-Hua; Liu, Xiang-De; Wang, Shu-Guang; Bie, Ping; Jiang, Peng; Dong, Jia-Hong; Li, Xiao-Wu

    2011-10-01

    The Src family kinase Fyn, heterogenous nuclear ribonucleoprotein (HnRNP) A2B1 and Sam68 are thought to be associated with the metastasis of tumors, but their roles in the regulation of apoptosis remain unclear. This study investigated the role of Fyn and its potential relationship with HnRNPA2B1 and Sam68 in the regulation of apoptosis in pancreatic cancer. Experimental design. We examined both the activity of Fyn and the expression of HnRNPA2B1 in human pancreatic cancer tissues and systematically investigated the apoptotic mechanisms induced by Fyn activity using multiple experimental approaches. We found that Fyn activity was increased in metastatic pancreatic cancer tissues. In the pancreatic cancer BxPc3 cell line, the inhibition of Fyn activity by kinase-dead Fyn downregulated HnRNPA2B1 expression. Further analysis showed that HnRNPA2B1 expression was associated with pancreatic cancer progression. In BxPc3 cells, HnRNPA2B1 bound to Bcl-x messenger RNA (mRNA), which affected splicing and therefore, the formation of Bcl-x(s). Downregulation of HnRNPA2B1 by RNA interference (RNAi) resulted in the increased formation of the pro-apoptotic Bcl-x(s) and promoted apoptosis of BxPc3 cells. In addition, deactivation of Fyn in BxPc3 cells reduced Sam68 phosphorylation. This resulted in increased binding between Sam68 and Bcl-x mRNA, promoting the formation of the anti-apoptotic Bcl-x(L). The knockdown of Sam68 by RNAi also increased the formation of Bcl-x(L). Finally, HnRNPA2B1 overexpression or Sam68 knockdown could rescue pancreatic cancer cells from apoptosis. Our results suggest a mechanism by which Fyn requires HnRNPA2B1 and Sam68 to coordinate and regulate apoptosis, thus promoting the proliferation and metastasis of pancreatic cancer.

  16. Zinc finger protein Loz1 is required for zinc-responsive regulation of gene expression in fission yeast

    Science.gov (United States)

    Corkins, Mark E.; May, Margot; Ehrensberger, Kate M.; Hu, Ya-Mei; Liu, Yi-Hsuan; Bloor, Sean D.; Jenkins, Blair; Runge, Kurt W.; Bird, Amanda J.

    2013-01-01

    In Schizosaccharomyces pombe, alcohol dehydrogenase 1 (Adh1) is an abundant zinc-requiring enzyme that catalyses the conversion of acetaldehyde to ethanol during fermentation. In a zinc-replete cell, adh1 is highly expressed. However, in zinc-limited cells, adh1 gene expression is repressed, and cells induce the expression of an alternative alcohol dehydrogenase encoded by the adh4 gene. In our studies examining this zinc-dependent switch in alcohol dehydrogenase gene expression, we isolated an adh1Δ strain containing a partial loss of function mutation that resulted in higher levels of adh4 transcripts in zinc-replete cells. This mutation also led to the aberrant expression of other genes that are typically regulated by zinc. Using linkage analysis, we have mapped the position of this mutation to a single gene called Loss Of Zinc sensing 1 (loz1). Loz1 is a 55-kDa protein that contains a double C2H2-type zinc finger domain. The mapped mutation that disrupts Loz1 function leads to an arginine to glycine substitution in the second zinc finger domain, suggesting that the double zinc finger domain is important for Loz1 function. We show that loz1Δ cells hyperaccumulate zinc and that Loz1 is required for gene repression in zinc-replete cells. We also have found that Loz1 negatively autoregulates its own expression. We propose that Loz1 is a unique metalloregulatory factor that plays a central role in zinc homeostasis in S. pombe. PMID:24003116

  17. Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

    Science.gov (United States)

    Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Sang-Hee; Kwon, Daeho; Shin, Ho-Joon

    2012-08-01

    Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

  18. International Regulations of Propolis Quality: Required Assays do not Necessarily Reflect their Polyphenolic-Related In Vitro Activities.

    Science.gov (United States)

    Bridi, Raquel; Montenegro, Gloria; Nuñez-Quijada, Gabriel; Giordano, Ady; Fernanda Morán-Romero, Maria; Jara-Pezoa, Isaac; Speisky, Hernán; Atala, Elias; López-Alarcón, Camilo

    2015-06-01

    Propolis has been proposed as a polyphenolic-rich natural product potentially able to be used for human consumption or even for medicinal proposes. To guarantee a minimum phenolic and flavonoid content and as consequence of their related-biological activities, international requirements of propolis quality are commonly applied. In this work we assessed phenolic and flavonoid contents of propolis; the antioxidant capacity (toward peroxyl radicals and hypochlorous acid); the ability to generate nitric oxide (NO); and, finally the antimicrobial activity of 6 propolis samples from the VI region of Chile. Our results show that the total phenolic and flavonoid content of propolis samples are not always in agreement with their polyphenolic-associated in vitro activities. For example, P03 and P06 samples showed the lowest (25 ± 4 GAE/g propolis) and the highest (105 ± 3 GAE/g propolis) total phenolic content, respectively. This was in agreement with flavonoid content and their Oxygen Radical Absorbance Capacity (ORAC) activity. However, this dependence was not observed toward HOCl, NO release and antimicrobial activity. Based on our results, we consider that, in order to guarantee the antioxidant or antimicrobial in vitro effects, the international regulations of propolis quality should contemplate the convenience of incorporating other simple analytical test such as ORAC or antimicrobial tests. © 2015 Institute of Food Technologists®

  19. The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation1[OPEN

    Science.gov (United States)

    Cheng, Jinkui; Lai, Jinsheng; Gong, Zhizhong

    2016-01-01

    DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α. The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression. PMID:27208288

  20. CD81 is a central regulator of cellular events required for hepatitis C virus infection of human hepatocytes.

    Science.gov (United States)

    Brazzoli, Michela; Bianchi, Alessia; Filippini, Sara; Weiner, Amy; Zhu, Qing; Pizza, Mariagrazia; Crotta, Stefania

    2008-09-01

    Infection with hepatitis C virus (HCV) is still a major public health problem, and the events leading to hepatocyte infection are not yet fully understood. Combining confocal microscopy with biochemical analysis and studies of infection requirements using pharmacological inhibitors and small interfering RNAs, we show here that engagement of CD81 activates the Rho GTPase family members Rac, Rho, and Cdc42 and that the block of these signaling pathways drastically reduces HCV infectivity. Activation of Rho GTPases mediates actin-dependent relocalization of the HCV E2/CD81 complex to cell-cell contact areas where CD81 comes into contact with the tight-junction proteins occludin, ZO-1, and claudin-1, which was recently described as an HCV coreceptor. Finally, we show that CD81 engagement activates the Raf/MEK/ERK signaling cascade and that this pathway affects postentry events of the virus life cycle. In conclusion, we describe a range of cellular events that are manipulated by HCV to coordinate interactions with its multiple coreceptors and to establish productive infections and find that CD81 is a central regulator of these events.

  1. Surface water mineralization of isopyrazam according to OECD 309: observations on implementation of the new data requirement within agrochemical regulation.

    Science.gov (United States)

    Hand, Laurence H; Moreland, Harriet J

    2014-03-01

    A surface water mineralization study (according to the Organisation for Economic Co-operation and Development [OECD] guideline OECD 309) is a new requirement in European Union agrochemical regulations; therefore, industry has little experience with this test. The guideline allows for a number of options within the test design, notably the options to conduct the study under diffuse light and to include an inoculum of suspended sediment. The present study was designed to investigate the potential impact of these options on the degradation rate of a representative compound. The fungicide, isopyrazam, was chosen as it was previously shown to be susceptible to metabolism by phototrophic organisms under a fluorescent light-dark cycle. The impact of diffuse light was investigated at light intensities representative of those at depth in large, open water bodies (test compound [DT50] < 50 d), whereas degradation in continuous darkness was negligible. Furthermore, investigation at 2 different light intensities resulted in similar degradation rates, indicating that this transformation mechanism was not proportional to light intensity, provided that there was sufficient light for photosynthesis to occur. Inclusion of suspended sediment did not have a significant impact on the degradation rate of isopyrazam, except at extremely high sediment concentrations, which were not considered representative of conditions in large, open water bodies.

  2. Guide to NRC reporting and recordkeeping requirements. Compiled from requirements in Title 10 of the U.S. Code of Federal Regulations as codified on December 31, 1993; Revision 1

    Energy Technology Data Exchange (ETDEWEB)

    Collins, M.; Shelton, B.

    1994-07-01

    This compilation includes in the first two sections the reporting and recordkeeping requirements applicable to US Nuclear Regulatory Commission (NRC) licensees and applicants and to members of the public. It includes those requirements codified in Title 10 of the code of Federal Regulations, Chapter 1, on December 31, 1993. It also includes, in a separate section, any of those requirements that were superseded or discontinued between January 1992 and December 1993. Finally, the appendix lists mailing and delivery addresses for NRC Headquarters and Regional Offices mentioned in the compilation. The Office of Information Resources Management staff compiled this listing of reporting and recordkeeping requirements to briefly describe each in a single document primarily to help licensees readily identify the requirements. The compilation is not a substitute for the regulations, and is not intended to impose any new requirements or technical positions. It is part of NRC`s continuing efforts to comply with the Paperwork Reduction Act of 1980 and the Office of Management and Budget regulations that mandate effective and efficient Federal information resources management programs.

  3. Nuclear localization of the transcriptional regulator MIER1α requires interaction with HDAC1/2 in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Shengnan Li

    Full Text Available MIER1α is a transcriptional regulator that functions in gene repression through its ability to interact with various chromatin modifiers and transcription factors. We have also shown that MIER1α interacts with ERα and inhibits estrogen-stimulated growth. While MIER1α is localized in the nucleus of MCF7 cells, previous studies have shown that it does not contain a nuclear localization signal. In this report, we investigate the mechanism involved in transporting MIER1α into the nucleus. We explored the possibility that MIER1α is transported into the nucleus through a 'piggyback' mechanism. One obvious choice is via interaction with ERα, however we demonstrate that nuclear targeting of MIER1α does not require ERα. Knockdown of ERα reduced protein expression to 22% of control, but did not alter the percentage of cells with nuclear MIER1α (98% nuclear with scrambled shRNA vs. 95% with ERα shRNA. Further evidence was obtained using two stable transfectants derived from the ER-negative MDA231 cell line: MC2 (ERα+ and VC5 (ERα-. Confocal analysis showed no difference in MIER1α localization (86% nuclear in MC2 vs. 89% in VC5. These data demonstrate that ERα is not involved in nuclear localization of MIER1α. To identify the critical MIER1α sequence, we performed a deletion analysis and determined that the ELM2 domain was necessary and sufficient for nuclear localization. This domain binds HDAC1 & 2, therefore we investigated their role. Confocal analysis of an MIER1α containing an ELM2 point mutation previously shown to abolish HDAC binding revealed that this mutation results in almost complete loss of nuclear targeting: 10% nuclear vs. 97% with WT-MIER1α. Moreover, double knockdown of HDAC1 and 2 caused a reduction in percent nuclear from 86% to 44%. The results of this study demonstrate that nuclear targeting of MIER1α requires an intact ELM2 domain and is dependent on interaction with HDAC1/2.

  4. RCN1-regulated phosphatase activity and EIN2 modulate hypocotyl gravitropism by a mechanism that does not require ethylene signaling.

    Science.gov (United States)

    Muday, Gloria K; Brady, Shari R; Argueso, Cristiana; Deruère, Jean; Kieber, Joseph J; DeLong, Alison

    2006-08-01

    The roots curl in naphthylphthalamic acid1 (rcn1) mutant of Arabidopsis (Arabidopsis thaliana) has altered auxin transport, gravitropism, and ethylene response, providing an opportunity to analyze the interplay between ethylene and auxin in control of seedling growth. Roots of rcn1 seedlings were previously shown to have altered auxin transport, growth, and gravitropism, while rcn1 hypocotyl elongation exhibited enhanced ethylene response. We have characterized auxin transport and gravitropism phenotypes of rcn1 hypocotyls and have explored the roles of auxin and ethylene in controlling these phenotypes. As in roots, auxin transport is increased in etiolated rcn1 hypocotyls. Hypocotyl gravity response is accelerated, although overall elongation is reduced, in etiolated rcn1 hypocotyls. Etiolated, but not light grown, rcn1 seedlings also overproduce ethylene, and mutations conferring ethylene insensitivity restore normal hypocotyl elongation to rcn1. Auxin transport is unaffected by treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid in etiolated hypocotyls of wild-type and rcn1 seedlings. Surprisingly, the ethylene insensitive2-1 (ein2-1) and ein2-5 mutations dramatically reduce gravitropic bending in hypocotyls. However, the ethylene resistant1-3 (etr1-3) mutation does not significantly affect hypocotyl gravity response. Furthermore, neither the etr1 nor the ein2 mutation abrogates the accelerated gravitropism observed in rcn1 hypocotyls, indicating that both wild-type gravity response and enhanced gravity response in rcn1 do not require an intact ethylene-signaling pathway. We therefore conclude that the RCN1 protein affects overall hypocotyl elongation via negative regulation of ethylene synthesis in etiolated seedlings, and that RCN1 and EIN2 modulate hypocotyl gravitropism and ethylene responses through independent pathways.

  5. Sterol regulation of human fatty acid synthase promoter I requires nuclear factor-Y- and Sp-1-binding sites.

    Science.gov (United States)

    Xiong, S; Chirala, S S; Wakil, S J

    2000-04-11

    To understand cholesterol-mediated regulation of human fatty acid synthase promoter I, we tested various 5'-deletion constructs of promoter I-luciferase reporter gene constructs in HepG2 cells. The reporter gene constructs that contained only the Sp-1-binding site (nucleotides -82 to -74) and the two tandem sterol regulatory elements (SREs; nucleotides -63 to -46) did not respond to cholesterol. Only the reporter gene constructs containing a nuclear factor-Y (NF-Y) sequence, the CCAAT sequence (nucleotides -90 to -86), an Sp-1 sequence, and the two tandem SREs responded to cholesterol. The NF-Y-binding site, therefore, is essential for cholesterol response. Mutating the SREs or the NF-Y site and inserting 4 bp between the Sp-1- and NF-Y-binding sites both resulted in a minimal cholesterol response of the reporter genes. Electrophoretic mobility-shift assays using anti-SRE-binding protein (SREBP) and anti-NF-Ya antibodies confirmed that these SREs and the NF-Y site bind the respective factors. We also identified a second Sp-1 site located between nucleotides -40 and -30 that can substitute for the mutated Sp-1 site located between nucleotides -82 and -74. The reporter gene expression of the wild-type promoter and the Sp-1 site (nucleotides -82 to -74) mutant promoter was similar when SREBP1a [the N-terminal domain of SREBP (amino acids 1-520)] was constitutively overexpressed, suggesting that Sp-1 recruits SREBP to the SREs. Under the same conditions, an NF-Y site mutation resulted in significant loss of reporter gene expression, suggesting that NF-Y is required to activate the cholesterol response.

  6. Cellular zinc is required for intestinal epithelial barrier maintenance via the regulation of claudin-3 and occludin expression.

    Science.gov (United States)

    Miyoshi, Yuka; Tanabe, Soichi; Suzuki, Takuya

    2016-07-01

    Intracellular zinc is required for a variety of cell functions, but its precise roles in the maintenance of the intestinal tight junction (TJ) barrier remain unclear. The present study investigated the essential roles of intracellular zinc in the preservation of intestinal TJ integrity and the underlying molecular mechanisms. Depletion of intracellular zinc in both intestinal Caco-2 cells and mouse colons through the application of a cell-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) induced a disruption of the TJ barrier, as indicated by increased FITC-labeled dextran flux and decreased transepithelial electrical resistance. The TPEN-induced TJ disruption is associated with downregulation of two TJ proteins, occludin and claudin-3. Biotinylation of cell surface proteins revealed that the zinc depletion induced the proteolysis of occludin but not claudin-3. Occludin proteolysis was sensitive to the inhibition of calpain activity, and increased calpain activity was observed in the zinc-depleted cells. Although quantitative PCR analysis and promoter reporter assay have demonstrated that the zinc depletion-induced claudin-3 downregulation occurred at transcriptional levels, a site-directed mutation in the egr1 binding site in the claudin-3 promoter sequence induced loss of both the basal promoter activity and the TPEN-induced decreases. Reduced egr1 expression by a specific siRNA also inhibited claudin-3 expression and transepithelial electrical resistance maintenance in cells. This study shows that intracellular zinc has an essential role in the maintenance of the intestinal epithelial TJ barrier through regulation of occludin proteolysis and claudin-3 transcription.

  7. Endothelial-derived tissue factor pathway inhibitor regulates arterial thrombosis but is not required for development or hemostasis.

    NARCIS (Netherlands)

    White, T.A.; Johnson, T.; Zarzhevsky, N.; Tom, C.; Delacroix, S.; Holroyd, E.W.; Maroney, S.A.; Singh, R.; Pan, S.; Fay, W.P.; Deursen, J.M.A. van; Mast, A.E.; Sandhu, G.S.; Simari, R.D.

    2010-01-01

    The antithrombotic surface of endothelium is regulated in a coordinated manner. Tissue factor pathway inhibitor (TFPI) localized at the endothelial cell surface regulates the production of FXa by inhibiting the TF/VIIa complex. Systemic homozygotic deletion of the first Kunitz (K1) domain of TFPI

  8. The chemotaxis regulator pilG of Xylella fastidiosa is required for virulence in Vitis vinifera grapevines

    Science.gov (United States)

    Xylella fastidiosa is a Gram-negative, xylem-limited pathogenic bacterium that causes Pierce’s disease of grapevines. Type IV pili of X. fastidiosa are regulated by pilG, a chemotaxis regulator in Pil-Chp operon involving signal transduction pathways. To elucidate the role of pilG in twitching motil...

  9. ArgR and AhrC are both required for regulation of arginine metabolism in Lactococcus lactis

    NARCIS (Netherlands)

    Larsen, R; Buist, G; Kuipers, OP; Kok, J

    2004-01-01

    The DNA binding proteins ArgR and AhrC are essential for regulation of arginine metabolism in Escherichia Coli and Bacillus subtilis, respectively. A unique property of these regulators is that they form hexameric protein complexes, mediating repression of arginine biosynthetic pathways as well as a

  10. The chemotaxis regulator pilG of Xylella fastidiosa is required for virulence in Vitis vinifera grapevines

    Science.gov (United States)

    Type IV pili of X. fastidiosa are regulated by pilG, a response regulator protein putatively involved in chemotaxis-like operon sensing stimuli through signal transduction pathways. To elucidate roles of pilG in pathogenicity of X. fastidiosa, the pilG-deletion mutant and complementary strain contai...

  11. Conforming STOP Violence Against Women Formula Grant Program Regulations to Statutory Change; Definitions and Confidentiality Requirements Applicable to All OVW Grant Programs. Final rule.

    Science.gov (United States)

    2016-11-29

    This rule amends the regulations for the STOP (ServicesTrainingOfficersProsecutors) Violence Against Women Formula Grant Program (STOP Program) and the general provisions governing Office on Violence Against Women (OVW) programs to comply with statutory changes and reduce repetition of statutory language. Also, this rule implements statutory requirements for nondisclosure of confidential or private information relating to all OVW grant programs.

  12. VirS, an OmpR/PhoB subfamily response regulator, is required for activation of vapA gene expression in Rhodococcus equi.

    Science.gov (United States)

    Kakuda, Tsutomu; Hirota, Takuya; Takeuchi, Tatsuya; Hagiuda, Hirofumi; Miyazaki, Shiko; Takai, Shinji

    2014-10-03

    Rhodococcus equi is an important pulmonary pathogen in foals and in immunocompromised individuals. Virulent R. equi strains carry an 80-90 kb virulence plasmid that expresses the virulence-associated protein A (VapA). VapA expression is regulated by temperature and pH. The LysR-type transcriptional regulator, VirR, is involved in the regulation of the vapA gene. To examine the mechanism underlying transcriptional regulation of vapA, we characterized an R. equi mutant in which another putative transcriptional regulator encoded on the virulence plasmid, VirS, was deleted. Deletion of virS reduced vapA promoter activity to non-inducible levels. Complementary expression of VirS in the virS deletion mutant restored transcription at the PvapA promoter, even under non-inducing conditions (30°C and pH 8.0). In addition, VirS expression increased PvapA promoter activity in the absence of functional VirR. Further, transcription of the icgA operon containing virS was regulated by pH and temperature in the same manner as vapA. This study suggests that VirS is required for VapA expression and that regulation of PvapA-promoter activity may be achieved by controlling VirS expression levels.

  13. Requirement of regulated endocrine-specific protein-18 for development and expression of regulated endocrine-specific protein-18 isoform c in mice.

    Science.gov (United States)

    Liang, Min; Yang, Jian Ling; Bian, Min Juan; Liu, Jie; Hong, Xiao Qi; Wang, Yan Cong; Huang, Yu Fang; Gu, Shu Ping; Yu, Mei; Huang, Fang; Fei, Jian

    2011-04-01

    Regulated endocrine-specific protein-18 (RESP18) is distributed mainly in the peripheral endocrine and neuroendocrine tissues. The expression of RESP18 protein is regulated by physiological factors, such as blood glucose or dopaminergic drugs, but its functions remain unclear. In this study, to explore the biological functions of RESP18 in vivo, we generated RESP18 heterozygous deficient mice, and further found RESP18 was essential for embryonic development. In addition, we cloned a new isoform of mouse RESP18 by reverse transcription-polymerase chain reaction (RT-PCR), and denominated it as RESP18-c. Mouse RESP18-c, by skipping exon4 (43 bp in length), encodes a shorter protein of 120 amino acid residues. The distribution of RESP18-c mRNA is similar with that of RESP18 mRNA in the peripheral tissues and brains of mice.

  14. Growth Arrest Specific 2 Is Up-Regulated in Chronic Myeloid Leukemia Cells and Required for Their Growth

    OpenAIRE

    Haixia Zhou; Yue Ge; Lili Sun; Wenjuan Ma; Jie Wu; Xiuyan Zhang; Xiaohui Hu; Eaves, Connie J; Depei Wu; Yun Zhao

    2014-01-01

    Although the generation of BCR-ABL is the molecular hallmark of chronic myeloid leukemia (CML), the comprehensive molecular mechanisms of the disease remain unclear yet. Growth arrest specific 2 (GAS2) regulates multiple cellular functions including cell cycle, apoptosis and calpain activities. In the present study, we found GAS2 was up-regulated in CML cells including CD34+ progenitor cells compared to their normal counterparts. We utilized RNAi and the expression of dominant negative form o...

  15. Balancing spatially regulated β-actin translation and dynamin-mediated endocytosis is required to assemble functional epithelial monolayers.

    Science.gov (United States)

    Cruz, Lissette A; Vedula, Pavan; Gutierrez, Natasha; Shah, Neel; Rodriguez, Steven; Ayee, Brian; Davis, Justin; Rodriguez, Alexis J

    2015-12-01

    Regulating adherens junction complex assembly/disassembly is critical to maintaining epithelial homeostasis in healthy epithelial tissues. Consequently, adherens junction structure and function is often perturbed in clinically advanced tumors of epithelial origin. Some of the most studied factors driving adherens junction complex perturbation in epithelial cancers are transcriptional and epigenetic down-regulation of E-cadherin expression. However, numerous reports demonstrate that post-translational regulatory mechanisms such as endocytosis also regulate early phases of epithelial-mesenchymal transition and metastatic progression. In already assembled healthy epithelia, E-cadherin endocytosis recycles cadherin-catenin complexes to regulate the number of mature adherens junctions found at cell-cell contact sites. However, following de novo epithelial cell-cell contact, endocytosis negatively regulates adherens junction assembly by removing E-cadherin from the cell surface. By contrast, following de novo epithelial cell-cell contact, spatially localized β-actin translation drives cytoskeletal remodeling and consequently E-cadherin clustering at cell-cell contact sites and therefore positively regulates adherens junction assembly. In this report we demonstrate that dynamin-mediated endocytosis and β-actin translation-dependent cadherin-catenin complex anchoring oppose each other following epithelial cell-cell contact. Consequently, the final extent of adherens junction assembly depends on which of these processes is dominant following epithelial cell-cell contact. We expressed β-actin transcripts impaired in their ability to properly localize monomer synthesis (Δ3'UTR) in MDCK cells to perturb actin filament remodeling and anchoring, and demonstrate the resulting defect in adherens junction structure and function is rescued by inhibiting dynamin mediated endocytosis. Therefore, we demonstrate balancing spatially regulated β-actin translation and dynamin

  16. Adjustment of Energy requirements in TEK; TEK= Technical Regulations under the Norwegian Planning and Building Act; Justering av energikrav i TEK

    Energy Technology Data Exchange (ETDEWEB)

    Thyholt, Marit; Dokka, Tor Helge; Schild, Peter; Grini, Catherine; Mysen, Mads; Sartori, Igor

    2008-07-01

    The National Office of Building Technology and Administration (BE) desired to review the consequences of different levels of ambition for requirements for heat gaining from vent air, as well as a possible requirement for energy efficient design of building fronts. In addition the energy scope in the regulation (TEK2007) should be adjusted according to the final establishment of a new calculation standard (Norwegian Standard - NS 3031:2007). A statement on these subjects has been carried out at SINTEF Byggforsk, and is described in this report. Adjustments of framework regulations.There are only minor differences between adjusted calculations according to NS 3031 and the original energy framework calculations, i.e. the difference for net energy need amounts to the size of 0 to 6 percent. Heat gain.The report shows that it is possible - both from techical and financial considerations - to increase the requirement level for heat gain from vent air for most categories of buildings. This implies a sharpening of the annual median temperature efficiency from 70 % to 80 %, for all building categories, except from hospitals, institutions and light industry/workshops. A possible sharpening of regulations for heat gain in houses has not been evaluated. Depending on building category a sharpening of regulations for heat gain from vent air will imply that net energy need will be reduced on a scale of 20 to 30 kWh/m2 per annum. The report demonstrates that despite a possible sharpening of the requirements on energy efficiency for heat recovery devices does not prevent the use of large areas of windows and window panes. Vulnerability analyses show that deviations from the prerequisites in the basis for the energy framework concerning air quantities and air temperatures give the possibility of weakening the building's heating characteristics. Building fronts. Different methods for added requirements for building fronts have been examined. The aim has been to find methods and

  17. Clavibacter michiganensis subsp. michiganensis Vatr1 and Vatr2 transcriptional regulators are required for virulence in tomato.

    Science.gov (United States)

    Savidor, Alon; Chalupowicz, Laura; Teper, Doron; Gartemann, Karl-Heinz; Eichenlaub, Rudolf; Manulis-Sasson, Shulamit; Barash, Isaac; Sessa, Guido

    2014-10-01

    The plant pathogen Clavibacter michiganensis subsp. michiganensis is a gram-positive bacterium responsible for wilt and canker disease of tomato. Although disease development is well characterized and diagnosed, molecular mechanisms of C. michiganensis subsp. michiganensis virulence are poorly understood. Here, we identified and characterized two C. michiganensis subsp. michiganensis transcriptional regulators, Vatr1 and Vatr2, that are involved in pathogenicity of C. michiganensis subsp. michiganensis. Vatr1 and Vatr2 belong to TetR and MocR families of transcriptional regulators, respectively. Mutations in their corresponding genes caused attenuated virulence, with the Δvatr2 mutant showing a more dramatic effect than Δvatr1. Although both mutants grew well in vitro and reached a high titer in planta, they caused reduced wilting and canker development in infected plants compared with the wild-type bacterium. They also led to a reduced expression of the ethylene-synthesizing tomato enzyme ACC-oxidase compared with wild-type C. michiganensis subsp. michiganensis and to reduced ethylene production in the plant. Transcriptomic analysis of wild-type C. michiganensis subsp. michiganensis and the two mutants under infection-mimicking conditions revealed that Vatr1 and Vatr2 regulate expression of virulence factors, membrane and secreted proteins, and signal-transducing proteins. A 70% overlap between the sets of genes positively regulated by Vatr1 and Vatr2 suggests that these transcriptional regulators are on the same molecular pathway responsible for C. michiganensis subsp. michiganensis virulence.

  18. JMJD-5/KDM8 regulates H3K36me2 and is required for late steps of homologous recombination and genome integrity

    DEFF Research Database (Denmark)

    Amendola, Pier Giorgio; Zaghet, Nico; Ramalho, João J

    2017-01-01

    The eukaryotic genome is organized in a three-dimensional structure called chromatin, constituted by DNA and associated proteins, the majority of which are histones. Post-translational modifications of histone proteins greatly influence chromatin structure and regulate many DNA-based biological...... processes. Methylation of lysine 36 of histone 3 (H3K36) is a post-translational modification functionally relevant during early steps of DNA damage repair. Here, we show that the JMJD-5 regulates H3K36 di-methylation and it is required at late stages of double strand break repair mediated by homologous...

  19. Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

    Science.gov (United States)

    Cui, Cheng; Chatterjee, Bishwanath; Lozito, Thomas P; Zhang, Zhen; Francis, Richard J; Yagi, Hisato; Swanhart, Lisa M; Sanker, Subramaniam; Francis, Deanne; Yu, Qing; San Agustin, Jovenal T; Puligilla, Chandrakala; Chatterjee, Tania; Tansey, Terry; Liu, Xiaoqin; Kelley, Matthew W; Spiliotis, Elias T; Kwiatkowski, Adam V; Tuan, Rocky; Pazour, Gregory J; Hukriede, Neil A; Lo, Cecilia W

    2013-11-01

    Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to

  20. Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Cheng Cui

    2013-11-01

    Full Text Available Planar cell polarity (PCP regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin

  1. BolA Is Required for the Accurate Regulation of c-di-GMP, a Central Player in Biofilm Formation.

    Science.gov (United States)

    Moreira, Ricardo N; Dressaire, Clémentine; Barahona, Susana; Galego, Lisete; Kaever, Volkhard; Jenal, Urs; Arraiano, Cecília M

    2017-09-19

    The bacterial second messenger cyclic dimeric GMP (c-di-GMP) is a nearly ubiquitous intracellular signaling molecule involved in the transition from the motile to the sessile/biofilm state in bacteria. C-di-GMP regulates various cellular processes, including biofilm formation, motility, and virulence. BolA is a transcription factor that promotes survival in different stresses and is also involved in biofilm formation. Both BolA and c-di-GMP participate in the regulation of motility mechanisms leading to similar phenotypes. Here, we establish the importance of the balance between these two factors for accurate regulation of the transition between the planktonic and sessile lifestyles. This balance is achieved by negative-feedback regulation of BolA and c-di-GMP. BolA not only contributes directly to the motility of bacteria but also regulates the expression of diguanylate cyclases and phosphodiesterases. This expression modulation influences the synthesis and degradation of c-di-GMP, while this signaling metabolite has a negative influence in bolA mRNA transcription. Finally, we present evidence of the dominant role of BolA in biofilm, showing that, even in the presence of elevated c-di-GMP levels, biofilm formation is reduced in the absence of BolA. C-di-GMP is one of the most important bacterial second messengers involved in several cellular processes, including virulence, cell cycle regulation, biofilm formation, and flagellar synthesis. In this study, we unravelled a direct connection between the bolA morphogene and the c-di-GMP signaling molecule. We show the important cross-talk that occurs between these two molecular regulators during the transition between the motile/planktonic and adhesive/sessile lifestyles in Escherichia coli This work provides important clues that can be helpful in the development of new strategies, and the results can be applied to other organisms with relevance for human health.IMPORTANCE Bacterial cells have evolved several mechanisms

  2. Regulation of Telomere Length Requires a Conserved N-Terminal Domain of Rif2 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kaizer, Hannah; Connelly, Carla J; Bettridge, Kelsey; Viggiani, Christopher; Greider, Carol W

    2015-10-01

    The regulation of telomere length equilibrium is essential for cell growth and survival since critically short telomeres signal DNA damage and cell cycle arrest. While the broad principles of length regulation are well established, the molecular mechanism of how these steps occur is not fully understood. We mutagenized the RIF2 gene in Saccharomyces cerevisiae to understand how this protein blocks excess telomere elongation. We identified an N-terminal domain in Rif2 that is essential for length regulation, which we have termed BAT domain for Blocks Addition of Telomeres. Tethering this BAT domain to Rap1 blocked telomere elongation not only in rif2Δ mutants but also in rif1Δ and rap1C-terminal deletion mutants. Mutation of a single amino acid in the BAT domain, phenylalanine at position 8 to alanine, recapitulated the rif2Δ mutant phenotype. Substitution of F8 with tryptophan mimicked the wild-type phenylalanine, suggesting the aromatic amino acid represents a protein interaction site that is essential for telomere length regulation.

  3. Three fim genes required for the regulation of length and mediation of adhesion of Escherichia coli type 1 fimbriae

    DEFF Research Database (Denmark)

    Klemm, P; Christiansen, Gunna

    1987-01-01

    Three novel fim genes of Escherichia coli, fimF, fimG and fimH, were characterized. These genes were not necessary for the production of fimbriae but were shown to be involved in the adhesive property and longitudinal regulation of these structures. Complementation experiments indicated that both...

  4. 77 FR 33635 - Amendment to the Bank Secrecy Act Regulations-Requirement That Clerks of Court Report Certain...

    Science.gov (United States)

    2012-06-07

    ... Revenue Code of 1986'' file reports with FinCEN in the time and manner prescribed by regulation. Section... because of the applicable restrictions on disclosure in the U.S. tax code. During calendar year 2010... statement under section 202. List of Subjects in 31 CFR Part 1010 Administrative practice and procedure...

  5. The H-loop in the Second Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator is Required for Efficient Chloride Channel Closing

    OpenAIRE

    Kloch, Monika; Milewski, Michał; Nurowska, Ewa; Dworakowska, Beata; Cutting, Garry R.; Dołowy, Krzysztof

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine res...

  6. Binding of a 100-kDa ubiquitous factor to the human prolactin promoter is required for its basal and hormone-regulated activity.

    Science.gov (United States)

    Peers, B; Nalda, A M; Monget, P; Voz, M L; Belayew, A; Martial, J A

    1992-11-15

    cAMP strongly stimulates the activity of the human prolactin (hPRL) promoter. We have previously shown that two types of cis-element are required for this cAMP regulation; binding sites for the pituitary-specific factor Pit-1, and the sequence spanning nucleotides -115 to -85 (named sequence A). Sequence A contains the TGACG motif found in the consensus sequence of the cAMP-responsive element (CRE). In this study, we show that a mutation in the TGACG motif of sequence A strongly reduces not only the cAMP regulation but also the Ca2+ regulation and basal activity of the hPRL promoter. Furthermore, gel-shift assays indicate that the mutation prevents binding of a ubiquitous factor which is not the CRE-binding protein. Southwestern experiments suggest that this ubiquitous factor's molecular mass is approximately 100 kDa. We conclude that binding of a 100-kDa ubiquitous factor to sequence A is required for full basal and hormonal regulation of hPRL-promoter activity.

  7. Pyrrolizidine alkaloids (PAs) in honey and pollen-legal regulation of PA levels in food and animal feed required.

    Science.gov (United States)

    Kempf, Michael; Reinhard, Annika; Beuerle, Till

    2010-01-01

    Pyrrolizidine alkaloids (PAs) are secondary plant constituents that comprise about 400 different structures and occur in two major forms, a tertiary form and the corresponding N-oxide. PAs containing a 1,2-double bond are pre-toxins and metabolically activated by the action of hepatic P-450 enzymes to toxic pyrroles. Besides the acute toxic effects, the genotoxic and tumorigenicity potential of PAs was demonstrated in some eukaryotic model systems. Recently, the potential PA contamination of food and feeding stuff attracted recurrent great deals of attention. Humans are exposed to these toxins by consumption of herbal medicine, herbal teas, dietary supplements or food containing PA plant material. In numerous studies the potential threat to human health by PAs is stated. In pharmaceuticals, the use of these plants is regulated. Considering the PA concentrations observed especially in authentic honey from PA producing plants and pollen products, the results provoke an international regulation of PAs in food.

  8. RTN3 Regulates the Expression Level of Chemokine Receptor CXCR4 and is Required for Migration of Primordial Germ Cells

    Directory of Open Access Journals (Sweden)

    Haitao Li

    2016-04-01

    Full Text Available CXCR4 is a crucial chemokine receptor that plays key roles in primordial germ cell (PGC homing. To further characterize the CXCR4-mediated migration of PGCs, we screened CXCR4-interacting proteins using yeast two-hybrid screening. We identified reticulon3 (RTN3, a member of the reticulon family, and considered an apoptotic signal transducer, as able to interact directly with CXCR4. Furthermore, we discovered that the mRNA and protein expression levels of CXCR4 could be regulated by RTN3. We also found that RTN3 altered CXCR4 translocation and localization. Moreover, increasing the signaling of either CXCR4b or RTN3 produced similar PGC mislocalization phenotypes in zebrafish. These results suggested that RTN3 modulates PGC migration through interaction with, and regulation of, CXCR4.

  9. Arabidopsis thaliana VOZ (Vascular plant One-Zinc finger) transcription factors are required for proper regulation of flowering time.

    Science.gov (United States)

    Celesnik, Helena; Ali, Gul S; Robison, Faith M; Reddy, Anireddy S N

    2013-04-15

    Transition to flowering in plants is tightly controlled by environmental cues, which regulate the photoperiod and vernalization pathways, and endogenous signals, which mediate the autonomous and gibberellin pathways. In this work, we investigated the role of two Zn(2+)-finger transcription factors, the paralogues AtVOZ1 and AtVOZ2, in Arabidopsis thaliana flowering. Single atvoz1-1 and atvoz2-1 mutants showed no significant phenotypes as compared to wild type. However, atvoz1-1 atvoz2-1 double mutant plants exhibited several phenotypes characteristic of flowering-time mutants. The double mutant displayed a severe delay in flowering, together with additional pleiotropic phenotypes. Late flowering correlated with elevated expression of FLOWERING LOCUS C (FLC), which encodes a potent floral repressor, and decreased expression of its target, the floral promoter FD. Vernalization rescued delayed flowering of atvoz1-1 atvoz2-1 and reversed elevated FLC levels. Accumulation of FLC transcripts in atvoz1-1 atvoz2-1 correlated with increased expression of several FLC activators, including components of the PAF1 and SWR1 chromatin-modifying complexes. Additionally, AtVOZs were shown to bind the promoter of MOS3/SAR3 and directly regulate expression of this nuclear pore protein, which is known to participate in the regulation of flowering time, suggesting that AtVOZs exert at least some of their flowering regulation by influencing the nuclear pore function. Complementation of atvoz1-1 atvoz2-1 with AtVOZ2 reversed all double mutant phenotypes, confirming that the observed morphological and molecular changes arise from the absence of functional AtVOZ proteins, and validating the functional redundancy between AtVOZ1 and AtVOZ2.

  10. TyrR, the regulator of aromatic amino acid metabolism, is required for mice infection of Yersinia pestis

    Directory of Open Access Journals (Sweden)

    Zhongliang eDeng

    2015-02-01

    Full Text Available Yersinia pestis, the causative agent of plague, poses a serious health threat to rodents and human beings. TyrR is a transcriptional regulator that controls the metabolism of aromatic amino acids in Escherichia coli. In this paper, TyrR played an important role in Y. pestis virulence. Inactivation of tyrR did not seem to affect the in vitro growth of this organism, but resulted in at least 10,000-fold attenuation compared with the wild-type (WT strain upon subcutaneous infection to mice. In addition, loads of tyrR mutant within mice livers and spleens significantly decreased compared with the WT strain. Transcriptome analysis revealed that TyrR, directly or indirectly, regulated 29 genes encoded on Y. pestis chromosome or plasmids under in vitro growth condition. Similar to the regulatory function of this protein in E. coli, five aromatic-pathway genes (aroF-tyrA, aroP, aroL, and tyrP were significantly reduced upon deletion of the tyrR gene. Two genes (glnL and glnG that encode sensory histidine kinase and regulator in a two-component regulatory system involved in nitrogen assimilation were downregulated in the tyrR mutant. Several genes encoding type III secretion proteins were transcribed by 2.0- to 4.2-fold in a tyrR mutant relative to the WT strain. Interestingly, the acid-stressed genes, hdeB and hdeD, were downregulated, and such downregulation partly accounted for the decrease in tolerance of the tyrR mutant under acidic conditions. In conclusion, regulation of TyrR in Y. pestis is similar to, but distinct from, that in E. coli. TyrR is a metabolic virulence determinant in Y. pestis that is important for extracellular survival and/or proliferation.

  11. Cartilage development requires the function of Estrogen-related receptor alpha that directly regulates sox9 expression in zebrafish.

    Science.gov (United States)

    Kim, Yong-Il; No Lee, Joon; Bhandari, Sushil; Nam, In-Koo; Yoo, Kyeong-Won; Kim, Se-Jin; Oh, Gi-Su; Kim, Hyung-Jin; So, Hong-Seob; Choe, Seong-Kyu; Park, Raekil

    2015-12-10

    Estrogen-related receptor alpha (ESRRa) regulates a number of cellular processes including development of bone and muscles. However, direct evidence regarding its involvement in cartilage development remains elusive. In this report, we establish an in vivo role of Esrra in cartilage development during embryogenesis in zebrafish. Gene expression analysis indicates that esrra is expressed in developing pharyngeal arches where genes necessary for cartilage development are also expressed. Loss of function analysis shows that knockdown of esrra impairs expression of genes including sox9, col2a1, sox5, sox6, runx2 and col10a1 thus induces abnormally formed cartilage in pharyngeal arches. Importantly, we identify putative ESRRa binding elements in upstream regions of sox9 to which ESRRa can directly bind, indicating that Esrra may directly regulate sox9 expression. Accordingly, ectopic expression of sox9 rescues defective formation of cartilage induced by the knockdown of esrra. Taken together, our results indicate for the first time that ESRRa is essential for cartilage development by regulating sox9 expression during vertebrate development.

  12. TALEN Gene Knockouts Reveal No Requirement for the Conserved Human Shelterin Protein Rap1 in Telomere Protection and Length Regulation

    Directory of Open Access Journals (Sweden)

    Shaheen Kabir

    2014-11-01

    Full Text Available The conserved protein Rap1 functions at telomeres in fungi, protozoa, and vertebrates. Like yeast Rap1, human Rap1 has been implicated in telomere length regulation and repression of nonhomologous end-joining (NHEJ at telomeres. However, mouse telomeres lacking Rap1 do not succumb to NHEJ. To determine the functions of human Rap1, we generated several transcription activator-like effector nuclease (TALEN-mediated human cell lines lacking Rap1. Loss of Rap1 did not affect the other components of shelterin, the modification of telomeric histones, the subnuclear position of telomeres, or the 3′ telomeric overhang. Telomeres lacking Rap1 did not show a DNA damage response, NHEJ, or consistent changes in their length, indicating that Rap1 does not have an important function in protection or length regulation of human telomeres. As human Rap1, like its mouse and unicellular orthologs, affects gene expression, we propose that the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres.

  13. Suppressor of fusion, a Fusarium oxysporum homolog of Ndt80, is required for nutrient-dependent regulation of anastomosis.

    Science.gov (United States)

    Shahi, Shermineh; Fokkens, Like; Houterman, Petra M; Rep, Martijn

    2016-10-01

    Heterokaryon formation is an essential step in asexual recombination in Fusarium oxysporum. Filamentous fungi have an elaborate nonself recognition machinery to prevent formation and proliferation of heterokaryotic cells, called heterokaryon incompatibility (HI). In F. oxysporum the regulation of this machinery is not well understood. In Neurospora crassa, Vib-1, a putative transcription factor of the p53-like Ndt80 family of transcription factors, has been identified as global regulator of HI. In this study we investigated the role of the F. oxysporum homolog of Vib-1, called Suf, in vegetative hyphal and conidial anastomosis tube (CAT) fusion and HI. We identified a novel function for an Ndt80 homolog as a nutrient-dependent regulator of anastomosis. Strains carrying the SUF deletion mutation display a hyper-fusion phenotype during vegetative growth as well as germling development. In addition, conidial paring of incompatible SUF deletion strains led to more heterokaryon formation, which is independent of suppression of HI. Our data provides further proof for the divergence in the functions of different members Ndt80 family. We propose that Ndt80 homologs mediate responses to nutrient quality and quantity, with specific responses varying between species.

  14. Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity

    DEFF Research Database (Denmark)

    Burbage, Marianne; Keppler, Selina J; Gasparrini, Francesca

    2015-01-01

    The small Rho GTPase Cdc42, known to interact with Wiskott-Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-de...... and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell-intrinsic block in plasma cell differentiation. Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology.......The small Rho GTPase Cdc42, known to interact with Wiskott-Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42......-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization. Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling...

  15. Growth arrest specific 2 is up-regulated in chronic myeloid leukemia cells and required for their growth.

    Directory of Open Access Journals (Sweden)

    Haixia Zhou

    Full Text Available Although the generation of BCR-ABL is the molecular hallmark of chronic myeloid leukemia (CML, the comprehensive molecular mechanisms of the disease remain unclear yet. Growth arrest specific 2 (GAS2 regulates multiple cellular functions including cell cycle, apoptosis and calpain activities. In the present study, we found GAS2 was up-regulated in CML cells including CD34+ progenitor cells compared to their normal counterparts. We utilized RNAi and the expression of dominant negative form of GAS2 (GAS2DN to target GAS2, which resulted in calpain activity enhancement and growth inhibition of both K562 and MEG-01 cells. Targeting GAS2 also sensitized K562 cells to Imatinib mesylate (IM. GAS2DN suppressed the tumorigenic ability of MEG-01 cells and impaired the tumour growth as well. Moreover, the CD34+ cells from CML patients and healthy donors were transduced with control and GAS2DN lentiviral vectors, and the CD34+ transduced (YFP+ progeny cells (CD34+YFP+ were plated for colony-forming cell (CFC assay. The results showed that GAS2DN inhibited the CFC production of CML cells by 57±3% (n = 3, while affected those of normal hematopoietic cells by 31±1% (n = 2. Next, we found the inhibition of CML cells by GAS2DN was dependent on calpain activity but not the degradation of beta-catenin. Lastly, we generated microarray data to identify the differentially expressed genes upon GAS2DN and validated that the expression of HNRPDL, PTK7 and UCHL5 was suppressed by GAS2DN. These 3 genes were up-regulated in CML cells compared to normal control cells and the growth of K562 cells was inhibited upon HNRPDL silence. Taken together, we have demonstrated that GAS2 is up-regulated in CML cells and the inhibition of GAS2 impairs the growth of CML cells, which indicates GAS2 is a novel regulator of CML cells and a potential therapeutic target of this disease.

  16. The H-loop in the second nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator is required for efficient chloride channel closing.

    Science.gov (United States)

    Kloch, Monika; Milewski, Michał; Nurowska, Ewa; Dworakowska, Beata; Cutting, Garry R; Dołowy, Krzysztof

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR-->AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR-->AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.

  17. Ripening-regulated susceptibility of tomato fruit to Botrytis cinerea requires NOR but not RIN or ethylene.

    Science.gov (United States)

    Cantu, Dario; Blanco-Ulate, Barbara; Yang, Liya; Labavitch, John M; Bennett, Alan B; Powell, Ann L T

    2009-07-01

    Fruit ripening is a developmental process that is associated with increased susceptibility to the necrotrophic pathogen Botrytis cinerea. Histochemical observations demonstrate that unripe tomato (Solanum lycopersicum) fruit activate pathogen defense responses, but these responses are attenuated in ripe fruit infected by B. cinerea. Tomato fruit ripening is regulated independently and cooperatively by ethylene and transcription factors, including NON-RIPENING (NOR) and RIPENING-INHIBITOR (RIN). Mutations in NOR or RIN or interference with ethylene perception prevent fruit from ripening and, thereby, would be expected to influence susceptibility. We show, however, that the susceptibility of ripe fruit is dependent on NOR but not on RIN and only partially on ethylene perception, leading to the conclusion that not all of the pathways and events that constitute ripening render fruit susceptible. Additionally, on unripe fruit, B. cinerea induces the expression of genes also expressed as uninfected fruit ripen. Among the ripening-associated genes induced by B. cinerea are LePG (for polygalacturonase) and LeExp1 (for expansin), which encode cell wall-modifying proteins and have been shown to facilitate susceptibility. LePG and LeExp1 are induced only in susceptible rin fruit and not in resistant nor fruit. Thus, to infect fruit, B. cinerea relies on some of the processes and events that occur during ripening, and the fungus induces these pathways in unripe fruit, suggesting that the pathogen itself can initiate the induction of susceptibility by exploiting endogenous developmental programs. These results demonstrate the developmental plasticity of plant responses to the fungus and indicate how known regulators of fruit ripening participate in regulating ripening-associated pathogen susceptibility.

  18. The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation

    DEFF Research Database (Denmark)

    Desai, D M; Sap, J; Silvennoinen, O;

    1994-01-01

    Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not...

  19. 77 FR 30473 - Notice of Intent To Revise Stormwater Regulations To Specify That an NPDES Permit Is Not Required...

    Science.gov (United States)

    2012-05-23

    ... programs, including forest stewardship and sustainability initiatives. Under these required or voluntary... performance standards are encouraged to be abandoned. Abandonment strategies may involve the removal of stream... order by forest industry land, corporate non-industrial land, and private non-industrial land (Prud...

  20. 7 CFR 319.5 - Requirements for submitting requests to change the regulations in 7 CFR part 319.

    Science.gov (United States)

    2010-01-01

    ... required in order to complete a pest risk analysis in accordance with international standards for pest risk... implementation. (8) Existing documentation: Relevant pest risk analyses, environmental assessment(s), biological... Analysis and Operations, PPQ, APHIS, 4700 River Road Unit 140, Riverdale, MD 20737. (d) Information....

  1. 76 FR 14812 - Final Regulation Extending the Reporting Deadline for Year 2010 Data Elements Required Under the...

    Science.gov (United States)

    2011-03-18

    ... Review B. Paperwork Reduction Act C. Regulatory Flexibility Act (RFA) D. Unfunded Mandates Reform Act... facilities. 331492 Zinc dust reclaiming facilities, recovering from scrap and/or alloying purchased metals... addition of facility- and parent-level reporting requirements (75 FR 57669, September 22, 2010); technical...

  2. Inhibition of seed germination and induction of systemic disease resistance by Pseudomonas chlororaphis O6 requires phenazine production regulated by the global regulator, gacS.

    Science.gov (United States)

    Kang, Beom Ryong; Han, Song Hee; Zdor, Rob E; Anderson, Anne J; Spencer, Matt; Yang, Kwang Yeol; Kim, Yong Hwan; Lee, Myung Chul; Cho, Baik Ho; Kim, Young Cheol

    2007-04-01

    Seed coating by a phenazine-producing bacterium, Pseudomonas chlororaphis O6, induced dose-dependent inhibition of germination in wheat and barley seeds, but did not inhibit germination of rice or cucumber seeds. In wheat seedlings grown from inoculated seeds, phenazine production levels near the seed were higher than in the roots. Deletion of the gacS gene reduced transcription from the genes required for phenazine synthesis, the regulatory phzI gene and the biosynthetic phzA gene. The inhibition of seed germination and the induction of systemic disease resistance against a bacterial soft-rot pathogen, Erwinia carotovora subsp. carotovora, were impaired in the gacS and phzA mutants of P chlororaphis O6. Culture filtrates of the gacS and phzA mutants of P chlororaphis 06 did not inhibit seed germination of wheat, whereas that of the wild-type was inhibitory. Our results showed that the production of phenazines by P chlororaphis O6 was correlated with reduced germination of barley and wheat seeds, and the level of systemic resistance in tobacco against E. carotovora.

  3. Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition

    Science.gov (United States)

    Hafner, Markus; Max, Klaas E.A.; Bandaru, Pradeep; Morozov, Pavel; Gerstberger, Stefanie; Brown, Miguel; Molina, Henrik; Tuschl, Thomas

    2013-01-01

    Human LIN28A and LIN28B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ∼3000 mRNAs at ∼9500 sites located in the 3′ UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors. PMID:23481595

  4. Regulation of cell divisions and differentiation by MALE STERILITY32 is required for anther development in maize.

    Science.gov (United States)

    Moon, Jihyun; Skibbe, David; Timofejeva, Ljudmilla; Wang, Chung-Ju Rachel; Kelliher, Timothy; Kremling, Karl; Walbot, Virginia; Cande, William Zacheus

    2013-11-01

    Male fertility in flowering plants relies on proper division and differentiation of cells in the anther, a process that gives rise to four somatic layers surrounding central germinal cells. The maize gene male sterility32 (ms32) encodes a basic helix-loop-helix (bHLH) transcription factor, which functions as an important regulator of both division and differentiation during anther development. After the four somatic cell layers are generated properly through successive periclinal divisions, in the ms32 mutant, tapetal precursor cells fail to differentiate, and, instead, undergo additional periclinal divisions to form extra layers of cells. These cells become vacuolated and expand, and lead to failure in pollen mother cell development. ms32 expression is specific to the pre-meiotic anthers and is distributed initially broadly in the four lobes, but as the anther develops, its expression becomes restricted to the innermost somatic layer, the tapetum. The ms32-ref mac1-1 double mutant is unable to form tapetal precursors and also exhibits excessive somatic proliferation leading to numerous, disorganized cell layers, suggesting a synergistic interaction between ms32 and mac1. Altogether, our results show that MS32 is a major regulator in maize anther development that promotes tapetum differentiation and inhibits periclinal division once a tapetal cell is specified. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  5. Opposite-sex attraction in male mice requires testosterone-dependent regulation of adult olfactory bulb neurogenesis

    Science.gov (United States)

    Schellino, Roberta; Trova, Sara; Cimino, Irene; Farinetti, Alice; Jongbloets, Bart C.; Pasterkamp, R. Jeroen; Panzica, Giancarlo; Giacobini, Paolo; De Marchis, Silvia; Peretto, Paolo

    2016-01-01

    Opposite-sex attraction in most mammals depends on the fine-tuned integration of pheromonal stimuli with gonadal hormones in the brain circuits underlying sexual behaviour. Neural activity in these circuits is regulated by sensory processing in the accessory olfactory bulb (AOB), the first central station of the vomeronasal system. Recent evidence indicates adult neurogenesis in the AOB is involved in sex behaviour; however, the mechanisms underlying this function are unknown. By using Semaphorin 7A knockout (Sema7A ko) mice, which show a reduced number of gonadotropin-releasing-hormone neurons, small testicles and subfertility, and wild-type males castrated during adulthood, we demonstrate that the level of circulating testosterone regulates the sex-specific control of AOB neurogenesis and the vomeronasal system activation, which influences opposite-sex cue preference/attraction in mice. Overall, these data highlight adult neurogenesis as a hub for the integration of pheromonal and hormonal cues that control sex-specific responses in brain circuits. PMID:27782186

  6. Serum inducible kinase is a positive regulator of cortical dendrite development and is required for BDNF-promoted dendritic arborization

    Institute of Scientific and Technical Information of China (English)

    Shun-Ling Guo; Guo-He Tan; Shuai Li; Xue-Wen Cheng; Ya Zhou; Yun-Fang Jia; Hui Xiong; Jiong Tao; Zhi-Qi Xiong

    2012-01-01

    Serum inducible kinase (SNK),also known as (p)olo-(l)ike (k)inase 2 (PLK2),is a known regulator of mitosis,synaptogenesis and synaptic homeostasis.However,its role in early cortical development is unknown.Herein,we show that snk is expressed in the cortical plate from embryonic day 14,but not in the ventricular/subventricular zones (VZ/SVZ),and SNK protein localizes to the soma and dendrites of cultured immature cortical neurons.Loss of SNK impaired dendritic but not axonal arborization in a dose-dependent manner and overexpression had opposite effects,both in vitro and in vivo.Overexpression of SNK also caused abnormal branching of the leading process of migrating cortical neurons in electroporated cortices.The kinase activity was necessary for these effects.Extracellular signalregulated kinase (ERK) pathway activity downstream of brain-derived neurotrophic factor (BDNF) stimulation led to increases in SNK protein expression via transcriptional regulation,and this upregulation was necessary for the growth-promoting effect of BDNF on dendritic arborization.Taken together,our results indicate that SNK is essential for dendrite morphogenesis in cortical neurons.

  7. A DNA2 Homolog Is Required for DNA Damage Repair, Cell Cycle Regulation, and Meristem Maintenance in Plants.

    Science.gov (United States)

    Jia, Ning; Liu, Xiaomin; Gao, Hongbo

    2016-05-01

    Plant meristem cells divide and differentiate in a spatially and temporally regulated manner, ultimately giving rise to organs. In this study, we isolated the Arabidopsis jing he sheng 1 (jhs1) mutant, which exhibited retarded growth, an abnormal pattern of meristem cell division and differentiation, and morphological defects such as fasciation, an irregular arrangement of siliques, and short roots. We identified JHS1 as a homolog of human and yeast DNA Replication Helicase/Nuclease2, which is known to be involved in DNA replication and damage repair. JHS1 is strongly expressed in the meristem of Arabidopsis. The jhs1 mutant was sensitive to DNA damage stress and had an increased DNA damage response, including increased expression of genes involved in DNA damage repair and cell cycle regulation, and a higher frequency of homologous recombination. In the meristem of the mutant plants, cell cycle progression was delayed at the G2 or late S phase and genes essential for meristem maintenance were misregulated. These results suggest that JHS1 plays an important role in DNA replication and damage repair, meristem maintenance, and development in plants. © 2016 American Society of Plant Biologists. All Rights Reserved.

  8. JMJD-5/KDM8 regulates H3K36me2 and is required for late steps of homologous recombination and genome integrity

    Science.gov (United States)

    Zaghet, Nico; Ramalho, João J.; Vilstrup Johansen, Jens; Salcini, Anna Elisabetta

    2017-01-01

    The eukaryotic genome is organized in a three-dimensional structure called chromatin, constituted by DNA and associated proteins, the majority of which are histones. Post-translational modifications of histone proteins greatly influence chromatin structure and regulate many DNA-based biological processes. Methylation of lysine 36 of histone 3 (H3K36) is a post-translational modification functionally relevant during early steps of DNA damage repair. Here, we show that the JMJD-5 regulates H3K36 di-methylation and it is required at late stages of double strand break repair mediated by homologous recombination. Loss of jmjd-5 results in hypersensitivity to ionizing radiation and in meiotic defects, and it is associated with aberrant retention of RAD-51 at sites of double strand breaks. Analyses of jmjd-5 genetic interactions with genes required for resolving recombination intermediates (rtel-1) or promoting the resolution of RAD-51 double stranded DNA filaments (rfs-1 and helq-1) suggest that jmjd-5 prevents the formation of stalled postsynaptic recombination intermediates and favors RAD-51 removal. As these phenotypes are all recapitulated by a catalytically inactive jmjd-5 mutant, we propose a novel role for H3K36me2 regulation during late steps of homologous recombination critical to preserve genome integrity. PMID:28207814

  9. CD81 Is a Central Regulator of Cellular Events Required for Hepatitis C Virus Infection of Human Hepatocytes▿ †

    OpenAIRE

    Brazzoli, Michela; Bianchi, Alessia; Filippini, Sara; Weiner, Amy; Zhu, Qing; Pizza, Mariagrazia; Crotta, Stefania

    2008-01-01

    Infection with hepatitis C virus (HCV) is still a major public health problem, and the events leading to hepatocyte infection are not yet fully understood. Combining confocal microscopy with biochemical analysis and studies of infection requirements using pharmacological inhibitors and small interfering RNAs, we show here that engagement of CD81 activates the Rho GTPase family members Rac, Rho, and Cdc42 and that the block of these signaling pathways drastically reduces HCV infectivity. Activ...

  10. Engineering requirements due to the ESP/ESPN regulation apply at the port plug for ITER diagnostic system

    Energy Technology Data Exchange (ETDEWEB)

    Giacomin, T., E-mail: Thibaud.Giacomin@iter.org [ITER Organization, Route de Vinon sur Verdon, 13115 St Paul-lez-Durance (France); Delhom, D.; Drevon, J.-M. [Bertin Technologies, Aix en Provence (France); Guirao, J.; Iglesias, S. [NATEC Ingenieros (Spain); Jourdan, T. [ITER Organization, Route de Vinon sur Verdon, 13115 St Paul-lez-Durance (France); Loesser, D. [Princeton Plasma Physics Laboratory (United States); Maquet, P. [ITER Organization, Route de Vinon sur Verdon, 13115 St Paul-lez-Durance (France); Ordieres, J. [NATEC Ingenieros (Spain); Pak, S. [ITER Organization, Route de Vinon sur Verdon, 13115 St Paul-lez-Durance (France); Proust, M. [CEA Cadarache, St. Paul-lez-Durance (France); Smith, M. [Princeton Plasma Physics Laboratory (United States); Udintsev, V.S. [ITER Organization, Route de Vinon sur Verdon, 13115 St Paul-lez-Durance (France); Vacas, C. [NATEC Ingenieros (Spain); Walsh, M.J. [ITER Organization, Route de Vinon sur Verdon, 13115 St Paul-lez-Durance (France); Zhai, Y. [Princeton Plasma Physics Laboratory (United States)

    2015-10-15

    Highlights: • The ESP/ESPN regulation is applied to all equatorial and upper port-based diagnostic systems. • The EPP and UPP structure provides a flexible platform for a variety of diagnostics. • The EPP and UPP structure provide a support the diagnostic shielding module. - Abstract: Due to this position close to the plasma, the port plug structure and the diagnostic first wall (DFW) contain water to allow cooling during operation and for heating during bake-out. To remove the heat coming from the plasma due to radiation and neutrons, the pressure inside these structures should be up to 44 bars. On the other hand, the dominant load expected to drive the design of these structures is of electromagnetic origin during the plasma disruption. Description of the loads acting on DFWs and generic port plug structures and the significance of the load due to the water pressure, with implications on the design and inspection, are discussed in this paper.

  11. CLNR1, the AREA/NIT2-like global nitrogen regulator of the plant fungal pathogen Colletotrichum lindemuthianum is required for the infection cycle.

    Science.gov (United States)

    Pellier, Anne-Laure; Laugé, Richard; Veneault-Fourrey, Claire; Langin, Thierry

    2003-05-01

    Nitrogen starvation is generally assumed to be encountered by biotrophic and hemibiotrophic plant fungal pathogens at the beginning of their infection cycle. We tested whether nitrogen starvation constitutes a cue regulating genes that are required for pathogenicity of Colletotrichum lindemuthianum, a fungal pathogen of common bean. The clnr1 (C. lindemuthianumnitrogen regulator 1) gene, the areA/nit-2 orthologue of C. lindemuthianum, was isolated. The predicted CLNR1 protein exhibits high amino acid sequence similarities with the AREA and NIT2 global fungal nitrogen regulators. Targeted clnr1- mutants are unable to use a wide array of nitrogen sources, indicating that clnr1 is the C. lindemuthianum major nitrogen regulatory gene. The clnr1- mutants are non-pathogenic, although few anthracnose lesions seldom occur on whole plantlets. Surprisingly, cytological analysis reveals that the clnr1- mutants are not disturbed from the penetration stage until the end of the biotrophic phase, but that they are impaired during the setting up of the necrotrophic phase. Thus, through CLNR1, nitrogen starvation constitutes a cue for the regulation of genes that are compulsory for this stage of the C. lindemuthianum infection process. Additionally, clnr1- mutants complemented with the Aspergillus nidulans areA gene are fully pathogenic, indicating that areA is able to activate the C. lindemuthianum suited genes, normally under the control of clnr1.

  12. Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

    Science.gov (United States)

    Clemens, Michael J; Elia, Androulla; Morley, Simon J

    2013-01-01

    The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

  13. Growth regulator requirement for in vitro embryogenic cultures of snowdrop (Galanthus nivalis L.) suitable for germplasm preservation.

    Science.gov (United States)

    Resetár, Anna; Demeter, Zita; Ficsor, Emese; Balázs, Andrea; Mosolygó, Agnes; Szőke, Eva; Gonda, S; Papp, L; Surányi, G; Máthé, C

    2014-06-01

    In this study, we report on the production of bulb scale-derived tissue cultures capable of efficient shoot and plant regeneration in three genotypes of snowdrop (Galanthus nivalis L., Amaryllidaceae), a protected ornamental plant. For culture line A, high auxin and low cytokinin concentration is required for callus production and plant regeneration. The type of auxin is of key importance: α-naphthaleneacetic acid (NAA) in combination with indole-3-acetic acid (IAA) at concentrations of 2 mg L-1 or 2-10 mg L-1 NAA with 1 mg L-1 N6-benzyladenine (BA), a cytokinin on full-strength media are required for regeneration. Cultures showing regeneration were embryogenic. When lines B and C were induced and maintained with 2 mg L-1 NAA and 1 mg L-1 BA, they produced mature bulblets with shoots, without roots. Line A produced immature bulblets with shoots under the above culture condition. Amplified Fragment Length Polymorphism (AFLP) analysis showed that (i) genetic differences between line A and its bulb explants were not significant, therefore these tissue cultures are suitable for germplasm preservation, and (ii) different morphogenetic responses of lines A, B and C originated from genetic differences. Culture line A is suitable for field-growing, cultivation and germplasm preservation of G. nivalis and for the production of Amaryllidaceae alkaloids.

  14. Shape-induced terminal differentiation of human epidermal stem cells requires p38 and is regulated by histone acetylation.

    Directory of Open Access Journals (Sweden)

    John T Connelly

    Full Text Available Engineered model substrates are powerful tools for examining interactions between stem cells and their microenvironment. Using this approach, we have previously shown that restricted cell adhesion promotes terminal differentiation of human epidermal stem cells via activation of serum response factor (SRF and transcription of AP-1 genes. Here we investigate the roles of p38 MAPK and histone acetylation. Inhibition of p38 activity impaired SRF transcriptional activity and shape-induced terminal differentiation of human keratinocytes. In addition, inhibiting p38 reduced histone H3 acetylation at the promoters of SRF target genes, FOS and JUNB. Although histone acetylation correlated with SRF transcriptional activity and target gene expression, treatment with the histone de-acetylase inhibitor, trichostatin A (TSA blocked terminal differentiation on micro-patterned substrates and in suspension. TSA treatment simultaneously maintained expression of LRIG1, TP63, and ITGB1. Therefore, global histone de-acetylation represses stem cell maintenance genes independent of SRF. Our studies establish a novel role for extrinsic physical cues in the regulation of chromatin remodeling, transcription, and differentiation of human epidermal stem cells.

  15. Mammalian diaphanous-related formin 1 regulates GSK3β-dependent microtubule dynamics required for T cell migratory polarization.

    Directory of Open Access Journals (Sweden)

    Baoxia Dong

    Full Text Available The mammalian diaphanous-related formin (mDia1, a Rho-regulated cytoskeletal modulator, has been shown to promote T lymphocyte chemotaxis and interaction with antigen presenting cells, but the mechanisms underpinning mDia1 roles in these processes have not been defined. Here we show that mDia1(-/- T cells exhibit impaired lymphocyte function-associated antigen 1 (LFA-1-mediated T cell adhesion, migration and in vivo trafficking. These defects are associated with impaired microtubule (MT polarization and stabilization, altered MT dynamics and reduced peripheral clustering of the MT plus-end-protein, adenomatous polyposis coli (APC in migrating T cells following LFA-1-engagement. Loss of mDia1 also leads to impaired inducible inactivation of the glycogen synthase kinase (GSK 3β as well as hyperphosphorylation and reduced levels of APC in migrating T cells. These findings identify essential roles for the mDia1 formin in modulating GSK3β-dependent MT contributions to induction of T-cell polarity, adhesion and motility.

  16. Mammalian diaphanous-related formin 1 regulates GSK3β-dependent microtubule dynamics required for T cell migratory polarization.

    Science.gov (United States)

    Dong, Baoxia; Zhang, Steven S; Gao, Wen; Su, Haichun; Chen, Jun; Jin, Fuzi; Bhargava, Ajay; Chen, Xiequn; Jorgensen, Lars; Alberts, Arthur S; Zhang, Jinyi; Siminovitch, Katherine A

    2013-01-01

    The mammalian diaphanous-related formin (mDia1), a Rho-regulated cytoskeletal modulator, has been shown to promote T lymphocyte chemotaxis and interaction with antigen presenting cells, but the mechanisms underpinning mDia1 roles in these processes have not been defined. Here we show that mDia1(-/-) T cells exhibit impaired lymphocyte function-associated antigen 1 (LFA-1)-mediated T cell adhesion, migration and in vivo trafficking. These defects are associated with impaired microtubule (MT) polarization and stabilization, altered MT dynamics and reduced peripheral clustering of the MT plus-end-protein, adenomatous polyposis coli (APC) in migrating T cells following LFA-1-engagement. Loss of mDia1 also leads to impaired inducible inactivation of the glycogen synthase kinase (GSK) 3β as well as hyperphosphorylation and reduced levels of APC in migrating T cells. These findings identify essential roles for the mDia1 formin in modulating GSK3β-dependent MT contributions to induction of T-cell polarity, adhesion and motility.

  17. The plant cuticle is required for osmotic stress regulation of abscisic acid biosynthesis and osmotic stress tolerance in Arabidopsis

    KAUST Repository

    Wang, Zhenyu

    2011-05-01

    Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA biosynthesis, we screened for Arabidopsis thaliana mutants that failed to induce the NCED3 genee xpression in response to osmotic stress treatments. The ced1 (for 9-cis epoxycarotenoid dioxy genase defective 1) mutant isolated in this study showed markedly reduced expression of NCED3 in response to osmotic stress (polyethylene glycol)treatments compared with the wild type. Other ABA biosynthesis genes are also greatly reduced in ced1 under osmotic stress. ced1 mutant plants are very sensitive to even mild osmotic stress. Map-based cloning revealed unexpectedly thatCED1 encodes a putative a/b hydrolase domain-containing protein and is allelic to the BODYGUARD gene that was recently shown to be essential for cuticle biogenesis. Further studies discovered that other cut in biosynthesis mutants are also impaired in osmotic stress induction of ABA biosynthesis genes and are sensitive to osmotic stress. Our work demonstrates that the cuticle functions not merely as a physical barrier to minimize water loss but also mediates osmotic stress signaling and tolerance by regulating ABA biosynthesis and signaling. © 2011 American Society of Plant Biologists. All rights reserved.

  18. The imitation switch ATPase Snf2l is required for superovulation and regulates Fgl2 in differentiating mouse granulosa cells.

    Science.gov (United States)

    Pépin, David; Paradis, François; Perez-Iratxeta, Carol; Picketts, David J; Vanderhyden, Barbara C

    2013-06-01

    Imitation switch (ISWI) proteins are catalytic subunits of chromatin remodeling complexes that alter nucleosome positioning by hydrolyzing ATP to regulate access to DNA. In mice, there are two paralogs, SNF2-homolog (SNF2H) and SNF2-like (SNF2L), which participate in different complexes and have contrasting patterns of expression. Here we investigate the role of SNF2L in ovaries by characterizing a mouse bearing an inactivating deletion of exon 6 that disrupts the ATPase domain. Snf2l mutant mice produce significantly fewer eggs than control mice when superovulated. Gonadotropin stimulation leads to a significant deficit in secondary follicles and an increase in abnormal antral follicles. Mutant females also failed to induce fibrinogen-like 2 (Fgl2) in response to human chorionic gonadotropin (hCG) stimulation, while overexpression of SNF2L was sufficient to drive its expression in granulosa cells. SNF2L was also shown to directly interact with the nuclear receptor co-activator flightless I (FLI-I) as shown by immunoprecipitation. These results begin to establish a role for SNF2L in the precise coordination of gene expression in granulosa cells during folliculogenesis and its broader implications in fertility.

  19. The bristle patterning genes hairy and extramacrochaetae regulate the development of structures required for flight in Diptera☆

    Science.gov (United States)

    Costa, Marta; Calleja, Manuel; Alonso, Claudio R.; Simpson, Pat

    2014-01-01

    The distribution of sensory bristles on the thorax of Diptera (true flies) provides a useful model for the study of the evolution of spatial patterns. Large bristles called macrochaetes are arranged into species-specific stereotypical patterns determined via spatially discrete expression of the proneural genes achaete–scute (ac–sc). In Drosophila ac-sc expression is regulated by transcriptional activation at sites where bristle precursors develop and by repression outside of these sites. Three genes, extramacrochaetae (emc), hairy (h) and stripe (sr), involved in repression have been documented. Here we demonstrate that in Drosophila, the repressor genes emc and h, like sr, play an essential role in the development of structures forming part of the flight apparatus. In addition we find that, in Calliphora vicina a species diverged from D. melanogaster by about 100 Myr, spatial expression of emc, h and sr is conserved at the location of development of those structures. Based on these findings we argue, first, that the role emc, h and sr in development of the flight apparatus preceded their activities for macrochaete patterning; second, that species-specific variation in activation and repression of ac-sc expression is evolving in parallel to establish a unique distribution of macrochaetes in each species. PMID:24384389

  20. Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo

    Science.gov (United States)

    Goody, Michelle F.; Kelly, Meghan W.; Lessard, Kevin N.; Khalil, Andre; Henry, Clarissa A.

    2010-01-01

    Cell-matrix adhesion complexes (CMACs) play fundamental roles during morphogenesis. Given the ubiquitous nature of CMACs and their roles in many cellular processes, one question is how specificity of CMAC function is modulated. The clearly defined cell behaviors that generate segmentally reiterated axial skeletal muscle during zebrafish development comprise an ideal system with which to investigate CMAC function during morphogenesis. We found that Nicotinamide riboside kinase 2b (Nrk2b) cell autonomously modulates the molecular composition of CMACs in vivo. Nrk2b is required for normal Laminin polymerization at the myotendinous junction (MTJ). In Nrk2b-deficient embryos, at MTJ loci where Laminin is not properly polymerized, muscle fibers elongate into adjacent myotomes and are abnormally long. In yeast and human cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative salvage pathway. Exogenous NAD+ treatment rescues MTJ development in Nrk2b-deficient embryos, but not in laminin mutant embryos. Both Nrk2b and Laminin are required for localization of Paxillin, but not β-Dystroglycan, to CMACs at the MTJ. Overexpression of Paxillin in Nrk2b-deficient embryos is sufficient to rescue MTJ integrity. Taken together, these data show that Nrk2b plays a specific role in modulating subcellular localization of discrete CMAC components that in turn play roles in musculoskeletal development. Furthermore, these data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development. These results indicate a previously unrecognized complexity to CMAC assembly in vivo and also elucidate a novel role for NAD+ during morphogenesis. PMID:20566368

  1. HCN channel dendritic targeting requires bipartite interaction with TRIP8b and regulates antidepressant-like behavioral effects

    Science.gov (United States)

    Han, Ye; Heuermann, Robert J.; Lyman, Kyle A.; Fisher, Daniel; Ismail, Quratul-Ain; Chetkovich, Dane M.

    2016-01-01

    Major Depressive Disorder is a prevalent psychiatric condition with limited therapeutic options beyond monoaminergic therapies. Although effective in some individuals, many patients fail to respond adequately to existing treatments and new pharmacologic targets are needed. HCN channels regulate excitability in neurons and blocking HCN channel function has been proposed as a novel antidepressant strategy. However, systemic blockade of HCN channels produces cardiac effects that limit this approach. Knockout (KO) of the brain-specific HCN channel auxiliary subunit TRIP8b also produces antidepressant-like behavioral effects and suggests that inhibiting TRIP8b function could produce antidepressant-like effects without affecting the heart. We examined the structural basis of TRIP8b-mediated HCN channel trafficking and its relationship to antidepressant-like behavior using a viral rescue approach in TRIP8b KO mice. We found that restoring TRIP8b to the hippocampus was sufficient to reverse the impaired HCN channel trafficking and antidepressant-like behavioral effects caused by TRIP8b KO. Moreover, we found that hippocampal expression of a mutated version of TRIP8b further impaired HCN channel trafficking and increased the antidepressant-like behavioral phenotype of TRIP8b KO mice. Thus, modulating the TRIP8b-HCN interaction bidirectionally influences channel trafficking and antidepressant-like behavior. Overall, our work suggests that small molecule inhibitors of the interaction between TRIP8b and HCN should produce antidepressant-like behaviors and could represent a new paradigm for the treatment of Major Depressive Disorder. PMID:27400855

  2. Palmitoylation on the carboxyl terminus tail is required for the selective regulation of dopamine D2 versus D3 receptors.

    Science.gov (United States)

    Zhang, Xiaowei; Le, Hang Thi; Zhang, Xiaohan; Zheng, Mei; Choi, Bo-Gil; Kim, Kyeong-Man

    2016-09-01

    Dopamine D2 receptor (D2R) and D3 receptor (D3R) possess highly conserved amino acid sequences but this study showed that D3R was more extensively palmitoylated than D2R. Based on this finding, the molecular basis of this selective palmitoylation of D3R was determined and the roles of palmitoylation in the regulation of D3R functions were investigated. D3R was palmitoylated on the cysteine residue on its carboxyl terminus tail, the last amino acid residue of D3R, and an exchange of the carboxyl terminus tail between D2R and D3R (D2R-D3C and D3R-D2C) resulted in the switching of the palmitoylation phenotype. When the consensus site for palmitoylation was mutated or the palmitoylation of D3R was inhibited by treatment with 2-bromopalmitate (2BP), a palmitoylation blocker, cell-surface expression, PKC-mediated endocytosis, agonist affinity, and agonist-induced tolerance of D3R were all inhibited. However, these changes were not observed when D3R palmitoylation was inhibited by replacing its carboxyl tail with that of D2R (D3R-D2C) or when the palmitoylation of D2R-D3C was inhibited by treatment with 2BP. Overall, this study shows that D3R is palmitoylated more extensively than D2R even though the carboxyl terminus tails of D2R and D3R are highly homologous, and thus provides a new clue regarding the consensus sequence for palmitoylation. This study also shows that palmitoylation controls various functionalities of D3R only when the receptor is in the intact D3R configuration. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Cue-elicited reward-seeking requires extracellular signal-regulated kinase activation in the nucleus accumbens.

    Science.gov (United States)

    Shiflett, Michael W; Martini, Ross P; Mauna, Jocelyn C; Foster, Rebecca L; Peet, Eloise; Thiels, Edda

    2008-02-01

    The motivation to seek out rewards can come under the control of stimuli associated with reward delivery. The ability of cues to motivate reward-seeking behavior depends on the nucleus accumbens (NAcc). The molecular mechanisms in the NAcc that underlie the ability of a cue to motivate reward-seeking are not well understood. We examined whether extracellular signal-regulated kinase (ERK), an important intracellular signaling pathway in learning and memory, has a role in these motivational processes. We first examined p42 ERK (ERK2) activation in the NAcc after rats were trained to associate an auditory stimulus with food delivery and found that, as a consequence of training, presentation of the auditory cue itself was sufficient to increase ERK2 activation in the NAcc. To examine whether inhibition of ERK in the NAcc prevents cue-induced reward-seeking, we infused an inhibitor of ERK, U0126, into the NAcc before assessing rats' instrumental responding in the presence versus absence of the conditioned cue. We found that, whereas vehicle-infused rats showed increased instrumental responding during cue presentation, rats infused with U0126 showed a profound impairment in cue-induced instrumental responding. In contrast, intra-NAcc U0126 infusion had no effect on rats' food-reinforced instrumental responding or their ability to execute conditioned approach behavior. Our results demonstrate learning-related changes in ERK signaling in the NAcc, and that disruption of ERK activation in this structure interferes with the incentive-motivational effects of conditioned stimuli. The molecular mechanisms described here may have implications for cue-elicited drug craving after repeated exposure to drugs of abuse.

  4. HCN-channel dendritic targeting requires bipartite interaction with TRIP8b and regulates antidepressant-like behavioral effects.

    Science.gov (United States)

    Han, Y; Heuermann, R J; Lyman, K A; Fisher, D; Ismail, Q-A; Chetkovich, D M

    2017-03-01

    Major depressive disorder (MDD) is a prevalent psychiatric condition with limited therapeutic options beyond monoaminergic therapies. Although effective in some individuals, many patients fail to respond adequately to existing treatments, and new pharmacologic targets are needed. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate excitability in neurons, and blocking HCN channel function has been proposed as a novel antidepressant strategy. However, systemic blockade of HCN channels produces cardiac effects that limit this approach. Knockout (KO) of the brain-specific HCN-channel auxiliary subunit tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) also produces antidepressant-like behavioral effects and suggests that inhibiting TRIP8b function could produce antidepressant-like effects without affecting the heart. We examined the structural basis of TRIP8b-mediated HCN-channel trafficking and its relationship with antidepressant-like behavior using a viral rescue approach in TRIP8b KO mice. We found that restoring TRIP8b to the hippocampus was sufficient to reverse the impaired HCN-channel trafficking and antidepressant-like behavioral effects caused by TRIP8b KO. Moreover, we found that hippocampal expression of a mutated version of TRIP8b further impaired HCN-channel trafficking and increased the antidepressant-like behavioral phenotype of TRIP8b KO mice. Thus, modulating the TRIP8b-HCN interaction bidirectionally influences channel trafficking and antidepressant-like behavior. Overall, our work suggests that small-molecule inhibitors of the interaction between TRIP8b and HCN should produce antidepressant-like behaviors and could represent a new paradigm for the treatment of MDD.

  5. Development and application of a permit information system for shale oil (PERMISSO). Final report appendix: summary sheets of regulations required for oil shale development, June 1978--May 1979

    Energy Technology Data Exchange (ETDEWEB)

    1979-07-01

    This appendix is comprised of summaries of various governmental permits, licenses and other approvals required for oil shale development. The summaries were completed during the period June--October 1978, and are current as of July 1, 1978, although more recent authority was cited in some cases. One of the major purposes of Phase II of the project will be to update these summaries as statutes and regulations are added, changed or eliminated. This updating will be particularly important in the case of environmental permits and approvals. Many legislative and regulatory changes affecting environmental requirements are pending at this time and will alter many of the summaries herein. In addition, many regulatory proposals have been or likely will be challenged in the courts. When such conflicts are resolved further changes may be in order.

  6. The Carboxy-terminus of BAK1 regulates kinase activity and is required for normal growth of Arabidopsis

    Directory of Open Access Journals (Sweden)

    Man-Ho eOh

    2014-02-01

    Full Text Available Binding of brassinolide to the BRASSINOSTEROID-INSENSTIVE 1 (BRI1 receptor kinase promotes interaction with its co-receptor, BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1. Juxtaposition of the kinase domains that occurs then allows reciprocal transphosphorylation and activation of both kinases, but details of that process are not entirely clear. In the present study we show that the carboxy (C - terminal polypeptide of BAK1 may play a role. First, we demonstrate that the C-terminal domain is a strong inhibitor of the transphosphorylation activity of the recombinant BAK1 cytoplasmic domain protein. However, recombinant BAK1 lacking the C-terminal domain is unable to transactivate the peptide kinase activity of BRI1 in vitro. Thus, the C-terminal domain may play both a positive and negative role. Interestingly, a synthetic peptide corresponding to the full C-terminal domain (residues 576 to 615 of BAK1 interacted with recombinant BRI1 in vitro, and that interaction was enhanced by phosphorylation at the Tyr-610 site. Expression of a BAK1 C-terminal domain truncation (designated BAK1-ΔCT-Flag in transgenic Arabidopsis plants lacking endogenous bak1 and its functional paralog, bkk1, produced plants that were wild type in appearance but much smaller than plants expressing full-length BAK1-Flag. The reduction in growth may be attributed to a partial inhibition of BR signaling in vivo as reflected in root growth assays but other factors are likely involved as well. Our working model is that in vivo, the inhibitory action of the C-terminal domain of BAK1 is relieved by binding to BRI1. However, that interaction is not essential for BR signaling, but other aspects of cellular signaling are impacted when the C-terminal domain is truncated and result in inhibition of growth. These results increase the molecular understanding of the C-terminal domain of BAK1 as a regulator of kinase activity that may serve as a model for other receptor kinases.

  7. Transcriptional regulation by triiodothyronine requires synergistic action of the thyroid receptor with another trans-acting factor.

    Science.gov (United States)

    Voz, M L; Peers, B; Wiedig, M J; Jacquemin, P; Belayew, A; Martial, J A

    1992-09-01

    Human placental lactogen B (hCS-B) promoter activity is strongly stimulated by triiodothyronine (T3) in pituitary GC cells through interaction between the thyroid receptor and a thyroid receptor-binding element (TBE) spanning coordinates -67 to -41. This TBE is adjacent to the binding site for pituitary factor GHF1 (-95 to -68) which seems necessary for T3 stimulation of hCS-B promoter activity (M. L. Voz, B. Peers, A. Belayew, and J. A. Martial, J. Biol. Chem. 266:13397-13404, 1991). We here demonstrate actual synergy between the thyroid receptor and GHF1. Indeed, in placental JEG-3 cells devoid of factor GHF1, hCS promoter activity is barely stimulated by T3, while a strong response is observed in pituitary GC cells. In the latter, furthermore, neither the TBE nor the GHF1-binding site alone is sufficient to render the thymidine kinase promoter responsive to T3, while in combination they promote strong T3 stimulation. Close proximity between these sites is required for optimal synergy: T3 stimulation globally decreases with increased spacing. Furthermore, synergy occurs not only with a GHF1-binding site but also with all other factor recognition sequences tested (Sp1, NF1, CP1, Oct1, and CACCC boxes) and even with two other copies of the TBE. Nor is it specific to hCS TBE, since the palindromic sequence TCAGGTCA TGACCTGA (TREpal) also exhibits cooperativity.

  8. A Myb transcription factor of Phytophthora sojae, regulated by MAP kinase PsSAK1, is required for zoospore development.

    Directory of Open Access Journals (Sweden)

    Meng Zhang

    Full Text Available PsSAK1, a mitogen-activated protein (MAP kinase from Phytophthora sojae, plays an important role in host infection and zoospore viability. However, the downstream mechanism of PsSAK1 remains unclear. In this study, the 3'-tag digital gene expression (DGE profiling method was applied to sequence the global transcriptional sequence of PsSAK1-silenced mutants during the cysts stage and 1.5 h after inoculation onto susceptible soybean leaf tissues. Compared with the gene expression levels of the recipient P. sojae strain, several candidates of Myb family were differentially expressed (up or down in response to the loss of PsSAK1, including of a R2R3-type Myb transcription factor, PsMYB1. qRT-PCR indicated that the transcriptional level of PsMYB1 decreased due to PsSAK1 silencing. The transcriptional level of PsMYB1 increased during sporulating hyphae, in germinated cysts, and early infection. Silencing of PsMYB1 results in three phenotypes: a no cleavage of the cytoplasm into uninucleate zoospores or release of normal zoospores, b direct germination of sporangia, and c afunction in zoospore-mediated plant infection. Our data indicate that the PsMYB1 transcription factor functions downstream of MAP kinase PsSAK1 and is required for zoospore development of P. sojae.

  9. Part I---Evaluating Effects of Oligomer Formation on Cytochrome P450 2C9 Electron Transfer and Drug Metabolism, Part II---Utilizing Molecular Modeling Techniques to Study the Src-Interacting Proteins Actin Filament Associated Protein of 110 kDa (AFAP-110) and Cortactin

    Science.gov (United States)

    Jett, John Edward, Jr.

    nanopillars, the immobilization of CYP2C9 enzymes to those nanopillars, and the utilization of the array to perform conductive probe atomic force microscopy experiments examining the electron transfer process of CYP2C9 in the absence and presence of substrate molecules. Part II. The Src protein has been known to play a role in cancer cell progression for over 30 years. The function of a non-receptor tyrosine kinase such as Src is to relay extracellular signals through intracellular tyrosine phosphorylation. As a tyrosine kinase, Src and the cellular signaling pathways it is involved in play many functional roles in the cell, both in cellular proliferation and in cytoskeletal dynamics, cell adhesion, motility and invasion. Two of the many proteins comprising Src cellular signaling pathways are actin filament associated protein of 110 kDa (AFAP-110) and cortactin. AFAP-110 is a known activator of Src; one mechanism to abrogate the AFAP-110-induced activation of Src is to inhibit their colocalization within the cell. This colocalization is expected to occur when the pleckstrin homology (PH1 and PH2) domains of AFAP-110 are allowed to interact with membrane-bound phospholipids. Cortactin, on the other hand, is a cytosolic protein capable of being phosphorylated on various tyrosine residues, activating it and allowing it to interact with actin. The Src homology 2 (SH2) domain of Src has been shown to be capable of interacting with cortactin, an association which will be probed here. This section of the dissertation will discuss the use of molecular modeling techniques to develop structural models of the AFAP-110 PH1 and PH2 domains and use them to make predictions about how the protein interacts with phospholipids in the plasma membrane and how they might be stabilized to interact with other proteins. Structural models were designed using homology modeling methods, docking programs were used to predict key residues of AFAP-110 involved in binding to phospholipids and mutational

  10. A Conserved C-Terminal Domain of the Aspergillus fumigatus Developmental Regulator MedA Is Required for Nuclear Localization, Adhesion and Virulence

    Science.gov (United States)

    Al Abdallah, Qusai; Choe, Se-In; Campoli, Paolo; Baptista, Stefanie; Gravelat, Fabrice N.; Lee, Mark J.; Sheppard, Donald C.

    2012-01-01

    MedA is a developmental regulator that is conserved in the genome of most filamentous fungi. In the pathogenic fungus Aspergillus fumigatus MedA regulates conidiogenesis, adherence to host cells, and pathogenicity. The mechanism by which MedA governs these phenotypes remains unknown. Although the nuclear import of MedA orthologues has been reported in other fungi, no nuclear localization signal, DNA-binding domain or other conserved motifs have been identified within MedA. In this work, we performed a deletion analysis of MedA and identified a novel domain within the C-terminal region of the protein, designated MedA346–557, that is necessary and sufficient for nuclear localization of MedA. We further demonstrate that MedA nuclear localization is required for the function of MedA. Surprisingly, expression of the minimal nuclear localization fragment MedA346–557 alone was sufficient to restore conidogenesis, biofilm formation and virulence to the medA mutant strain. Collectively these results suggest that MedA functions in the regulation of transcription, and that the MedA346–557 domain is both necessary and sufficient to mediate MedA function. PMID:23185496

  11. NF1 regulation of RAS/ERK signaling is required for appropriate granule neuron progenitor expansion and migration in cerebellar development.

    Science.gov (United States)

    Sanchez-Ortiz, Efrain; Cho, Woosung; Nazarenko, Inga; Mo, Wei; Chen, Jian; Parada, Luis F

    2014-11-01

    Cerebellar development is regulated by a coordinated spatiotemporal interplay between granule neuron progenitors (GNPs), Purkinje neurons, and glia. Abnormal development can trigger motor deficits, and more recent data indicate important roles in aspects of memory, behavior, and autism spectrum disorders (ASDs). Germline mutation in the NF1 tumor suppressor gene underlies Neurofibromatosis type 1, a complex disease that enhances susceptibility to certain cancers and neurological disorders, including intellectual deficits and ASD. The NF1 gene encodes for neurofibromin, a RAS GTPase-activating protein, and thus negatively regulates the RAS signaling pathway. Here, using mouse models to direct conditional NF1 ablation in either embryonic cerebellar progenitors or neonatal GNPs, we show that neurofibromin is required for appropriate development of cerebellar folia layering and structure. Remarkably, neonatal administration of inhibitors of the ERK pathway reversed the morphological defects. Thus, our findings establish a critical cell-autonomous role for the NF1-RAS-ERK pathway in the appropriate regulation of cerebellar development and provide a basis for using neonatal ERK inhibitor-based therapies to treat NF1-induced cerebellar disorders.

  12. A LysR-family transcriptional regulator required for virulence in Brucella abortus is highly conserved among the α-proteobacteria.

    Science.gov (United States)

    Sheehan, Lauren M; Budnick, James A; Blanchard, Catlyn; Dunman, Paul M; Caswell, Clayton C

    2015-10-01

    Small RNAs are principal elements of bacterial gene regulation and physiology. Two small RNAs in Brucella abortus, AbcR1 and AbcR2, are required for wild-type virulence. Examination of the abcR loci revealed the presence of a gene encoding a LysR-type transcriptional regulator flanking abcR2 on chromosome 1. Deletion of this lysR gene (bab1_1517) resulted in the complete loss of abcR2 expression while no difference in abcR1 expression was observed. The B. abortus bab1_1517 mutant strain was significantly attenuated in macrophages and mice, and bab1_1517 was subsequently named vtlR for virulence-associated transcriptional LysR-family regulator. Microarray analysis revealed three additional genes encoding small hypothetical proteins also under the control of VtlR. Electrophoretic mobility shift assays demonstrated that VtlR binds directly to the promoter regions of abcR2 and the three hypothetical protein-encoding genes, and DNase I footprint analysis identified the specific nucleotide sequence in these promoters that VtlR binds to and drives gene expression. Strikingly, orthologs of VtlR are encoded in a wide range of host-associated α-proteobacteria, and it is likely that the VtlR genetic system represents a common regulatory circuit critical for host-bacterium interactions.

  13. Requirements for Foreign and Domestic Establishment Registration and Listing for Human Drugs, Including Drugs That Are Regulated Under a Biologics License Application, and Animal Drugs. Final rule.

    Science.gov (United States)

    2016-08-31

    The Food and Drug Administration (FDA) is amending its regulations governing drug establishment registration and drug listing. These amendments reorganize, modify, and clarify current regulations concerning who must register establishments and list human drugs, human drugs that are also biological products, and animal drugs. The final rule requires electronic submission, unless waived in certain circumstances, of registration and listing information. This rulemaking pertains to finished drug products and to active pharmaceutical ingredients (APIs) alone or together with one or more other ingredients. The final rule describes how and when owners or operators of establishments at which drugs are manufactured or processed must register their establishments with FDA and list the drugs they manufacture or process. In addition, the rule makes certain changes to the National Drug Code (NDC) system. We are taking this action to improve management of drug establishment registration and drug listing requirements and make these processes more efficient and effective for industry and for us. This action also supports implementation of the electronic prescribing provisions of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (MMA) and the availability of current drug labeling information through DailyMed, a computerized repository of drug information maintained by the National Library of Medicine.

  14. The Drosophila deoxyhypusine hydroxylase homologue nero and its target eIF5A are required for cell growth and the regulation of autophagy.

    Science.gov (United States)

    Patel, Prajal H; Costa-Mattioli, Mauro; Schulze, Karen L; Bellen, Hugo J

    2009-06-29

    Hypusination is a unique posttranslational modification by which lysine is transformed into the atypical amino acid hypusine. eIF5A (eukaryotic initiation factor 5A) is the only known protein to contain hypusine. In this study, we describe the identification and characterization of nero, the Drosophila melanogaster deoxyhypusine hydroxylase (DOHH) homologue. nero mutations affect cell and organ size, bromodeoxyuridine incorporation, and autophagy. Knockdown of the hypusination target eIF5A via RNA interference causes phenotypes similar to nero mutations. However, loss of nero appears to cause milder phenotypes than loss of eIF5A. This is partially explained through a potential compensatory mechanism by which nero mutant cells up-regulate eIF5A levels. The failure of eIF5A up-regulation to rescue nero mutant phenotypes suggests that hypusination is required for eIF5A function. Furthermore, expression of enzymatically impaired forms of DOHH fails to rescue nero clones, indicating that hypusination activity is important for nero function. Our data also indicate that nero and eIF5A are required for cell growth and affect autophagy and protein synthesis.

  15. Novel promoter sequence required for manganese regulation of manganese peroxidase isozyme 1 gene expression in Phanerochaete chrysosporium.

    Science.gov (United States)

    Ma, Biao; Mayfield, Mary B; Godfrey, Bruce J; Gold, Michael H

    2004-06-01

    Manganese peroxidase (MnP) is a major, extracellular component of the lignin-degrading system produced by the wood-rotting basidiomycetous fungus Phanerochaete chrysosporium. The transcription of MnP-encoding genes (mnps) in P. chrysosporium occurs as a secondary metabolic event, triggered by nutrient-nitrogen limitation. In addition, mnp expression occurs only under Mn2+ supplementation. Using a reporter system based on the enhanced green fluorescent protein gene (egfp), we have characterized the P. chrysosporium mnp1 promoter by examining the effects of deletion, replacement, and translocation mutations on mnp1 promoter-directed egfp expression. The 1,528-bp mnp1 promoter fragment drives egfp expression only under Mn2+-sufficient, nitrogen-limiting conditions, as required for endogenous MnP production. However, deletion of a 48-bp fragment, residing 521 bp upstream of the translation start codon in the mnp1 promoter, or replacement of this fragment with an unrelated sequence resulted in egfp expression under nitrogen limitation, both in the absence and presence of exogenous Mn2+. Translocation of the 48-bp fragment to a site 120 bp downstream of its original location resulted in Mn2+-dependent egfp expression under conditions similar to those observed with the wild-type mnp1 promoter. These results suggest that the 48-bp fragment contains at least one Mn2+-responsive cis element. Additional promoter-deletion experiments suggested that the Mn2+ element(s) is located within the 33-bp sequence at the 3' end of the 48-bp fragment. This is the first promoter sequence containing a Mn2+-responsive element(s) to be characterized in any eukaryotic organism. Copyright 2004 American Society for Microbiology

  16. A dietary polyphenol resveratrol acts to provide neuroprotection in recurrent stroke models by regulating AMPK and SIRT1 signaling, thereby reducing energy requirements during ischemia.

    Science.gov (United States)

    2016-04-01

    The above article from European Journal of Neuroscience, published online on 5 March 2013 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1111/ejn.12162/full), has been retracted by agreement between the Editors-in-Chief, Paul Bolam and John Foxe, the authors and John Wiley & Sons Ltd. The retraction has been agreed as Dr Phillip Barber has informed the publisher that he had seen neither the original data nor any version of the manuscript, and had not been involved in the work reported. A subsequent Institutional investigation found evidence of misconduct on the part of the submitting author. Reference Wang, L.-M., Wang, Y.-J., Cui, M., Luo, W.-J., Wang, X.-J., Barber, P.A. & Chen, Z.-Y. (2013) A dietary polyphenol resveratrol acts to provide neuroprotection in recurrent stroke models by regulating AMPK and SIRT1 signaling, thereby reducing energy requirements during ischemia.

  17. The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and filamin A.

    Science.gov (United States)

    Tu, Chia-Ling; Chang, Wenhan; Bikle, Daniel D

    2011-05-01

    Extracellular Ca(2+) (Ca(2+)(o)) functioning through the calcium-sensing receptor (CaR) induces E-cadherin-mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that CaR regulates cell-cell adhesion through Fyn/Src tyrosine kinases. In this study, we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca(2+)(o)-induced association of Fyn with E-cadherin and suppressed the Ca(2+)(o)-induced tyrosine phosphorylation of β-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca(2+)(o)-stimulated expression of terminal differentiation markers. Elevating the Ca(2+)(o) level induced interactions among CaR, Rho A, E-cadherin, and the scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense complementary DNA inhibited Ca(2+)(o)-induced activation of endogenous Rho. Ca(2+)(o) activation of Rho required a direct interaction between CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca(2+)(o)-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca(2+)(o)-induced E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.

  18. Proper actin ring formation and septum constriction requires coordinated regulation of SIN and MOR pathways through the germinal centre kinase MST-1.

    Science.gov (United States)

    Heilig, Yvonne; Dettmann, Anne; Mouriño-Pérez, Rosa R; Schmitt, Kerstin; Valerius, Oliver; Seiler, Stephan

    2014-04-01

    Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in Δmst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1-dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes.

  19. Proper actin ring formation and septum constriction requires coordinated regulation of SIN and MOR pathways through the germinal centre kinase MST-1.

    Directory of Open Access Journals (Sweden)

    Yvonne Heilig

    2014-04-01

    Full Text Available Nuclear DBF2p-related (NDR kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR and septation initiation (SIN networks and are activated by pathway-specific germinal centre (GC kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in Δmst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1-dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes.

  20. Regulation of abiotic stress signalling by Arabidopsis C-terminal domain phosphatase-like 1 requires interaction with a k-homology domain-containing protein.

    Directory of Open Access Journals (Sweden)

    In Sil Jeong

    Full Text Available Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD PHOSPHATASE-LIKE 1 (CPL1 regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds RNA binding motifs (dsRBMs at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3 as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.

  1. Regulation of Monocarboxylic Acid Transporter 1 Trafficking by the Canonical Wnt/β-Catenin Pathway in Rat Brain Endothelial Cells Requires Cross-talk with Notch Signaling.

    Science.gov (United States)

    Liu, Zejian; Sneve, Mary; Haroldson, Thomas A; Smith, Jeffrey P; Drewes, Lester R

    2016-04-01

    The transport of monocarboxylate fuels such as lactate, pyruvate, and ketone bodies across brain endothelial cells is mediated by monocarboxylic acid transporter 1 (MCT1). Although the canonical Wnt/β-catenin pathway is required for rodent blood-brain barrier development and for the expression of associated nutrient transporters, the role of this pathway in the regulation of brain endothelial MCT1 is unknown. Here we report expression of nine members of the frizzled receptor family by the RBE4 rat brain endothelial cell line. Furthermore, activation of the canonical Wnt/β-catenin pathway in RBE4 cells via nuclear β-catenin signaling with LiCl does not alter brain endothelialMct1mRNA but increases the amount of MCT1 transporter protein. Plasma membrane biotinylation studies and confocal microscopic examination of mCherry-tagged MCT1 indicate that increased transporter results from reduced MCT1 trafficking from the plasma membrane via the endosomal/lysosomal pathway and is facilitated by decreased MCT1 ubiquitination following LiCl treatment. Inhibition of the Notch pathway by the γ-secretase inhibitorN-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester negated the up-regulation of MCT1 by LiCl, demonstrating a cross-talk between the canonical Wnt/β-catenin and Notch pathways. Our results are important because they show, for the first time, the regulation of MCT1 in cerebrovascular endothelial cells by the multifunctional canonical Wnt/β-catenin and Notch signaling pathways.

  2. The master regulator for biofilm formation in Bacillus subtilis governs the expression of an operon encoding secreted proteins required for the assembly of complex multicellular communities.

    Energy Technology Data Exchange (ETDEWEB)

    Branda, Steven S. (Harvard Medical School, Cambridge, MA); Losick, Richard (Harvard University, Cambridge, MA); Kolter, Roberto (Harvard Medical School, Cambridge, MA); Kearns, Daniel B. (Harvard University, Cambridge, MA); Chu, Frances (Harvard University, Cambridge, MA)

    2005-08-01

    Wild strains of Bacillus subtilis are capable of forming architecturally complex communities of cells known as biofilms. Critical to biofilm formation is the eps operon, which is believed to be responsible for the biosynthesis of an exopolysaccharide that binds chains of cells together in bundles. We report that transcription of eps is under the negative regulation of SinR, a repressor that was found to bind to multiple sites in the regulatory region of the operon. Mutations in sinR bypassed the requirement in biofilm formation of two genes of unknown function, ylbF and ymcA, and sinI, which is known to encode an antagonist of SinR. We propose that these genes are members of a pathway that is responsible for counteracting SinR-mediated repression. We further propose that SinR is a master regulator that governs the transition between a planktonic state in which the bacteria swim as single cells in liquid or swarm in small groups over surfaces, and a sessile state in which the bacteria adhere to each other to form bundled chains and assemble into multicellular communities.

  3. Casein kinase II is required for proper cell division and acts as a negative regulator of centrosome duplication in Caenorhabditis elegans embryos

    Science.gov (United States)

    Medley, Jeffrey C.; Kabara, Megan M.; Stubenvoll, Michael D.; DeMeyer, Lauren E.

    2017-01-01

    ABSTRACT Centrosomes are the primary microtubule-organizing centers that orchestrate microtubule dynamics during the cell cycle. The correct number of centrosomes is pivotal for establishing bipolar mitotic spindles that ensure accurate segregation of chromosomes. Thus, centrioles must duplicate once per cell cycle, one daughter per mother centriole, the process of which requires highly coordinated actions among core factors and modulators. Protein phosphorylation is shown to regulate the stability, localization and activity of centrosome proteins. Here, we report the function of Casein kinase II (CK2) in early Caenorhabditis elegans embryos. The catalytic subunit (KIN-3/CK2α) of CK2 localizes to nuclei, centrosomes and midbodies. Inactivating CK2 leads to cell division defects, including chromosome missegregation, cytokinesis failure and aberrant centrosome behavior. Furthermore, depletion or inhibiting kinase activity of CK2 results in elevated ZYG-1 levels at centrosomes, restoring centrosome duplication and embryonic viability to zyg-1 mutants. Our data suggest that CK2 functions in cell division and negatively regulates centrosome duplication in a kinase-dependent manner. PMID:27881437

  4. Sgs1's roles in DNA end resection, HJ dissolution, and crossover suppression require a two-step SUMO regulation dependent on Smc5/6.

    Science.gov (United States)

    Bermúdez-López, Marcelino; Villoria, María Teresa; Esteras, Miguel; Jarmuz, Adam; Torres-Rosell, Jordi; Clemente-Blanco, Andres; Aragon, Luis

    2016-06-01

    The RecQ helicase Sgs1 plays critical roles during DNA repair by homologous recombination, from end resection to Holliday junction (HJ) dissolution. Sgs1 has both pro- and anti-recombinogenic roles, and therefore its activity must be tightly regulated. However, the controls involved in recruitment and activation of Sgs1 at damaged sites are unknown. Here we show a two-step role for Smc5/6 in recruiting and activating Sgs1 through SUMOylation. First, auto-SUMOylation of Smc5/6 subunits leads to recruitment of Sgs1 as part of the STR (Sgs1-Top3-Rmi1) complex, mediated by two SUMO-interacting motifs (SIMs) on Sgs1 that specifically recognize SUMOylated Smc5/6. Second, Smc5/6-dependent SUMOylation of Sgs1 and Top3 is required for the efficient function of STR. Sgs1 mutants impaired in recognition of SUMOylated Smc5/6 (sgs1-SIMΔ) or SUMO-dead alleles (sgs1-KR) exhibit unprocessed HJs at damaged replication forks, increased crossover frequencies during double-strand break repair, and severe impairment in DNA end resection. Smc5/6 is a key regulator of Sgs1's recombination functions.

  5. The Acinetobacter baumannii Two-Component System AdeRS Regulates Genes Required for Multidrug Efflux, Biofilm Formation, and Virulence in a Strain-Specific Manner

    Directory of Open Access Journals (Sweden)

    Grace E. Richmond

    2016-04-01

    Full Text Available The opportunistic pathogen Acinetobacter baumannii is able to persist in the environment and is often multidrug resistant (MDR, causing difficulties in the treatment of infections. Here, we show that the two-component system AdeRS, which regulates the production of the AdeABC multidrug resistance efflux pump, is required for the formation of a protective biofilm in an ex vivo porcine mucosal model, which mimics a natural infection of the human epithelium. Interestingly, deletion of adeB impacted only on the ability of strain AYE to form a biofilm on plastic and only on the virulence of strain Singapore 1 for Galleria mellonella. RNA-Seq revealed that loss of AdeRS or AdeB significantly altered the transcriptional landscape, resulting in the changed expression of many genes, notably those associated with antimicrobial resistance and virulence interactions. For example, A. baumannii lacking AdeRS displayed decreased expression of adeABC, pil genes, com genes, and a pgaC-like gene, whereas loss of AdeB resulted in increased expression of pil and com genes and decreased expression of ferric acinetobactin transport system genes. These data define the scope of AdeRS-mediated regulation, show that changes in the production of AdeABC mediate important phenotypes controlled by AdeRS, and suggest that AdeABC is a viable target for antimicrobial drug and antibiofilm discovery.

  6. The Acinetobacter baumannii Two-Component System AdeRS Regulates Genes Required for Multidrug Efflux, Biofilm Formation, and Virulence in a Strain-Specific Manner

    Science.gov (United States)

    Richmond, Grace E.; Evans, Laura P.; Anderson, Michele J.; Wand, Matthew E.; Bonney, Laura C.; Ivens, Alasdair; Chua, Kim Lee; Webber, Mark A.; Sutton, J. Mark; Peterson, Marnie L.

    2016-01-01

    ABSTRACT The opportunistic pathogen Acinetobacter baumannii is able to persist in the environment and is often multidrug resistant (MDR), causing difficulties in the treatment of infections. Here, we show that the two-component system AdeRS, which regulates the production of the AdeABC multidrug resistance efflux pump, is required for the formation of a protective biofilm in an ex vivo porcine mucosal model, which mimics a natural infection of the human epithelium. Interestingly, deletion of adeB impacted only on the ability of strain AYE to form a biofilm on plastic and only on the virulence of strain Singapore 1 for Galleria mellonella. RNA-Seq revealed that loss of AdeRS or AdeB significantly altered the transcriptional landscape, resulting in the changed expression of many genes, notably those associated with antimicrobial resistance and virulence interactions. For example, A. baumannii lacking AdeRS displayed decreased expression of adeABC, pil genes, com genes, and a pgaC-like gene, whereas loss of AdeB resulted in increased expression of pil and com genes and decreased expression of ferric acinetobactin transport system genes. These data define the scope of AdeRS-mediated regulation, show that changes in the production of AdeABC mediate important phenotypes controlled by AdeRS, and suggest that AdeABC is a viable target for antimicrobial drug and antibiofilm discovery. PMID:27094331

  7. Zebrafish cyclin Dx is required for development of motor neuron progenitors, and its expression is regulated by hypoxia-inducible factor 2α.

    Science.gov (United States)

    Lien, Huang-Wei; Yuan, Rey-Yue; Chou, Chih-Ming; Chen, Yi-Chung; Hung, Chin-Chun; Hu, Chin-Hwa; Hwang, Sheng-Ping L; Hwang, Pung-Pung; Shen, Chia-Ning; Chen, Chih-Lung; Cheng, Chia-Hsiung; Huang, Chang-Jen

    2016-06-21

    Cyclins play a central role in cell-cycle regulation; in mammals, the D family of cyclins consists of cyclin D1, D2, and D3. In Xenopus, only homologs of cyclins D1 and D2 have been reported, while a novel cyclin, cyclin Dx (ccndx), was found to be required for the maintenance of motor neuron progenitors during embryogenesis. It remains unknown whether zebrafish possess cyclin D3 or cyclin Dx. In this study, we identified a zebrafish ccndx gene encoding a protein which can form a complex with Cdk4. Through whole-mount in situ hybridization, we observed that zccndx mRNA is expressed in the motor neurons of hindbrain and spinal cord during development. Analysis of a 4-kb promoter sequence of the zccndx gene revealed the presence of HRE sites, which can be regulated by HIF2α. Morpholino knockdown of zebrafish Hif2α and cyclin Dx resulted in the abolishment of isl1 and oligo2 expression in the precursors of motor neurons, and also disrupted axon growth. Overexpression of cyclin Dx mRNA in Hif2α morphants partially rescued zccndx expression. Taken together, our data indicate that zebrafish cyclin Dx plays a role in maintaining the precursors of motor neurons.

  8. AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation.

    Science.gov (United States)

    Ghaffari, Saghi; Kitidis, Claire; Zhao, Wei; Marinkovic, Dragan; Fleming, Mark D; Luo, Biao; Marszalek, Joseph; Lodish, Harvey F

    2006-03-01

    AKT serine threonine kinase of the protein kinase B (PKB) family plays essential roles in cell survival, growth, metabolism, and differentiation. In the erythroid system, AKT is known to be rapidly phosphorylated and activated in response to erythropoietin (Epo) engagement of Epo receptor (EpoR) and to sustain survival signals in cultured erythroid cells. Here we demonstrate that activated AKT complements EpoR signaling and supports erythroid-cell differentiation in wild-type and JAK2-deficient fetal liver cells. We show that erythroid maturation of AKT-transduced cells is not solely dependent on AKT-induced cell survival or proliferation signals, suggesting that AKT transduces also a differentiation-specific signal downstream of EpoR in erythroid cells. Down-regulation of expression of AKT kinase by RNA interference, or AKT activity by expression of dominant negative forms, inhibits significantly fetal liver-derived erythroid-cell colony formation and gene expression, demonstrating that AKT is required for Epo regulation of erythroid-cell maturation.

  9. Fibroblast growth factor 21 regulates lipolysis in white adipose tissue but is not required for ketogenesis and triglyceride clearance in liver.

    Science.gov (United States)

    Hotta, Yuhei; Nakamura, Hirotoshi; Konishi, Morichika; Murata, Yusuke; Takagi, Hiroyuki; Matsumura, Shigenobu; Inoue, Kazuo; Fushiki, Tohru; Itoh, Nobuyuki

    2009-10-01

    Fibroblast growth factors (Fgfs) are polypeptide growth factors with diverse functions. Fgf21, a unique member of the Fgf family, is expected to function as a metabolic regulator in an endocrine manner. Hepatic Fgf21 expression was increased by fasting. The phenotypes of hepatic Fgf21 transgenic or knockdown mice and high-fat, low-carbohydrate ketogenic diet-fed mice suggests that Fgf21 stimulates lipolysis in the white adipose tissue during normal feeding and is required for ketogenesis and triglyceride clearance in the liver during fasting. However, the physiological roles of Fgf21 remain unclear. To elucidate the physiological roles of Fgf21, we generated Fgf21 knockout (KO) mice by targeted disruption. Fgf21 KO mice were viable, fertile, and seemingly normal. Food intake, oxygen consumption, and energy expenditure were also essentially unchanged in Fgf21 KO mice. However, hypertrophy of adipocytes, decreased lipolysis in adipocytes, and decreased blood nonesterified fatty acid levels were observed when Fgf21 KO mice were fed normally. In contrast, increased lipolysis in adipocytes and increased blood nonesterified fatty acid levels were observed in Fgf21 KO mice by fasting for 24 h, indicating that Fgf21 stimulates lipolysis in the white adipose tissue during feeding but inhibits it during fasting. In contrast, unexpectedly, hepatic triglyceride levels were essentially unchanged in Fgf21 KO mice. In addition, ketogenesis in Fgf21 KO mice was not impaired by fasting for 24 h. The present results indicate that Fgf21 regulates lipolysis in adipocytes in response to the metabolic state but is not required for ketogenesis and triglyceride clearance in the liver.

  10. Calcium-regulation of mitochondrial respiration maintains ATP homeostasis and requires ARALAR/AGC1-malate aspartate shuttle in intact cortical neurons.

    Science.gov (United States)

    Llorente-Folch, Irene; Rueda, Carlos B; Amigo, Ignacio; del Arco, Araceli; Saheki, Takeyori; Pardo, Beatriz; Satrústegui, Jorgina

    2013-08-28

    Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse cortical neurons in basal conditions and in response to increased workload caused by increases in [Na+]cyt (veratridine, high-K+ depolarization) and/or [Ca2+]cyt (carbachol). Respiration in nonstimulated neurons on 2.5-5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in [Ca2+]mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads.

  11. Tomato LeTHIC is an Fe-requiring HMP-P synthase involved in thiamine synthesis and regulated by multiple factors.

    Science.gov (United States)

    Zhao, Weina; Cheng, Xudong; Huang, Zongan; Fan, Huajie; Wu, Huilan; Ling, Hong-Qing

    2011-06-01

    Thiamine is a key primary metabolite which is necessary for the viability of all organisms. It is a dietary requirement for mammals because only prokaryotes, fungi and plants are thiamine prototrophs. In contrast to the well documented biosynthetic mechanism in bacteria, much remains to be deciphered in plants. In this work, a tomato thiamine-auxotrophic (thiamineless, tl) mutant was characterized. The tl mutant occurs due to inactivation of LeTHIC transcription as a result of insertion of a large unknown DNA fragment in its 5'-untranslated region. Expression of wild-type LeTHIC in tl plants was able to complement the mutant to wild type. LeTHIC possessed the same function as E.cTHIC [an Escherichia coli 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase involved in synthesis of the pyrimidine moiety of thiamine] because expression of LeTHIC rescued THIC-deficient strains of E. coli under culture conditions without thiamine supplementation, suggesting that plants employ a bacteria-like route of pyrimidine moiety synthesis. LeTHIC is an Fe-S cluster protein localized in chloroplasts, and Fe is required for maintenance of its enzyme activity because Fe deficiency resulted in a significant reduction of thiamine content in tomato leaves. Further, we also showed that the expression of LeTHIC is tightly regulated at the transcriptional and post-transcriptional level by multiple factors, such as light, Fe status and thiamine pyrophosphate (TPP)-riboswitch. The results clearly demonstrated that a feedback regulation mechanism is involved in synthesis of the pyrimidine moiety for controlling thiamine synthesis in tomato. Our results provide a new insight into understanding the molecular mechanism of thiamine biosynthesis in plants.

  12. TtsI, a key regulator of Rhizobium species NGR234 is required for type III-dependent protein secretion and synthesis of rhamnose-rich polysaccharides.

    Science.gov (United States)

    Marie, Corinne; Deakin, William J; Ojanen-Reuhs, Tuula; Diallo, Ericka; Reuhs, Brad; Broughton, William J; Perret, Xavier

    2004-09-01

    Formation of nitrogen-fixing nodules on legume roots by Rhizobium sp. NGR234 requires an array of bacterial factors, including nodulation outer proteins (Nops) secreted through a type III secretion system (TTSS). Secretion of Nops is abolished upon inactivation of ttsI (formerly y4xI), a protein with characteristics of two-component response regulators that was predicted to activate transcription of TTSS-related genes. During the symbiotic interaction, the phenotype of NGR omega ttsI differs from that of a mutant with a nonfunctional secretion machine, however. This indicated that TtsI regulates the synthesis of other symbiotic factors as well. Conserved sequences, called tts boxes, proposed to act as binding sites for TtsI, were identified not only within the TTSS cluster but also in the promoter regions of i) genes predicted to encode homologs of virulence factors secreted by pathogenic bacteria, ii) loci involved in the synthesis of a rhamnose-rich component (rhamnan) of the lipopolysaccharides (LPS), and iii) open reading frames that play roles in plasmid partitioning. Transcription studies showed that TtsI and tts boxes are required for the activation of TTSS-related genes and those involved in rhamnose synthesis. Furthermore, extraction of polysaccharides revealed that inactivation of ttsI abolishes the synthesis of the rhamnan component of the LPS. The phenotypes of mutants impaired in TTSS-dependent protein secretion, rhamnan synthesis, or in both functions were compared to assess the roles of some of the TtsI-controlled factors during symbiosis.

  13. Fragile X mental retardation protein is required for rapid experience-dependent regulation of the potassium channel Kv3.1b.

    Science.gov (United States)

    Strumbos, John G; Brown, Maile R; Kronengold, Jack; Polley, Daniel B; Kaczmarek, Leonard K

    2010-08-04

    Fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates synaptic plasticity by repressing translation of specific mRNAs. We found that FMRP binds mRNA encoding the voltage-gated potassium channel Kv3.1b in brainstem synaptosomes. To explore the regulation of Kv3.1b by FMRP, we investigated Kv3.1b immunoreactivity and potassium currents in the auditory brainstem sound localization circuit of male mice. The unique features of this circuit allowed us to control neuronal activity in vivo by exposing animals to high-frequency, amplitude-modulated stimuli, which elicit predictable and stereotyped patterns of input to the anterior ventral cochlear nucleus (AVCN) and medial nucleus of the trapezoid body (MNTB). In wild-type (WT) animals, Kv3.1b is expressed along a tonotopic gradient in the MNTB, with highest levels in neurons at the medial, high-frequency end. At baseline, Fmr1(-/-) mice, which lack FMRP, displayed dramatically flattened tonotopicity in Kv3.1b immunoreactivity and K(+) currents relative to WT controls. Moreover, after 30 min of acoustic stimulation, levels of Kv3.1b immunoreactivity were significantly elevated in both the MNTB and AVCN of WT, but not Fmr1(-/-), mice. These results suggest that FMRP is necessary for maintenance of the gradient in Kv3.1b protein levels across the tonotopic axis of the MNTB, and are consistent with a role for FMRP as a repressor of protein translation. Using numerical simulations, we demonstrate that Kv3.1b tonotopicity may be required for accurate encoding of stimulus features such as modulation rate, and that disruption of this gradient, as occurs in Fmr1(-/-) animals, degrades processing of this information.

  14. Requirements of Multiple Domains of SLI-1, a Caenorhabditis elegans Homologue of c-Cbl, and an Inhibitory Tyrosine in LET-23 in Regulating Vulval Differentiation

    Science.gov (United States)

    Yoon, Charles H.; Chang, Chieh; Hopper, Neil A.; Lesa, Giovanni M.; Sternberg, Paul W.

    2000-01-01

    SLI-1, a Caenorhabditis elegans homologue of the proto-oncogene product c-Cbl, is a negative regulator of LET-23-mediated vulval differentiation. Lack of SLI-1 activity can compensate for decreased function of the LET-23 epidermal growth factor receptor, the SEM-5 adaptor, but not the LET-60 RAS, suggesting that SLI-1 acts before RAS activation. SLI-1 and c-Cbl comprise an N-terminal region (termed SLI-1:N/Cbl-N, containing a four-helix bundle, an EF hand calcium-binding domain, and a divergent SH2 domain) followed by a RING finger domain and a proline-rich C-terminus. In a transgenic functional assay, the proline-rich C-terminal domain is not essential for sli-1(+) function. A protein lacking the SH2 and RING finger domains has no activity, but a chimeric protein with the SH2 and RING finger domains of SLI-1 replaced by the equivalent domains of c-Cbl has activity. The RING finger domain of c-Cbl has been shown recently to enhance ubiquitination of active RTKs by acting as an E3 ubiquitin–protein ligase. We find that the RING finger domain of SLI-1 is partially dispensable. Further, we identify an inhibitory tyrosine of LET-23 requiring sli-1(+) for its effects: removal of this tyrosine closely mimics the loss of sli-1 but not of another negative regulator, ark-1. Thus, we suggest that this inhibitory tyrosine mediates its effects through SLI-1, which in turn inhibits signaling upstream of LET-60 RAS in a manner not wholly dependent on the ubiquitin–ligase domain. PMID:11071924

  15. A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

    Science.gov (United States)

    Catling, A D; Schaeffer, H J; Reuter, C W; Reddy, G R; Weber, M J

    1995-10-01

    Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

  16. CYB5D2 requires heme-binding to regulate HeLa cell growth and confer survival from chemotherapeutic agents.

    Directory of Open Access Journals (Sweden)

    Anthony Bruce

    Full Text Available The cytochrome b5 domain containing 2 (CYB5D2; Neuferricin protein has been reported to bind heme, however, the critical residues responsible for heme-binding are undefined. Furthermore, the relationship between heme-binding and CYB5D2-mediated intracellular functions remains unknown. Previous studies examining heme-binding in two cytochrome b5 heme-binding domain-containing proteins, damage-associated protein 1 (Dap1; Saccharomyces cerevisiae and human progesterone receptor membrane component 1 (PGRMC1, have revealed that conserved tyrosine (Y 73, Y79, aspartic acid (D 86, and Y127 residues present in human CYB5D2 may be involved in heme-binding. CYB5D2 binds to type b heme, however, only the substitution of glycine (G at D86 (D86G within its cytochrome b5 heme-binding (cyt-b5 domain abolished its heme-binding ability. Both CYB5D2 and CYB5D2(D86G localize to the endoplasmic reticulum. Ectopic CYB5D2 expression inhibited cell proliferation and anchorage-independent colony growth of HeLa cells. Conversely, CYB5D2 knockdown and ectopic CYB5D2(D86G expression increased cell proliferation and colony growth. As PGRMC1 has been reported to regulate the expression and activities of cytochrome P450 proteins (CYPs, we examined the role of CYB5D2 in regulating the activities of CYPs involved in sterol synthesis (CYP51A1 and drug metabolism (CYP3A4. CYB5D2 co-localizes with cytochrome P450 reductase (CYPOR, while CYB5D2 knockdown reduced lanosterol demethylase (CYP51A1 levels and rendered HeLa cells sensitive to mevalonate. Additionally, knockdown of CYB5D2 reduced CYP3A4 activity. Lastly, CYB5D2 expression conferred HeLa cell survival from chemotherapeutic agents (paclitaxel, cisplatin and doxorubicin, with its ability to promote survival being dependent on its heme-binding ability. Taken together, this study provides evidence that heme-binding is critical for CYB5D2 in regulating HeLa cell growth and survival, with endogenous CYB5D2 being required to

  17. Iron-regulated metabolites produced by Pseudomonas fluorescens WCS374r are not required for eliciting induced systemic resistance against Pseudomonas syringae pv. tomato in Arabidopsis.

    Science.gov (United States)

    Djavaheri, Mohammad; Mercado-Blanco, Jesús; Versluis, C; Meyer, J-M; Loon, L C; Bakker, Peter A H M

    2012-09-01

    The plant growth-promoting rhizobacterium Pseudomonas fluorescens WCS374r produces several iron-regulated metabolites, including the fluorescent siderophore pseudobactin (Psb374), salicylic acid (SA), and pseudomonine (Psm), a siderophore that contains a SA moiety. After purification of Psb374 from culture supernatant of WCS374r, its structure was determined following isoelectrofocusing and tandem mass spectrometry, and found to be identical to the fluorescent siderophore produced by P. fluorescens ATCC 13525. To study the role of SA and Psm production in colonization of Arabidopsis thaliana roots and in induced systemic resistance (ISR) against Pseudomonas syringae pv. tomato (Pst) by strain WCS374r, mutants disrupted in the production of these metabolites were obtained by homologous recombination. These mutants were further subjected to transposon Tn5 mutagenesis to generate mutants also deficient in Psb374 production. The mutants behaved similar to the wild type in both their Arabidopsis rhizosphere-colonizing capacity and their ability to elicit ISR against Pst. We conclude that Psb374, SA, and Psm production by P. fluorescens WCS374r are not required for eliciting ISR in Arabidopsis.

  18. Efficient design and simulation of an expandable hybrid (wind-photovoltaic) power system with MPPT and inverter input voltage regulation features in compliance with electric grid requirements

    Energy Technology Data Exchange (ETDEWEB)

    Skretas, Sotirios B.; Papadopoulos, Demetrios P. [Electrical Machines Laboratory, Department of Electrical and Computer Engineering, Democritos University of Thrace (DUTH), 12 V. Sofias, 67100 Xanthi (Greece)

    2009-09-15

    In this paper an efficient design along with modeling and simulation of a transformer-less small-scale centralized DC - bus Grid Connected Hybrid (Wind-PV) power system for supplying electric power to a single phase of a three phase low voltage (LV) strong distribution grid are proposed and presented. The main components of the hybrid system are: a PV generator (PVG); and an array of horizontal-axis, fixed-pitch, small-size, variable-speed wind turbines (WTs) with direct-driven permanent magnet synchronous generator (PMSG) having an embedded uncontrolled bridge rectifier. An overview of the basic theory of such systems along with their modeling and simulation via Simulink/MATLAB software package are presented. An intelligent control method is applied to the proposed configuration to simultaneously achieve three desired goals: to extract maximum power from each hybrid power system component (PVG and WTs); to guarantee DC voltage regulation/stabilization at the input of the inverter; to transfer the total produced electric power to the electric grid, while fulfilling all necessary interconnection requirements. Finally, a practical case study is conducted for the purpose of fully evaluating a possible installation in a city site of Xanthi/Greece, and the practical results of the simulations are presented. (author)

  19. Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA.

    Science.gov (United States)

    Schertzer, Michael; Jouravleva, Karina; Perderiset, Mylene; Dingli, Florent; Loew, Damarys; Le Guen, Tangui; Bardoni, Barbara; de Villartay, Jean-Pierre; Revy, Patrick; Londoño-Vallejo, Arturo

    2015-02-18

    Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Enterohemorrhagic Escherichia coli O157:H7 requires quorum sensing transcriptional regulators QseA and SdiA for colonization and persistence in the bovine intestinal tract

    Science.gov (United States)

    QseA and SdiA are two of several transcriptional regulators that regulate virulence gene expression of enterohemorrhagic Escherichia coli (EHEC) O157:H7 via quorum sensing (QS). QseA regulates the expression of the locus of enterocyte effacement (LEE). LEE encodes for a type III secretion (T3S) sys...

  1. The Epstein-Barr virus (EBV) glycoprotein B cytoplasmic C-terminal tail domain regulates the energy requirement for EBV-induced membrane fusion.

    Science.gov (United States)

    Chen, Jia; Zhang, Xianming; Jardetzky, Theodore S; Longnecker, Richard

    2014-10-01

    The entry of enveloped viruses into host cells is preceded by membrane fusion, which in Epstein-Barr virus (EBV) is thought to be mediated by the refolding of glycoprotein B (gB) from a prefusion to a postfusion state. In our current studies, we characterized a gB C-terminal tail domain (CTD) mutant truncated at amino acid 843 (gB843). This truncation mutant is hyperfusogenic as monitored by syncytium formation and in a quantitative fusion assay and is dependent on gH/gL for fusion activity. gB843 can rescue the fusion function of other glycoprotein mutants that have null or decreased fusion activity in epithelial and B cells. In addition, gB843 requires less gp42 and gH/gL for fusion, and can function in fusion at a lower temperature than wild-type gB, indicating a lower energy requirement for fusion activation. Since a key step in fusion is the conversion of gB from a prefusion to an active postfusion state by gH/gL, gB843 may access this activated gB state more readily. Our studies indicate that the gB CTD may participate in the fusion function by maintaining gB in an inactive prefusion form prior to activation by receptor binding. Importance: Diseases resulting from Epstein-Barr virus (EBV) infection in humans range from the fairly benign disease infectious mononucleosis to life-threatening cancer. As an enveloped virus, EBV must fuse with a host cell membrane for entry and infection by using glycoproteins gH/gL, gB, and gp42. Among these glycoproteins, gB is thought to be the protein that executes fusion. To further characterize the function of the EBV gB cytoplasmic C-terminal tail domain (CTD) in fusion, we used a previously constructed CTD truncation mutant and studied its fusion activity in the context of other EBV glycoprotein mutants. From these studies, we find that the gB CTD regulates fusion by altering the energy requirements for the triggering of fusion mediated by gH/gL or gp42. Overall, our studies may lead to a better understanding of EBV fusion

  2. CAP1, an adenylate cyclase-associated protein gene, regulates bud-hypha transitions, filamentous growth, and cyclic AMP levels and is required for virulence of Candida albicans.

    Science.gov (United States)

    Bahn, Y S; Sundstrom, P

    2001-05-01

    In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.

  3. Intermittent hypoxia-induced endothelial barrier dysfunction requires ROS-dependent MAP kinase activation.

    Science.gov (United States)

    Makarenko, Vladislav V; Usatyuk, Peter V; Yuan, Guoxiang; Lee, May M; Nanduri, Jayasri; Natarajan, Viswanathan; Kumar, Ganesh K; Prabhakar, Nanduri R

    2014-04-15

    The objective of the present study was to determine the impact of simulated apnea with intermittent hypoxia (IH) on endothelial barrier function and assess the underlying mechanism(s). Experiments were performed on human lung microvascular endothelial cells exposed to IH-consisting alternating cycles of 1.5% O2 for 30s followed by 20% O2 for 5 min. IH decreased transendothelial electrical resistance (TEER) suggesting attenuated endothelial barrier function. The effect of IH on TEER was stimulus dependent and reversible after reoxygenation. IH-exposed cells exhibited stress fiber formation and redistribution of cortactin, vascular endothelial-cadherins, and zona occludens-1 junction proteins along with increased intercellular gaps at cell-cell boundaries. Extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) were phosphorylated in IH-exposed cells. Inhibiting either ERK or JNK prevented the IH-induced decrease in TEER and the reorganization of the cytoskeleton and junction proteins. IH increased reactive oxygen species (ROS) levels, and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride, a membrane-permeable antioxidant, prevented ERK and JNK phosphorylation as well as IH-induced changes in endothelial barrier function. These results demonstrate that IH via ROS-dependent activation of MAP kinases leads to reorganization of cytoskeleton and junction proteins resulting in endothelial barrier dysfunction.

  4. Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation and is also required for efficient chemotaxis.

    Science.gov (United States)

    Mohanty, S; Jermyn, K A; Early, A; Kawata, T; Aubry, L; Ceccarelli, A; Schaap, P; Williams, J G; Firtel, R A

    1999-08-01

    Dd-STATa is a structural and functional homologue of the metazoan STAT (Signal Transducer and Activator of Transcription) proteins. We show that Dd-STATa null cells exhibit several distinct developmental phenotypes. The aggregation of Dd-STATa null cells is delayed and they chemotax slowly to a cyclic AMP source, suggesting a role for Dd-STATa in these early processes. In Dd-STATa null strains, slug-like structures are formed but they have an aberrant pattern of gene expression. In such slugs, ecmB/lacZ, a marker that is normally specific for cells on the stalk cell differentiation pathway, is expressed throughout the prestalk region. Stalk cell differentiation in Dictyostelium has been proposed to be under negative control, mediated by repressor elements present in the promoters of stalk cell-specific genes. Dd-STATa binds these repressor elements in vitro and the ectopic expression of ecmB/lacZ in the null strain provides in vivo evidence that Dd-STATa is the repressor protein that regulates commitment to stalk cell differentiation. Dd-STATa null cells display aberrant behavior in a monolayer assay wherein stalk cell differentiation is induced using the stalk cell morphogen DIF. The ecmB gene, a general marker for stalk cell differentiation, is greatly overinduced by DIF in Dd-STATa null cells. Also, Dd-STATa null cells are hypersensitive to DIF for expression of ST/lacZ, a marker for the earliest stages in the differentiation of one of the stalk cell sub-types. We suggest that both these manifestations of DIF hypersensitivity in the null strain result from the balance between activation and repression of the promoter elements being tipped in favor of activation when the repressor is absent. Paradoxically, although Dd-STATa null cells are hypersensitive to the inducing effects of DIF and readily form stalk cells in monolayer assay, the Dd-STATa null cells show little or no terminal stalk cell differentiation within the slug. Dd-STATa null slugs remain

  5. Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6

    OpenAIRE

    Hidalgo-Carcedo, Cristina; Hooper, Steven; Chaudhry, Shahid I.; Williamson, Peter; Harrington, Kevin; Leitinger, Birgit; Sahai, Erik

    2010-01-01

    Collective cell migration occurs in a range of contexts: cancer cells frequently invade in cohorts while retaining cell-cell junctions. Here we show that collective cancer cell invasion depends on reducing actomyosin contractility at sites of cell-cell contact. When actomyosin is not down-regulated at cell-cell contacts migrating cells lose cohesion. We provide a novel molecular mechanism for this down-regulation. Depletion of Discoidin Domain Receptor 1 (DDR1) blocks collective cancer cell i...

  6. Regulation of cell growth by Notch signaling and its differential requirement in normal vs. tumor-forming stem cells in Drosophila.

    Science.gov (United States)

    Song, Yan; Lu, Bingwei

    2011-12-15

    Cancer stem cells (CSCs) are postulated to be a small subset of tumor cells with tumor-initiating ability that shares features with normal tissue-specific stem cells. The origin of CSCs and the mechanisms underlying their genesis are poorly understood, and it is uncertain whether it is possible to obliterate CSCs without inadvertently damaging normal stem cells. Here we show that a functional reduction of eukaryotic translation initiation factor 4E (eIF4E) in Drosophila specifically eliminates CSC-like cells in the brain and ovary without having discernable effects on normal stem cells. Brain CSC-like cells can arise from dedifferentiation of transit-amplifying progenitors upon Notch hyperactivation. eIF4E is up-regulated in these dedifferentiating progenitors, where it forms a feedback regulatory loop with the growth regulator dMyc to promote cell growth, particularly nucleolar growth, and subsequent ectopic neural stem cell (NSC) formation. Cell growth regulation is also a critical component of the mechanism by which Notch signaling regulates the self-renewal of normal NSCs. Our findings highlight the importance of Notch-regulated cell growth in stem cell maintenance and reveal a stronger dependence on eIF4E function and cell growth by CSCs, which might be exploited therapeutically.

  7. Common formin-regulating sequences in Smy1 and Bud14 are required for the control of actin cable assembly in vivo.

    Science.gov (United States)

    Eskin, Julian A; Rankova, Aneliya; Johnston, Adam B; Alioto, Salvatore L; Goode, Bruce L

    2016-03-01

    Formins comprise a large family of proteins with diverse roles in remodeling the actin cytoskeleton. However, the spatiotemporal mechanisms used by cells to control formin activities are only beginning to be understood. Here we dissected Smy1, which has dual roles in regulating formins and myosin. Using mutagenesis, we identified specific sequences in Smy1 critical for its in vitro inhibitory effects on the FH2 domain of the formin Bnr1. By integrating smy1 alleles targeting those sequences, we genetically uncoupled Smy1's functions in regulating formins and myosin. Quantitative imaging analysis further demonstrated that the ability of Smy1 to directly control Bnr1 activity is crucial in vivo for proper actin cable length, shape, and velocity and, in turn, efficient secretory vesicle transport. A Smy1-like sequence motif was also identified in a different Bnr1 regulator, Bud14, and found to be essential for Bud14 functions in regulating actin cable architecture and function in vivo. Together these observations reveal unanticipated mechanistic ties between two distinct formin regulators. Further, they emphasize the importance of tightly controlling formin activities in vivo to generate specialized geometries and dynamics of actin structures tailored to their physiological roles.

  8. ETS-1-mediated Transcriptional Up-regulation of CD44 Is Required for Sphingosine-1-phosphate Receptor Subtype 3-stimulated Chemotaxis*

    Science.gov (United States)

    Zhang, Wenliang; Zhao, Jiawei; Lee, Jen-Fu; Gartung, Allison; Jawadi, Hiba; Lambiv, Wanyu Louis; Honn, Kenneth V.; Lee, Menq-Jer

    2013-01-01

    Sphingosine-1-phosphate (S1P)-regulated chemotaxis plays critical roles in various physiological and pathophysiological conditions. S1P-regulated chemotaxis is mediated by the S1P family of G-protein-coupled receptors. However, molecular details of the S1P-regulated chemotaxis are incompletely understood. Cultured human lung adenocarcinoma cell lines abundantly express S1P receptor subtype 3 (S1P3), thus providing a tractable in vitro system to characterize molecular mechanism(s) underlying the S1P3 receptor-regulated chemotactic response. S1P treatment enhances CD44 expression and induces membrane localization of CD44 polypeptides via the S1P3/Rho kinase (ROCK) signaling pathway. Knockdown of CD44 completely diminishes the S1P-stimulated chemotaxis. Promoter analysis suggests that the CD44 promoter contains binding sites of the ETS-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) transcriptional factor. ChIP assay confirms that S1P treatment stimulates the binding of ETS-1 to the CD44 promoter region. Moreover, S1P induces the expression and nuclear translocation of ETS-1. Knockdown of S1P3 or inhibition of ROCK abrogates the S1P-induced ETS-1 expression. Furthermore, knockdown of ETS-1 inhibits the S1P-induced CD44 expression and cell migration. In addition, we showed that S1P3/ROCK signaling up-regulates ETS-1 via the activity of JNK. Collectively, we characterized a novel signaling axis, i.e., ROCK-JNK-ETS-1-CD44 pathway, which plays an essential role in the S1P3-regulated chemotactic response. PMID:24064218

  9. ETS-1-mediated transcriptional up-regulation of CD44 is required for sphingosine-1-phosphate receptor subtype 3-stimulated chemotaxis.

    Science.gov (United States)

    Zhang, Wenliang; Zhao, Jiawei; Lee, Jen-Fu; Gartung, Allison; Jawadi, Hiba; Lambiv, Wanyu Louis; Honn, Kenneth V; Lee, Menq-Jer

    2013-11-08

    Sphingosine-1-phosphate (S1P)-regulated chemotaxis plays critical roles in various physiological and pathophysiological conditions. S1P-regulated chemotaxis is mediated by the S1P family of G-protein-coupled receptors. However, molecular details of the S1P-regulated chemotaxis are incompletely understood. Cultured human lung adenocarcinoma cell lines abundantly express S1P receptor subtype 3 (S1P3), thus providing a tractable in vitro system to characterize molecular mechanism(s) underlying the S1P3 receptor-regulated chemotactic response. S1P treatment enhances CD44 expression and induces membrane localization of CD44 polypeptides via the S1P3/Rho kinase (ROCK) signaling pathway. Knockdown of CD44 completely diminishes the S1P-stimulated chemotaxis. Promoter analysis suggests that the CD44 promoter contains binding sites of the ETS-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) transcriptional factor. ChIP assay confirms that S1P treatment stimulates the binding of ETS-1 to the CD44 promoter region. Moreover, S1P induces the expression and nuclear translocation of ETS-1. Knockdown of S1P3 or inhibition of ROCK abrogates the S1P-induced ETS-1 expression. Furthermore, knockdown of ETS-1 inhibits the S1P-induced CD44 expression and cell migration. In addition, we showed that S1P3/ROCK signaling up-regulates ETS-1 via the activity of JNK. Collectively, we characterized a novel signaling axis, i.e., ROCK-JNK-ETS-1-CD44 pathway, which plays an essential role in the S1P3-regulated chemotactic response.

  10. A novel fur- and iron-regulated small RNA, NrrF, is required for indirect fur-mediated regulation of the sdhA and sdhC genes in Neisseria meningitidis.

    Science.gov (United States)

    Mellin, J R; Goswami, Sulip; Grogan, Susan; Tjaden, Brian; Genco, Caroline A

    2007-05-01

    Iron is both essential for bacterial growth and toxic at higher concentrations; thus, iron homeostasis is tightly regulated. In Neisseria meningitidis the majority of iron-responsive gene regulation is mediated by the ferric uptake regulator protein (Fur), a protein classically defined as a transcriptional repressor. Recently, however, microarray studies have identified a number of genes in N. meningitidis that are iron and Fur activated, demonstrating a new role for Fur as a transcriptional activator. Since Fur has been shown to indirectly activate gene transcription through the repression of small regulatory RNA molecules in other organisms, we hypothesized that a similar mechanism could account for Fur-dependent, iron-activated gene transcription in N. meningitidis. In this study, we used a bioinformatics approach to screen for the presence of Fur-regulated small RNA molecules in N. meningitidis MC58. This screen identified one small RNA, herein named NrrF (for neisserial regulatory RNA responsive to iron [Fe]), which was demonstrated to be both iron responsive and Fur regulated and which has a well-conserved orthologue in N. gonorrhoeae. In addition, this screen identified a number of other likely, novel small RNA transcripts. Lastly, we utilized a new bioinformatics approach to predict regulatory targets of the NrrF small RNA. This analysis led to the identification of the sdhA and sdhC genes, which were subsequently demonstrated to be under NrrF regulation in an nrrF mutant. This study is the first report of small RNAs in N. meningitidis and the first to use a bioinformatics approach to identify, a priori, regulatory targets of a small RNA.

  11. Differential requirement for NMDAR activity in SAP97β-mediated regulation of the number and strength of glutamatergic AMPAR-containing synapses.

    Science.gov (United States)

    Liu, Mingna; Lewis, Laura D; Shi, Rebecca; Brown, Emery N; Xu, Weifeng

    2014-02-01

    PSD-95-like, disc-large (DLG) family membrane-associated guanylate kinase proteins (PSD/DLG-MAGUKs) are essential for regulating synaptic AMPA receptor (AMPAR) function and activity-dependent trafficking of AMPARs. Using a molecular replacement strategy to replace endogenous PSD-95 with SAP97β, we show that the prototypic β-isoform of the PSD-MAGUKs, SAP97β, has distinct NMDA receptor (NMDAR)-dependent roles in regulating basic properties of AMPAR-containing synapses. SAP97β enhances the number of AMPAR-containing synapses in an NMDAR-dependent manner, whereas its effect on the size of unitary synaptic response is not fully dependent on NMDAR activity. These effects contrast with those of PSD-95α, which increases both the number of AMPAR-containing synapses and the size of unitary synaptic responses, with or without NMDAR activity. Our results suggest that SAP97β regulates synaptic AMPAR content by increasing surface expression of GluA1-containing AMPARs, whereas PSD-95α enhances synaptic AMPAR content presumably by increasing the synaptic scaffold capacity for synaptic AMPARs. Our approach delineates discrete effects of different PSD-MAGUKs on principal properties of glutamatergic synaptic transmission. Our results suggest that the molecular diversity of PSD-MAGUKs can provide rich molecular substrates for differential regulation of glutamatergic synapses in the brain.

  12. The CD3 gamma leucine-based receptor-sorting motif is required for efficient ligand-mediated TCR down-regulation

    DEFF Research Database (Denmark)

    von Essen, Marina; Menné, Charlotte; Nielsen, Bodil L

    2002-01-01

    that ligand- and PKC-induced TCR down-regulations are not interrelated. By analyses of a newly developed CD3 gamma-negative T cell variant, freshly isolated and PHA-activated PBMC, and a mouse T cell line, we challenged this dogma and demonstrate in this work that PKC activation and the CD3 gamma di...

  13. 40 CFR Appendix A to Part 282 - State Requirements Incorporated by Reference in Part 282 of the Code of Federal Regulations

    Science.gov (United States)

    2010-07-01

    ... certain storage tanks for heating oil. Section 590.890 Allocation of costs resulting from discharge from... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) APPROVED UNDERGROUND STORAGE TANK PROGRAMS... 1Short title. Section 2Definitions. Section 3Rules and regulations governing underground storage...

  14. Regulation of Lactobacillus casei Sorbitol Utilization Genes Requires DNA-Binding Transcriptional Activator GutR and the Conserved Protein GutM▿

    Science.gov (United States)

    Alcántara, Cristina; Sarmiento-Rubiano, Luz Adriana; Monedero, Vicente; Deutscher, Josef; Pérez-Martínez, Gaspar; Yebra, María J.

    2008-01-01

    Sequence analysis of the five genes (gutRMCBA) downstream from the previously described sorbitol-6-phosphate dehydrogenase-encoding Lactobacillus casei gutF gene revealed that they constitute a sorbitol (glucitol) utilization operon. The gutRM genes encode putative regulators, while the gutCBA genes encode the EIIC, EIIBC, and EIIA proteins of a phosphoenolpyruvate-dependent sorbitol phosphotransferase system (PTSGut). The gut operon is transcribed as a polycistronic gutFRMCBA messenger, the expression of which is induced by sorbitol and repressed by glucose. gutR encodes a transcriptional regulator with two PTS-regulated domains, a galactitol-specific EIIB-like domain (EIIBGat domain) and a mannitol/fructose-specific EIIA-like domain (EIIAMtl domain). Its inactivation abolished gut operon transcription and sorbitol uptake, indicating that it acts as a transcriptional activator. In contrast, cells carrying a gutB mutation expressed the gut operon constitutively, but they failed to transport sorbitol, indicating that EIIBCGut negatively regulates GutR. A footprint analysis showed that GutR binds to a 35-bp sequence upstream from the gut promoter. A sequence comparison with the presumed promoter region of gut operons from various firmicutes revealed a GutR consensus motif that includes an inverted repeat. The regulation mechanism of the L. casei gut operon is therefore likely to be operative in other firmicutes. Finally, gutM codes for a conserved protein of unknown function present in all sequenced gut operons. A gutM mutant, the first constructed in a firmicute, showed drastically reduced gut operon expression and sorbitol uptake, indicating a regulatory role also for GutM. PMID:18676710

  15. Integration Host Factor Is Required for RpoN-Dependent hrpL Gene Expression and Controls Motility by Positively Regulating rsmB sRNA in Erwinia amylovora.

    Science.gov (United States)

    Lee, Jae Hoon; Zhao, Youfu

    2016-01-01

    Erwinia amylovora requires an hrp-type III secretion system (T3SS) to cause disease. It has been reported that HrpL, the master regulator of T3SS, is transcriptionally regulated by sigma factor 54 (RpoN), YhbH, and HrpS. In this study, the role of integration host factor (IHF) in regulating hrpL and T3SS gene expression was investigated. IHF is a nucleoid-associated protein that regulates gene expression by influencing nucleoid structure and DNA bending. Our results showed that both ihfA and ihfB mutants of E. amylovora did not induce necrotic lesions on pear fruits. Growth of both mutants was greatly reduced, and expression of the hrpL and T3SS genes was significantly down-regulated as compared with those of the wild type. In addition, expression of the ihfA, but not the ihfB gene, was under auto-suppression by IHF. Furthermore, both ihfA and ihfB mutants were hypermotile, due to significantly reduced expression of small RNA (sRNA) rsmB. Electrophoresis mobility shift assay further confirmed that IHF binds to the promoters of the hrpL and ihfA genes, as well as the rsmB sRNA gene. These results indicate that IHF is required for RpoN-dependent hrpL gene expression and virulence, and controls motility by positively regulating the rsmB sRNA in E. amylovora.

  16. Consequences of introducing requirements for tanks prepared for solar heating in the building regulations including examinations of bacteria risks; Konsekvenser ved solvarmeforberedte beholdere i bygningsreglementet herunder undersoegelse af risici for bakteriegener

    Energy Technology Data Exchange (ETDEWEB)

    Ellehauge, K.; Kaersgaard, K. [Teknologisk Inst., SolEnergiCentret, Taastrup (Denmark); Bagh, L. [Teknologisk Inst., Miljoedivisionen (Denmark)

    2000-07-01

    A larger dissemination of solar heating units must be expected, if requirements for tanks prepared for solar heating are introduced in the building regulations. However, this may have effects, which have to be discussed beforehand, just as it has to be decided how the regulations can be put into practice. 1) The aim is to examine and discuss the consequences of introducing requirements for tanks prepared for solar heating in the building regulations including connections with other legislation, potential, consequences for the building services sector and proposals for rules in the building regulations (exceptions etc.) 2) Furthermore, the aim is to explain the risk of bacteria in tanks prepared for solar heating according to existing or new additional studies. It must be explained whether tanks prepared for solar heating will result in an increased number of bacteria in the water compared to traditional hot water tanks and - if possible - whether the change is caused by the increased volume of the tank (the water stays in the tank for a longer period) or changed temperature conditions which favours growth of bacteria at a certain incubation temperature. (EHS)

  17. Formation of proximal and anterior limb skeleton requires early function of Irx3 and Irx5 and is negatively regulated by Shh signaling.

    Science.gov (United States)

    Li, Danyi; Sakuma, Rui; Vakili, Niki A; Mo, Rong; Puviindran, Vijitha; Deimling, Steven; Zhang, Xiaoyun; Hopyan, Sevan; Hui, Chi-chung

    2014-04-28

    Limb skeletal pattern relies heavily on graded Sonic hedgehog (Shh) signaling. As a morphogen and growth cue, Shh regulates identities of posterior limb elements, including the ulna/fibula and digits 2 through 5. In contrast, proximal and anterior structures, including the humerus/femur, radius/tibia, and digit 1, are regarded as Shh independent, and mechanisms governing their specification are unclear. Here, we show that patterning of the proximal and anterior limb skeleton involves two phases. Irx3 and Irx5 (Irx3/5) are essential in the initiating limb bud to specify progenitors of the femur, tibia, and digit 1. However, these skeletal elements can be restored in Irx3/5 null mice when Shh signaling is diminished, indicating that Shh negatively regulates their formation after initiation. Our data provide genetic evidence supporting the concept of early specification and progressive determination of anterior limb pattern.

  18. Cloning and Characterization of an Outer Membrane Protein of Vibrio vulnificus Required for Heme Utilization: Regulation of Expression and Determination of the Gene Sequence

    OpenAIRE

    Litwin, Christine M.; Byrne, Burke L.

    1998-01-01

    Vibrio vulnificus is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in patients with iron overload and preexisting liver disease. For V. vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. V. vulnificus is able to use host iron sources such as hemoglobin and heme. We previously constructed a fur mutant of V. vulnificus which constitutively expresses at least two iron-regulated outer membrane pro...

  19. Wnt16 Signaling Is Required for IL-1β-Induced Matrix Metalloproteinase-13-Regulated Proliferation of Human Stem Cell-Derived Osteoblastic Cells

    Directory of Open Access Journals (Sweden)

    Nobuaki Ozeki

    2016-02-01

    Full Text Available We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7+hSMSC-derived osteoblast-like (α7+hSMSC-OB cells, and found that interleukin (IL-1β induces matrix metalloproteinase (MMP-13-regulated proliferation of these cells. These data suggest that MMP-13 plays a potentially unique physiological role in the regeneration of osteoblast-like cells. Here, we examined whether up-regulation of MMP-13 activity by IL-1β was mediated by Wingless/int1 (Wnt signaling and increased the proliferation of osteoblast-like cells. IL-1β increased the mRNA and protein levels of Wnt16 and the Wnt receptor Lrp5/Fzd2. Exogenous Wnt16 was found to increase MMP-13 mRNA, protein and activity, and interestingly, the proliferation rate of these cells. Treatment with small interfering RNAs against Wnt16 and Lrp5 suppressed the IL-1β-induced increase in cell proliferation. We revealed that a unique signaling cascade IL-1β→Wnt16→Lrp5→MMP-13, was intimately involved in the proliferation of osteoblast-like cells, and suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation are regulated by Wnt16.

  20. Novelty-induced activity-regulated cytoskeletal-associated protein (Arc) expression in frontal cortex requires serotonin 2A receptor activation.

    Science.gov (United States)

    Santini, M A; Klein, A B; El-Sayed, M; Ratner, C; Knudsen, G M; Mikkelsen, J D; Aznar, S

    2011-09-08

    Many psychiatric disorders are characterized by cognitive and emotional alterations that are related to abnormal function of the frontal cortex (FC). FC is involved in working memory and decision making and is activated following exposure to a novel environment. The serotonin 2A receptor (5-HT(2A)R) is highly expressed in the FC where its activation induces hallucinations, while blockade of 5-HT(2A)Rs contributes to the therapeutic effects of atypical antipsychotic drugs. The purpose of the present study was to investigate the involvement of 5-HT(2A)R in FC activation following exposure to a novel environment. As an output of FC activation we measured expression of activity-regulated cytoskeletal-associated protein (Arc). Novelty-exposure (open-field arena) robustly up-regulated FC Arc mRNA expression (∼160%) in mice compared to home-cage controls. This response was inhibited with the 5-HT(2A)R antagonists ketanserin and MDL100907, but not with the selective 5-HT(2C)R antagonist SB242084. Novelty-exposure also induced Arc mRNA expression in hippocampus (∼150%), but not in cerebellum or brainstem. Pretreatment with 5-HT(2A)R antagonist ketanserin did not repress the Arc induction in hippocampus, indicating that the involvement of 5-HT(2A)R in this response is restricted to the FC. Similarly, the novelty-induced stress as determined by increasing levels of plasma corticosterone, was not influenced by 5-HT(2A)R antagonism suggesting that Arc mRNA and stress are activated via distinct mechanisms. Taken together, our results demonstrate that the induction of Arc in the FC following exposure to a novel environment is dependent on the 5-HT(2A)R, and that the simultaneous release of corticosterone is regulated via another system independent of 5-HT(2A)R activation.

  1. PGC-1α Is Required for Exercise- and Exercise Training-Induced UCP1 Up-Regulation in Mouse White Adipose Tissue

    DEFF Research Database (Denmark)

    Jørgensen, Stine Ringholm; Grunnet Knudsen, Jakob; Leick, Lotte;

    2013-01-01

    The aim of the present study was to test the hypotheses that 1) a single exercise bout increases UCP1 mRNA in both inguinal (i)WAT and epididymal (e)WAT, 2) UCP1 expression and responsiveness to exercise are different in iWAT and eWAT, 3) PGC-1α determines the basal levels of UCP1 and PRDM16 in WAT...... and 4) exercise and exercise training regulate UCP1 and PRDM16 expression in WAT in a PGC-1α-dependent manner....

  2. Specifications require a closer look. A critical comment on the new Regulation on Photovoltaic Energy Conversion; Empfehlungen nur eingeschraenkt empfehlenswert. Photovoltaik-Richtlinie auf dem Pruefstand

    Energy Technology Data Exchange (ETDEWEB)

    Haselhuhn, R. [Deutsche Gesellschaft fuer Sonnenenergie e.V. (DGS), Berlin (Germany). Landesverband Berlin Brandenburg; Seltmann, T.

    2001-06-01

    I April 2001, new European regulations for testing of solar collectors and complete solar systems came into force. This will do away with the need for multiple testing of solar products prior to exporting into other European states. Opinions differ, however, as to the efficiency of a new dynamic collector test. The author presents the new specifications. [German] Mit der Richtlinie VDI 6012 Blatt 2 will der Verein Deutscher Ingenieure Hinweise fuer die Einbindung von Solarstromanlagen in die Gebaeudetechnik geben. Wir nehmen den VDI-Entwurf kritisch unter die Lupe. (orig.)

  3. Expression of the 1-Aminocyclopropane-1-Carboxylic Acid Deaminase Gene Requires Symbiotic Nitrogen-Fixing Regulator Gene nifA2 in Mesorhizobium loti MAFF303099

    OpenAIRE

    Nukui, Noriyuki; MINAMISAWA, KIWAMU; Ayabe, Shin-Ichi; Aoki, Toshio

    2006-01-01

    Many soil bacteria contain 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which degrades ACC, a precursor of the phytohormone ethylene. In order to examine the regulation of the acdS gene encoding ACC deaminase in Mesorhizobium loti MAFF303099 during symbiosis with the host legume Lotus japonicus, we introduced the β-glucuronidase (GUS) gene into acdS so that GUS was expressed under control of the acdS promoter, and we also generated disruption mutants with mutations in a nitrogen fix...

  4. The PmrA/PmrB Two-Component System of Legionella pneumophila Is a Global Regulator Required for Intracellular Replication within Macrophages and Protozoa▿ †

    OpenAIRE

    Al-khodor, Souhaila; Kalachikov, Sergey; Morozova, Irina; Price, Christopher T.; Abu Kwaik, Yousef

    2008-01-01

    To examine the role of the PmrA/PmrB two-component system (TCS) of Legionella pneumophila in global gene regulation and in intracellular infection, we constructed pmrA and pmrB isogenic mutants by allelic exchange. Genome-wide microarray gene expression analyses of the pmrA and pmrB mutants at both the exponential and the postexponential phases have shown that the PmrA/PmrB TCS has a global effect on the expression of 279 genes classified into nine groups of genes encoding eukaryotic-like pro...

  5. Lotus japonicus nodulation requires two GRAS domain regulators, one of which is functionally conserved in a non-legume

    DEFF Research Database (Denmark)

    Heckmann, Anne Birgitte Lau; Lombardo, Fabien; Miwa, Hiroki

    2006-01-01

    formation in both Ljnsp1-1 and Mtnsp1-1 mutants, indicating that NSP1 regulators from legumes and non-legumes can propagate the Nod factor-induced signal, activating appropriate downstream targets. The L. japonicus nodules complemented with NbNSP1 contained some cells with abnormal bacteroids and could fix...... nitrogen. However, the NbNSP1-complemented M. truncatula nodules did not fix nitrogen and contained very few bacteria released from infection threads. These observations suggest that NSP1 is also involved in infection, bacterial release, and normal bacteroid formation in nodule cells....

  6. ADJUSTMENT OF THE ROMANIAN ACCOUNTING REGULATIONS ACCORDING TO THE NEW REQUIREMENTS ENTAILED BY GLOBALIZATION AND THE ACCESSION OF ROMANIA TO THE EUROPEAN UNION

    Directory of Open Access Journals (Sweden)

    Lucian Constantin Gabriel BUDACIA

    2010-03-01

    Full Text Available The integration of inter-state relations and, ultimately, globalization imply the removal of some restrictions, the harmonization of certain regulations which would favor the development of trade exchanges, the expansion of multilateral cooperation, the transfer of technologies, the improvement and accessibility of the international financial-monetary system. As each element of the economic system, accountancy has to undergo changes and adjustments to the new trend of globalization, namely harmonization and integration, try to firstly adjust to local changes generated by the accession and adjustment of Romania to the EU and to international changes.

  7. 40 CFR 122.34 - As an operator of a regulated small MS4, what will my NPDES MS4 storm water permit require?

    Science.gov (United States)

    2010-07-01

    ... sanctions to ensure compliance include non-monetary penalties, fines, bonding requirements and/or permit... process that identifies the municipality's program goals (e.g., minimize water quality impacts resulting...., adopt a combination of structural and/or non-structural BMPs), operation and maintenance policies...

  8. The Challenge of Self-Directed and Self-Regulated Learning in Vocational Education: A Theoretical Analysis and Synthesis of Requirements

    Science.gov (United States)

    Jossberger, Helen; Brand-Gruwel, Saskia; Boshuizen, Henny; van de Wiel, Margje

    2010-01-01

    Workplace simulations (WPS), authentic learning environments at school, are increasingly used in vocational education. This article provides a theoretical analysis and synthesis of requirements considering learner skills, characteristics of the learning environment and the role of the teacher that influence good functioning in WPS and foster…

  9. Involvement of ZFR1 of Fusarium verticilliodes in kernel colonization and the regulation of FST1, a putative sugar transporter gene required for fumonisin biosynthesis on maize kernels

    Science.gov (United States)

    Fumonisins comprise a class of carcinogenic mycotoxins produced by Fusarium verticillioides during colonization of maize kernels. In previous work, we identified ZFR1, which is predicted to encode a Zn(II)2Cys6 zinc finger transcription factor required for fumonisin B1 (FB1) production during growt...

  10. CtrA response regulator binding to the Caulobacter chromosome replication origin is required during nutrient and antibiotic stress as well as during cell cycle progression.

    Science.gov (United States)

    Bastedo, D Patrick; Marczynski, Gregory T

    2009-04-01

    The Caulobacter crescentus chromosome replication origin (Cori) has five binding sites for CtrA, an OmpR/PhoB family 'response regulator'. CtrA is degraded in replicating 'stalked' cells but is abundant in the non-replicating 'swarmer' cells, where it was proposed to repress replication by binding to Cori. We systematically mutated all Cori CtrA binding sites, and examined their consequences in the contexts of autonomous Cori-plasmid replication and in the natural chromosome locus. Remarkably, the C. crescentus chromosome tolerates severe mutations in all five CtrA binding sites, demonstrating that CtrA is not essential for replication. Further physiological and cell cycle experiments more rigorously supported the original hypothesis that CtrA represses replication. However, our experiments argued against another hypothesis that residual and/or replenished CtrA protein in stalked cells might prevent extra or unscheduled chromosome replication before cell division. Surprisingly, we also demonstrated that Cori CtrA binding sites are very advantageous and can become essential when cells encounter nutrients and antibiotics. Therefore, the CtrA cell cycle regulator co-ordinates replication with viable cell growth in stressful and rapidly changing environments. We argue that this new role for CtrA provided the primary selective pressure for evolving control by CtrA.

  11. Induction of dormancy in Arabidopsis summer annuals requires parallel regulation of DOG1 and hormone metabolism by low temperature and CBF transcription factors.

    Science.gov (United States)

    Kendall, Sarah L; Hellwege, Anja; Marriot, Poppy; Whalley, Celina; Graham, Ian A; Penfield, Steven

    2011-07-01

    Summer annuals overwinter as seeds in the soil seed bank. This is facilitated by a cold-induced increase in dormancy during seed maturation followed by a switch to a state during seed imbibition in which cold instead promotes germination. Here, we show that the seed maturation transcriptome in Arabidopsis thaliana is highly temperature sensitive and reveal that low temperature during seed maturation induces several genes associated with dormancy, including DELAY OF GERMINATION1 (DOG1), and influences gibberellin and abscisic acid levels in mature seeds. Mutants lacking DOG1, or with altered gibberellin or abscisic acid synthesis or signaling, in turn show reduced ability to enter the deeply dormant states in response to low seed maturation temperatures. In addition, we find that DOG1 promotes gibberellin catabolism during maturation. We show that C-REPEAT BINDING FACTORS (CBFs) are necessary for regulation of dormancy and of GA2OX6 and DOG1 expression caused by low temperatures. However, the temperature sensitivity of CBF transcription is markedly reduced in seeds and is absent in imbibed seeds. Our data demonstrate that inhibition of CBF expression is likely a critical feature allowing cold to promote rather than inhibit germination and support a model in which CBFs act in parallel to a low-temperature signaling pathway in the regulation of dormancy.

  12. Induction of Dormancy in Arabidopsis Summer Annuals Requires Parallel Regulation of DOG1 and Hormone Metabolism by Low Temperature and CBF Transcription Factors[W][OA

    Science.gov (United States)

    Kendall, Sarah L.; Hellwege, Anja; Marriot, Poppy; Whalley, Celina; Graham, Ian A.; Penfield, Steven

    2011-01-01

    Summer annuals overwinter as seeds in the soil seed bank. This is facilitated by a cold-induced increase in dormancy during seed maturation followed by a switch to a state during seed imbibition in which cold instead promotes germination. Here, we show that the seed maturation transcriptome in Arabidopsis thaliana is highly temperature sensitive and reveal that low temperature during seed maturation induces several genes associated with dormancy, including DELAY OF GERMINATION1 (DOG1), and influences gibberellin and abscisic acid levels in mature seeds. Mutants lacking DOG1, or with altered gibberellin or abscisic acid synthesis or signaling, in turn show reduced ability to enter the deeply dormant states in response to low seed maturation temperatures. In addition, we find that DOG1 promotes gibberellin catabolism during maturation. We show that C-REPEAT BINDING FACTORS (CBFs) are necessary for regulation of dormancy and of GA2OX6 and DOG1 expression caused by low temperatures. However, the temperature sensitivity of CBF transcription is markedly reduced in seeds and is absent in imbibed seeds. Our data demonstrate that inhibition of CBF expression is likely a critical feature allowing cold to promote rather than inhibit germination and support a model in which CBFs act in parallel to a low-temperature signaling pathway in the regulation of dormancy. PMID:21803937

  13. Novelty-induced activity-regulated cytoskeletal-associated protein (Arc) expression in frontal cortex requires serotonin 2A receptor activation

    DEFF Research Database (Denmark)

    Santini, Martin; Klein, A B; El-Sayed, M;

    2011-01-01

    )R antagonists ketanserin and MDL100907, but not with the selective 5-HT(2C)R antagonist SB242084. Novelty-exposure also induced Arc mRNA expression in hippocampus (∼150%), but not in cerebellum or brainstem. Pretreatment with 5-HT(2A)R antagonist ketanserin did not repress the Arc induction in hippocampus...... environment. As an output of FC activation we measured expression of activity-regulated cytoskeletal-associated protein (Arc). Novelty-exposure (open-field arena) robustly up-regulated FC Arc mRNA expression (∼160%) in mice compared to home-cage controls. This response was inhibited with the 5-HT(2A......, indicating that the involvement of 5-HT(2A)R in this response is restricted to the FC. Similarly, the novelty-induced stress as determined by increasing levels of plasma corticosterone, was not influenced by 5-HT(2A)R antagonism suggesting that Arc mRNA and stress are activated via distinct mechanisms. Taken...

  14. WRN translocation from nucleolus to nucleoplasm is regulated by SIRT1 and required for DNA repair and the development of chemoresistance.

    Science.gov (United States)

    Lee, Sun-Young; Lee, Hyunwoo; Kim, Eun-Sun; Park, Sojin; Lee, Jiyoen; Ahn, Byungchan

    2015-04-01

    When defective or absent, Werner syndrome protein (WRN) causes a genetic premature aging disorder called Werner syndrome. Several studies have reported that defects in WRN function are responsible for not only progeria syndrome but also genomic instability via the deregulation of DNA repair, replication, recombination, and telomere stability. Given the importance of WRN in the repair process, we herein investigated the potential role of WRN in drug response by evaluating the DNA repair following exposure to cisplatin in human cancer cell lines. We found that the down-regulation of SIRT1 and inhibition of SIRT1 deacetylase activity blocked the translocation of WRN from the nucleolus to the nucleoplasm in response to genotoxic stresses. In addition, cells expressing low levels of WRN responded favorably to cisplatin, whereas cells expressing high levels responded poorly to cisplatin. The forced expression of WRN protein in chemosensitive cells resulted in an approximately two-fold increase in cell viability in response to cisplatin compared with vector controls and promoted DNA repair, while WRN-deficient cells accumulate unrepaired double-strand breaks following cisplatin exposure. These results suggest that WRN is regulated by SIRT1 and increased expression of WRN might be one of the determinants for the development of chemotherapeutic drug resistance.

  15. Disruption of microtubule network rescues aberrant actin comets in dynamin2-depleted cells.

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    Yuji Henmi

    Full Text Available A large GTPase dynamin, which is required for endocytic vesicle formation, regulates the actin cytoskeleton through its interaction with cortactin. Dynamin2 mutants impair the formation of actin comets, which are induced by Listeria monocytogenes or phosphatidylinositol-4-phosphate 5-kinase. However, the role of dynamin2 in the regulation of the actin comet is still unclear. Here we show that aberrant actin comets in dynamin2-depleted cells were rescued by disrupting of microtubule networks. Depletion of dynamin2, but not cortactin, significantly reduced the length and the speed of actin comets induced by Listeria. This implies that dynamin2 may regulate the actin comet in a cortactin-independent manner. As dynamin regulates microtubules, we investigated whether perturbation of microtubules would rescue actin comet formation in dynamin2-depleted cells. Treatment with taxol or colchicine created a microtubule-free space in the cytoplasm, and made no difference between control and dynamin2 siRNA cells. This suggests that the alteration of microtubules by dynamin2 depletion reduced the length and the speed of the actin comet.

  16. The ChrSA and HrrSA Two-Component Systems Are Required for Transcriptional Regulation of the hemA Promoter in Corynebacterium diphtheriae

    Science.gov (United States)

    Burgos, Jonathan M.

    2016-01-01

    ABSTRACT Corynebacterium diphtheriae utilizes heme and hemoglobin (Hb) as iron sources for growth in low-iron environments. In C. diphtheriae, the two-component signal transduction systems (TCSs) ChrSA and HrrSA are responsive to Hb levels and regulate the transcription of promoters for hmuO, hrtAB, and hemA. ChrSA and HrrSA activate transcription at the hmuO promoter and repress transcription at hemA in an Hb-dependent manner. In this study, we show that HrrSA is the predominant repressor at hemA and that its activity results in transcriptional repression in the presence and absence of Hb, whereas repression of hemA by ChrSA is primarily responsive to Hb. DNA binding studies showed that both ChrA and HrrA bind to the hemA promoter region at virtually identical sequences. ChrA binding was enhanced by phosphorylation, while binding to DNA by HrrA was independent of its phosphorylation state. ChrA and HrrA are phosphorylated in vitro by the sensor kinase ChrS, whereas no kinase activity was observed with HrrS in vitro. Phosphorylated ChrA was not observed in vivo, even in the presence of Hb, which is likely due to the instability of the phosphate moiety on ChrA. However, phosphorylation of HrrA was observed in vivo regardless of the presence of the Hb inducer, and genetic analysis indicates that ChrS is responsible for most of the phosphorylation of HrrA in vivo. Phosphorylation studies strongly suggest that HrrS functions primarily as a phosphatase and has only minimal kinase activity. These findings collectively show a complex mechanism of regulation at the hemA promoter, where both two-component systems act in concert to optimize expression of heme biosynthetic enzymes. IMPORTANCE Understanding the mechanism by which two-component signal transduction systems function to respond to environmental stimuli is critical to the study of bacterial pathogenesis. The current study expands on the previous analyses of the ChrSA and HrrSA TCSs in the human pathogen C

  17. PGC-1α is required for exercise- and exercise training-induced UCP1 up-regulation in mouse white adipose tissue.

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    Stine Ringholm

    Full Text Available BACKGROUND: The aim of the present study was to test the hypotheses that 1 a single exercise bout increases UCP1 mRNA in both inguinal (iWAT and epididymal (eWAT, 2 UCP1 expression and responsiveness to exercise are different in iWAT and eWAT, 3 PGC-1α determines the basal levels of UCP1 and PRDM16 in WAT and 4 exercise and exercise training regulate UCP1 and PRDM16 expression in WAT in a PGC-1α-dependent manner. METHODS: Whole body PGC-1α knockout (KO and wildtype (WT littermate mice performed a single treadmill exercise bout at 14 m/min and 10% slope for 1 hour. Mice were sacrificed and iWAT, eWAT and quadriceps muscle were removed immediately after, 2, 6 and 10 hours after running, and from sedentary mice that served as controls. In addition, PGC-1α KO mice and WT littermates were exercise trained for 5 weeks with sedentary mice as untrained controls. Thirty-six-37 hours after the last exercise bout iWAT was removed. RESULTS: UCP1 mRNA content increased 19-fold in iWAT and 7.5-fold in eWAT peaking at 6 h and 0' of recovery, respectively, in WT but with no changes in PGC-1α KO mice. UCP1 protein was undetectable in eWAT and very low in iWAT of untrained mice but increased with exercise training to 4.4 (AU in iWAT from WT mice without significant effects in PGC-1α KO mice. CONCLUSION: The present observations provide evidence that exercise training increases UCP1 protein in iWAT through PGC-1α, likely as a cumulative effect of transient increases in UCP1 expression after each exercise bout. Moreover, the results suggest that iWAT is more responsive than eWAT in exercise-induced regulation of UCP1. In addition, as PRDM16 mRNA content decreased in recovery from acute exercise, the present findings suggest that acute exercise elicits regulation of several brown adipose tissue genes in mouse WAT.

  18. Regulation of Hxt3 and Hxt7 turnover converges on the Vid30 complex and requires inactivation of the Ras/cAMP/PKA pathway in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Chris Snowdon

    Full Text Available Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c, an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.

  19. Nonredundant requirement for multiple histone modifications for the early anaphase release of the mitotic exit regulator Cdc14 from nucleolar chromatin.

    Directory of Open Access Journals (Sweden)

    William W Hwang

    2009-08-01

    Full Text Available In Saccharomyces cerevisiae, the conserved phosphatase Cdc14 is required for the exit from mitosis. It is anchored on nucleolar chromatin by the Cfi1/Net1 protein until early anaphase, at which time it is released into the nucleoplasm. Two poorly understood, redundant pathways promote Cdc14 release, the FEAR (Cdc fourteen early release network and the MEN (mitotic exit network. Through the analysis of genetic interactions, we report here a novel requirement for the ubiquitination of histone H2B by the Bre1 ubiquitin ligase in the cell cycle-dependent release of Cdc14 from nucleolar chromatin when the MEN is inactivated. This function for H2B ubiquitination is mediated by its activation of histone H3 methylation on lysines 4 and 79 (meH3K4 and meH3K79 but, surprisingly, is not dependent on the histone deacetylase (HDAC Sir2, which associates with Cdc14 on nucleolar chromatin as part of the RENT complex. We also observed a defect in Cdc14 release in cells lacking H3 lysine 36 methylation (meH3K36 and in cells lacking an HDAC recruited by this modification. These histone modifications represent previously unappreciated factors required for the accessibility to and/or action on nucleolar chromatin of FEAR network components. The nonredundant role for these modifications in this context contrasts with the notion of a highly combinatorial code by which histone marks act to control biological processes.

  20. Redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase is required for high-light stress tolerance in Arabidopsis.

    Science.gov (United States)

    Noshi, Masahiro; Hatanaka, Risa; Tanabe, Noriaki; Terai, Yusuke; Maruta, Takanori; Shigeoka, Shigeru

    2016-05-01

    Chloroplasts are a significant site for reactive oxygen species production under illumination and, thus, possess a well-organized antioxidant system involving ascorbate. Ascorbate recycling occurs in different manners in this system, including a dehydroascorbate reductase (DHAR) reaction. We herein investigated the physiological significance of DHAR3 in photo-oxidative stress tolerance in Arabidopsis. GFP-fused DHAR3 protein was targeted to chloroplasts in Arabidopsis leaves. A DHAR3 knockout mutant exhibited sensitivity to high light (HL). Under HL, the ascorbate redox states were similar in mutant and wild-type plants, while total ascorbate content was significantly lower in the mutant, suggesting that DHAR3 contributes, at least to some extent, to ascorbate recycling. Activation of monodehydroascorbate reductase occurred in dhar3 mutant, which might compensate for the lack of DHAR3. Interestingly, glutathione oxidation was consistently inhibited in dhar3 mutant. These findings indicate that DHAR3 regulates both ascorbate and glutathione redox states to acclimate to HL.

  1. Transcription factor TFAP2C regulates major programs required for murine fetal germ cell maintenance and haploinsufficiency predisposes to teratomas in male mice.

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    Jana Schemmer

    Full Text Available Maintenance and maturation of primordial germ cells is controlled by complex genetic and epigenetic cascades, and disturbances in this network lead to either infertility or malignant aberration. Transcription factor TFAP2C has been described to be essential for primordial germ cell maintenance and to be upregulated in several human germ cell cancers. Using global gene expression profiling, we identified genes deregulated upon loss of Tfap2c in embryonic stem cells and primordial germ cell-like cells. We show that loss of Tfap2c affects many aspects of the genetic network regulating germ cell biology, such as downregulation of maturation markers and induction of markers indicative for somatic differentiation, cell cycle, epigenetic remodeling and pluripotency. Chromatin-immunoprecipitation analyses demonstrated binding of TFAP2C to regulatory regions of deregulated genes (Sfrp1, Dmrt1, Nanos3, c-Kit, Cdk6, Cdkn1a, Fgf4, Klf4, Dnmt3b and Dnmt3l suggesting that these genes are direct transcriptional targets of TFAP2C in primordial germ cells. Since Tfap2c deficient primordial germ cell-like cells display cancer related deregulations in epigenetic remodeling, cell cycle and pluripotency control, the Tfap2c-knockout allele was bred onto 129S2/Sv genetic background. There, mice heterozygous for Tfap2c develop with high incidence germ cell cancer resembling human pediatric germ cell tumors. Precursor lesions can be observed as early as E16.5 in developing testes displaying persisting expression of pluripotency markers. We further demonstrate that mice with a heterozygous deletion of the TFAP2C target gene Nanos3 are also prone to develop teratomas. These data highlight TFAP2C as a critical and dose-sensitive regulator of germ cell fate.

  2. Optimal metabolic regulation of the mammalian heart Na(+)/Ca(2+) exchanger requires a spacial arrangements with a PtdIns(4)-5kinase.

    Science.gov (United States)

    Forcato, Diego; Posada, Velia; Beaugé, Luis; Berberián, Graciela

    2010-11-05

    In inside-out bovine heart sarcolemmal vesicles, p-chloromercuribenzenesulfonate (PCMBS) and n-ethylmaleimide (NEM) fully inhibited MgATP up-regulation of the Na(+)/Ca(2+) exchanger (NCX1) and abolished the MgATP-dependent PtdIns-4,5P2 increase in the NCX1-PtdIns-4,5P2 complex; in addition, these compounds markedly reduced the activity of the PtdIns(4)-5kinase. After PCMBS or NEM treatment, addition of dithiothreitol (DTT) restored a large fraction of the MgATP stimulation of the exchange fluxes and almost fully restored PtdIns(4)-5kinase activity; however, in contrast to PCMBS, the effects of NEM did not seem related to the alkylation of protein SH groups. By itself DTT had no effect on the synthesis of PtdIns-4,5P2 but affected MgATP stimulation of NCX1: moderate inhibition at 1mM MgATP and 1μM Ca(2+) and full inhibition at 0.25mM MgATP and 0.2μM Ca(2+). In addition, DDT prevented coimmunoprecipitation of NCX1 and PtdIns(4)-5kinase. These results indicate that, for a proper MgATP up-regulation of NCX1, the enzyme responsible for PtdIns-4,5P2 synthesis must be (i) functionally competent and (ii) set in the NCX1 microenvironment closely associated to the exchanger. This kind of supramolecular structure is needed to optimize binding of the newly synthesized PtdIns-4,5P2 to its target region in the exchanger protein.

  3. Acyl-coenzyme A-binding protein regulates Beta-oxidation required for growth and survival of non-small cell lung cancer.

    Science.gov (United States)

    Harris, Fredrick T; Rahman, S M Jamshedur; Hassanein, Mohamed; Qian, Jun; Hoeksema, Megan D; Chen, Heidi; Eisenberg, Rosana; Chaurand, Pierre; Caprioli, Richard M; Shiota, Masakazu; Massion, Pierre P

    2014-07-01

    We identified acyl-coenzyme A-binding protein (ACBP) as part of a proteomic signature predicting the risk of having lung cancer. Because ACBP is known to regulate β-oxidation, which in turn controls cellular proliferation, we hypothesized that ACBP contributes to regulation of cellular proliferation and survival of non-small cell lung cancer (NSCLC) by modulating β-oxidation. We used matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) and immunohistochemistry (IHC) to confirm the tissue localization of ABCP in pre-invasive and invasive NSCLCs. We correlated ACBP gene expression levels in NSCLCs with clinical outcomes. In loss-of-function studies, we tested the effect of the downregulation of ACBP on cellular proliferation and apoptosis in normal bronchial and NSCLC cell lines. Using tritiated-palmitate ((3)H-palmitate), we measured β-oxidation levels and tested the effect of etomoxir, a β-oxidation inhibitor, on proliferation and apoptosis. MALDI-IMS and IHC analysis confirmed that ACBP is overexpressed in pre-invasive and invasive lung cancers. High ACBP gene expression levels in NSCLCs correlated with worse survival (HR = 1.73). We observed a 40% decrease in β-oxidation and concordant decreases in proliferation and increases in apoptosis in ACBP-depleted NSCLC cells as compared with bronchial airway epithelial cells. Inhibition of β-oxidation by etomoxir in ACBP-overexpressing cells produced dose-dependent decrease in proliferation and increase in apoptosis (P = 0.01 and P oxidation.

  4. TIF-IA-dependent regulation of ribosome synthesis in drosophila muscle is required to maintain systemic insulin signaling and larval growth.

    Science.gov (United States)

    Ghosh, Abhishek; Rideout, Elizabeth J; Grewal, Savraj S

    2014-10-01

    The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis-a limiting step in ribosome biogenesis-via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.

  5. A strategy to determine AGC regulation capacity requirement in electric market%电力市场下确定系统AGC调节容量的一种策略

    Institute of Scientific and Technical Information of China (English)

    刘颖利; 张洪波

    2011-01-01

    在电力市场环境下,系统AGC调节容量的确定涉及2个基本问题:①如何确保所获取的调节容量满足系统安全性要求;②如何体现在市场中所获取的调节容量的经济性.针对第1个问题:利用系统负荷及发电计划的历史数据建立负荷偏差覆盖率统计表,确保满足系统安全性的要求.针对第2个问题:提出一种通过权衡获取系统AGC调节容量的边际费率和省际联络线偏差处罚费率期望值确定系统AGC调节容量的策略,体现了市场的经济性.通过简单的仿真算例,验证了所提模型和方法的有效性.%In electric market, the determination of AGC regulation capacity is related with two issues: I. How to ensure the regulation capacity satisfies the requirements of power system security; II. How to embody economic benefit of market. For the first issue, a load deviation coverage rate statistical form is set up based on the historical data. For the second issue, a strategy to determine AGC regulation capacity requirement is proposed through balancing the marginal cost of regulation capacity and deviation punishing expectation cost. Finally, simple simulations are carried out, results show the proposed model and method are with validity.

  6. Up-regulation of Bcl-2 is required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage

    Institute of Scientific and Technical Information of China (English)

    Yuting Lin; Junichi Fukuchi; Richard A Hiipakka; John M Kokontis; Jialing Xiang

    2007-01-01

    Bcl-2 is an anti-apoptotic oncoprotein and its protein levels are inversely correlated with prognosis in many cancers.However, the role of Bcl-2 in the progression of prostate cancer is not clear. Here we report that Bcl-2 is required for the progression of LNCaP prostate cancer cells from an androgen-dependent to an androgen-independent growth stage. The mRNA and protein levels of Bcl-2 are significantly increased in androgen-independent prostate cancer cells, shRNA-mediated gene silencing of Bcl-2 in androgen-independent prostate cancer cells promotes UV-induced apoptosis and suppresses the growth of prostate tumors in vivo. Growing androgen-dependent cells under androgen-deprivation conditions results in formation of androgen-independent colonies; and the transition from androgen-dependent to androgen-independent growth is blocked by ectopic expression of the Bcl-2 antagonist Bax or Bcl-2 shRNA. Thus, our results demonstrate that Bcl-2 is not only critical for the survival of androgen-independent prostate cancer cells, but is also required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage.

  7. Ste12/Fab1 phosphatidylinositol-3-phosphate 5-kinase is required for nitrogen-regulated mitotic commitment and cell size control

    Science.gov (United States)

    Schauries, Marie; Kaczmarek, Adrian; Franz-Wachtel, Mirita; Du, Wei; Krug, Karsten; Maček, Boris; Petersen, Janni

    2017-01-01

    Tight coupling of cell growth and cell cycle progression enable cells to adjust their rate of division, and therefore size, to the demands of proliferation in varying nutritional environments. Nutrient stress promotes inhibition of Target Of Rapamycin Complex 1 (TORC1) activity. In fission yeast, reduced TORC1 activity advances mitotic onset and switches growth to a sustained proliferation at reduced cell size. A screen for mutants, that failed to advance mitosis upon nitrogen stress, identified a mutant in the PIKFYVE 1-phosphatidylinositol-3-phosphate 5-kinase fission yeast homolog Ste12. Ste12PIKFYVE deficient mutants were unable to advance the cell cycle to reduce cell size after a nitrogen downshift to poor nitrogen (proline) growth conditions. While it is well established that PI(3,5)P2 signalling is required for autophagy and that Ste12PIKFYVE mutants have enlarged vacuoles (yeast lysosomes), neither a block to autophagy or mutants that independently have enlarged vacuoles had any impact upon nitrogen control of mitotic commitment. The addition of rapamycin to Ste12PIKFYVE deficient mutants reduced cell size at division to suggest that Ste12PIKFYVE possibly functions upstream of TORC1. ste12 mutants display increased Torin1 (TOR inhibitor) sensitivity. However, no major impact on TORC1 or TORC2 activity was observed in the ste12 deficient mutants. In summary, Ste12PIKFYVE is required for nitrogen-stress mediated advancement of mitosis to reduce cell size at division. PMID:28273166

  8. A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9.

    Science.gov (United States)

    Demoulin, J B; Uyttenhove, C; Van Roost, E; DeLestré, B; Donckers, D; Van Snick, J; Renauld, J C

    1996-09-01

    Interleukin-9 (IL-9), a T-cell-derived cytokine, interacts with a specific receptor associated with the IL-2 receptor gamma chain. In this report, we analyze the functional domains of the human IL-9 receptor transfected into mouse lymphoid cell lines. Three different functions were examined: growth stimulation in factor-dependent pro-B Ba/F3 cells, protection against dexamethasone-induced apoptosis, and Ly-6A2 induction in BW5147 lymphoma cells. The results indicated that a single tyrosine, at position 116 in the cytoplasmic domain, was required for all three activities. In addition, we observed that human IL-9 reduced the proliferation rate of transfected BW5147 cells, an effect also dependent on the same tyrosine. This amino acid was necessary for IL-9-mediated tyrosine phosphorylation of the receptor and for STAT activation but not for IRS-2/4PS activation or for JAK1 phosphorylation, which depended on a domain closer to the plasma membrane. We also showed that JAK1 was constitutively associated with the IL-9 receptor. Activated STAT complexes induced by IL-9 were found to contain STAT1, STAT3, and STAT5 transcription factors. Moreover, sequence homologies between human IL-9 receptor tyrosine 116 and tyrosines (of other receptors activating STAT3 and STAT5 were observed. Taken together, these data indicate that a single tyrosine of the IL-9 receptor, required for activation of three different STAT proteins, is necessary for distinct activities of this cytokine, including proliferative responses.

  9. Lotus japonicus nodulation requires two GRAS domain regulators, one of which is functionally conserved in a non-legume

    DEFF Research Database (Denmark)

    Heckmann, Anne Birgitte Lau; Lombardo, Fabien; Miwa, Hiroki

    2006-01-01

    could be in the ortholog of the Medicago truncatula NSP1 gene (MtNSP1). The sequence of the orthologous gene (LjNSP1) in the L. japonicus mutant (Ljnsp1-1) revealed a mutation causing a premature stop resulting in loss of the C-terminal 23 amino acids. We also sequenced the NSP2 gene from L. japonicus...... (LjNSP2). A mutant (Ljnsp2-3) with a premature stop codon was identified by TILLING showing a similar phenotype to Ljnsp1-1. Both LjNSP1 and LjNSP2 are predicted GRAS (GAI, RGA, SCR) domain transcriptional regulators. Transcript steady-state levels of LjNSP1 and LjNSP2 initially decreased...... and then increased following infection by M. loti. In hairy root transformations, LjNSP1 and MtNSP1 complemented both Mtnsp1-1 and Ljnsp1-1 mutants, demonstrating that these orthologous proteins have a conserved biochemical function. A Nicotiana benthamiana NSP1-like gene (NbNSP1) was shown to restore nodule...

  10. Lotus japonicus nodulation requires two GRAS-domain regulators, NSP1 and NSP2

    DEFF Research Database (Denmark)

    Heckmann, Anne Birgitte Lau; Lombardo, Fabien; Miwa, Hiroki

    Signalling Pathway 1) gene (Ljnsp1-1) resulting in loss of the C-terminal 23 amino acids (aa) and we recently identified another mutant in NSP1 (Ljnsp1-2) with a truncated protein of 341 aa. Additionally, we have sequenced and isolated a mutant in LjNSP2 (Ljnsp2-3) that has a premature stop codon showing...... a similar phenotype to the Ljnsp1 mutants, whereas another mutant (Ljnsp2-4) showed increased nodulation. Both LjNSP1 and LjNSP2 are predicted GRAS (GAI, RGA, SCR) domain transcriptional regulators. Transcript steady-state levels of LjNSP1 and LjNSP2 initially decreased and then increased following...... infection by M. loti. In hairy root transformations, LjNSP1 and MtNSP1 complemented both Mtnsp1-1 and Ljnsp1-1 mutants, demonstrating that these orthologous proteins have a conserved biochemical function. A Nicotiana benthamiana NSP1-like gene (NbNSP1) was shown to restore nodule formation in both Ljnsp1...

  11. Ptc6 is required for proper rapamycin-induced down-regulation of the genes coding for ribosomal and rRNA processing proteins in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Asier González

    Full Text Available Ptc6 is one of the seven components (Ptc1-Ptc7 of the protein phosphatase 2C family in the yeast Saccharomyces cerevisiae. In contrast to other type 2C phosphatases, the cellular role of this isoform is poorly understood. We present here a comprehensive characterization of this gene product. Cells lacking Ptc6 are sensitive to zinc ions, and somewhat tolerant to cell-wall damaging agents and to Li(+. Ptc6 mutants are sensitive to rapamycin, albeit to lesser extent than ptc1 cells. This phenotype is not rescued by overexpression of PTC1 and mutation of ptc6 does not reproduce the characteristic genetic interactions of the ptc1 mutation with components of the TOR pathway, thus suggesting different cellular roles for both isoforms. We show here that the rapamycin-sensitive phenotype of ptc6 cells is unrelated to the reported role of Pt6 in controlling pyruvate dehydrogenase activity. Lack of Ptc6 results in substantial attenuation of the transcriptional response to rapamycin, particularly in the subset of repressed genes encoding ribosomal proteins or involved in rRNA processing. In contrast, repressed genes involved in translation are Ptc6-independent. These effects cannot be attributed to the regulation of the Sch9 kinase, but they could involve modulation of the binding of the Ifh1 co-activator to specific gene promoters.

  12. p140Cap regulates memory and synaptic plasticity through Src-mediated and citron-N-mediated actin reorganization.

    Science.gov (United States)

    Repetto, Daniele; Camera, Paola; Melani, Riccardo; Morello, Noemi; Russo, Isabella; Calcagno, Eleonora; Tomasoni, Romana; Bianchi, Federico; Berto, Gaia; Giustetto, Maurizio; Berardi, Nicoletta; Pizzorusso, Tommaso; Matteoli, Michela; Di Stefano, Paola; Missler, Markus; Turco, Emilia; Di Cunto, Ferdinando; Defilippi, Paola

    2014-01-22

    A major challenge in the neuroscience field is the identification of molecules and pathways that control synaptic plasticity and memory. Dendritic spines play a pivotal role in these processes, as the major sites of excitatory synapses in neuronal communication. Previous studies have shown that the scaffold protein p140Cap localizes into dendritic spines and that its knockdown negatively modulates spine shape in culture. However, so far, there is no information on its in vivo relevance. By using a knock-out mouse model, we here demonstrate that p140Cap is a key element for both learning and synaptic plasticity. Indeed, p140Cap(-/-) mice are impaired in object recognition test, as well as in LTP and in LTD measurements. The in vivo effects of p140Cap loss are presumably attenuated by noncell-autonomous events, since primary neurons obtained from p140Cap(-/-) mice show a strong reduction in number of mushroom spines and abnormal organization of synapse-associated F-actin. These phenotypes are most likely caused by a local reduction of the inhibitory control of RhoA and of cortactin toward the actin-depolymerizing factor cofilin. These events can be controlled by p140Cap through its capability to directly inhibit the activation of Src kinase and by its binding to the scaffold protein Citron-N. Altogether, our results provide new insight into how protein associated with dynamic microtubules may regulate spine actin organization through interaction with postsynaptic density components.

  13. The CtsR regulator controls the expression of clpC, clpE and clpP and is required for the virulence of Enterococcus faecalis in an invertebrate model.

    Science.gov (United States)

    Cassenego, Ana Paula Vaz; de Oliveira, Naira Elane Moreira; Laport, Marinella Silva; Abranches, Jaqueline; Lemos, José A; Giambiagi-deMarval, Marcia

    2016-09-01

    The intrinsic ruggedness of Enterococcus faecalis is responsible for its widespread distribution in nature and is often viewed as an important virulence determinant. Previously, we showed that the ClpB ATPase is negatively regulated by CtsR and is required for thermotolerance and virulence in a Galleria mellonella invertebrate model. Here, we used in silico, Northern blot and quantitative real-time PCR analyses to identify additional members of the CtsR regulon, namely the clpP peptidase and the clpC and clpE ATPases. When compared to the parent strain, virulence of the ΔctsR strain in G. mellonella was significantly attenuated.

  14. The new DIN EN 50379. Requirements and testing regulated throughout Europe for portable exhaust gas measuring devices; Die neue DIN EN 50379. Anforderungen und Pruefung an tragbare Abgasmessgeraete europaweit geregelt

    Energy Technology Data Exchange (ETDEWEB)

    Ester, S. [Woehler Messgeraete Kehrgeraete GmbH, Bad Wuennenberg (Germany); Schrameyer, A. [TUEV Sued Industrie Service GmbH, Muenchen (Germany)

    2006-03-15

    The requirements for measuring devices for monitoring of small combustion systems have, up to now, been regulated in Germany in the context of the Federal Prevention of Pollution Ordinance and the individual states' Street Hygiene and Inspection Ordinances. This article describes the new DIN EN 50 379 series of standards, which, for the first time ever, now also covers devices for determination of combustion parameters for installation and maintenance of heating equipment and thus goes well beyond the previous scope of applicability. The new series of standards will, in future, assure a high quality level throughout Europe. (orig.)

  15. MicroRNA167-Directed Regulation of the Auxin Response Factors GmARF8a and GmARF8b Is Required for Soybean Nodulation and Lateral Root Development.

    Science.gov (United States)

    Wang, Youning; Li, Kexue; Chen, Liang; Zou, Yanmin; Liu, Haipei; Tian, Yinping; Li, Dongxiao; Wang, Rui; Zhao, Fang; Ferguson, Brett J; Gresshoff, Peter M; Li, Xia

    2015-07-01

    Legume root nodules convert atmospheric nitrogen gas into ammonium through symbiosis with a prokaryotic microsymbiont broadly called rhizobia. Auxin signaling is required for determinant nodule development; however, the molecular mechanism of auxin-mediated nodule formation remains largely unknown. Here, we show in soybean (Glycine max) that the microRNA miR167 acts as a positive regulator of lateral root organs, namely nodules and lateral roots. miR167c expression was up-regulated in the vasculature, pericycle, and cortex of soybean roots following inoculation with Bradyrhizobium japonicum strain USDA110 (the microsymbiont). It was found to positively regulate nodule numbers directly by repressing the target genes GmARF8a and GmARF8b (homologous genes of Arabidopsis [Arabidopsis thaliana] AtARF8 that encode auxin response factors). Moreover, the expression of miR167 and its targets was up- and down-regulated by auxin, respectively. The miR167-GmARF8 module also positively regulated nodulation efficiency under low microsymbiont density, a condition often associated with environmental stress. The regulatory role of miR167 on nodule initiation was dependent on the Nod factor receptor GmNFR1α, and it acts upstream of the nodulation-associated genes nodule inception, nodulation signaling pathway1, early nodulin40-1, NF-YA1 (previously known as HAEM activator protein2-1), and NF-YA2. miR167 also promoted lateral root numbers. Collectively, our findings establish a key role for the miR167-GmARF8 module in auxin-mediated nodule and lateral root formation in soybean. © 2015 American Society of Plant Biologists. All Rights Reserved.

  16. MicroRNA167-Directed Regulation of the Auxin Response Factors GmARF8a and GmARF8b Is Required for Soybean Nodulation and Lateral Root Development1[OPEN

    Science.gov (United States)

    Wang, Youning; Li, Kexue; Chen, Liang; Zou, Yanmin; Tian, Yinping; Li, Dongxiao; Wang, Rui; Zhao, Fang; Ferguson, Brett J.; Gresshoff, Peter M.

    2015-01-01

    Legume root nodules convert atmospheric nitrogen gas into ammonium through symbiosis with a prokaryotic microsymbiont broadly called rhizobia. Auxin signaling is required for determinant nodule development; however, the molecular mechanism of auxin-mediated nodule formation remains largely unknown. Here, we show in soybean (Glycine max) that the microRNA miR167 acts as a positive regulator of lateral root organs, namely nodules and lateral roots. miR167c expression was up-regulated in the vasculature, pericycle, and cortex of soybean roots following inoculation with Bradyrhizobium japonicum strain USDA110 (the microsymbiont). It was found to positively regulate nodule numbers directly by repressing the target genes GmARF8a and GmARF8b (homologous genes of Arabidopsis [Arabidopsis thaliana] AtARF8 that encode auxin response factors). Moreover, the expression of miR167 and its targets was up- and down-regulated by auxin, respectively. The miR167-GmARF8 module also positively regulated nodulation efficiency under low microsymbiont density, a condition often associated with environmental stress. The regulatory role of miR167 on nodule initiation was dependent on the Nod factor receptor GmNFR1α, and it acts upstream of the nodulation-associated genes NODULE INCEPTION, NODULATION SIGNALING PATHWAY1, EARLY NODULIN40-1, NF-YA1 (previously known as HAEM ACTIVATOR PROTEIN2-1), and NF-YA2. miR167 also promoted lateral root numbers. Collectively, our findings establish a key role for the miR167-GmARF8 module in auxin-mediated nodule and lateral root formation in soybean. PMID:25941314

  17. Detecting Human Hydrologic Alteration from Diversion Hydropower Requires Universal Flow Prediction Tools: A Proposed Framework for Flow Prediction in Poorly-gauged, Regulated Rivers

    Science.gov (United States)

    Kibler, K. M.; Alipour, M.

    2016-12-01

    Achieving the universal energy access Sustainable Development Goal will require great investment in renewable energy infrastructure in the developing world. Much growth in the renewable sector will come from new hydropower projects, including small and diversion hydropower in remote and mountainous regions. Yet, human impacts to hydrological systems from diversion hydropower are poorly described. Diversion hydropower is often implemented in ungauged rivers, thus detection of impact requires flow analysis tools suited to prediction in poorly-gauged and human-altered catchments. We conduct a comprehensive analysis of hydrologic alteration in 32 rivers developed with diversion hydropower in southwestern China. As flow data are sparse, we devise an approach for estimating streamflow during pre- and post-development periods, drawing upon a decade of research into prediction in ungauged basins. We apply a rainfall-runoff model, parameterized and forced exclusively with global-scale data, in hydrologically-similar gauged and ungauged catchments. Uncertain "soft" data are incorporated through fuzzy numbers and confidence-based weighting, and a multi-criteria objective function is applied to evaluate model performance. Testing indicates that the proposed framework returns superior performance (NSE = 0.77) as compared to models parameterized by rote calibration (NSE = 0.62). Confident that the models are providing `the right answer for the right reasons', our analysis of hydrologic alteration based on simulated flows indicates statistically significant hydrologic effects of diversion hydropower across many rivers. Mean annual flows, 7-day minimum and 7-day maximum flows decreased. Frequency and duration of flow exceeding Q25 decreased while duration of flows sustained below the Q75 increased substantially. Hydrograph rise and fall rates and flow constancy increased. The proposed methodology may be applied to improve diversion hydropower design in data-limited regions.

  18. Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo: Nrk2b and NAD+ in muscle morphogenesis.

    Science.gov (United States)

    Goody, Michelle F; Kelly, Meghan W; Lessard, Kevin N; Khalil, Andre; Henry, Clarissa A

    2010-08-15

    Cell-matrix adhesion complexes (CMACs) play fundamental roles during morphogenesis. Given the ubiquitous nature of CMACs and their roles in many cellular processes, one question is how specificity of CMAC function is modulated. The clearly defined cell behaviors that generate segmentally reiterated axial skeletal muscle during zebrafish development comprise an ideal system with which to investigate CMAC function during morphogenesis. We found that Nicotinamide riboside kinase 2b (Nrk2b) cell autonomously modulates the molecular composition of CMACs in vivo. Nrk2b is required for normal Laminin polymerization at the myotendinous junction (MTJ). In Nrk2b-deficient embryos, at MTJ loci where Laminin is not properly polymerized, muscle fibers elongate into adjacent myotomes and are abnormally long. In yeast and human cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative salvage pathway. Exogenous NAD+ treatment rescues MTJ development in Nrk2b-deficient embryos, but not in laminin mutant embryos. Both Nrk2b and Laminin are required for localization of Paxillin, but not beta-Dystroglycan, to CMACs at the MTJ. Overexpression of Paxillin in Nrk2b-deficient embryos is sufficient to rescue MTJ integrity. Taken together, these data show that Nrk2b plays a specific role in modulating subcellular localization of discrete CMAC components that in turn plays roles in musculoskeletal development. Furthermore, these data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development. These results indicate a previously unrecognized complexity to CMAC assembly in vivo and also elucidate a novel role for NAD+ during morphogenesis. Copyright 2010 Elsevier Inc. All rights reserved.

  19. FDA & digital mammography: why has FDA required full field digital mammography systems to be regulated as potentially dangerous devices for more than 10 years?

    Science.gov (United States)

    Nields, Morgan W

    2010-05-01

    Digital mammography is routinely used in the US to screen asymptomatic women for breast cancer and currently over 50% of US screening centers employ the technology. In spite of FDAs knowledge that digital mammography requires less radiation than film mammography and that its equivalence has been proven in a prospective randomized trial, the agency has failed to allow the technology market access via the 510(k) pre market clearance pathway. As a result of the restrictive Pre Market Approval process, only four suppliers have received FDA approval. The resulting lack of a competitive market has kept costs high, restricted technological innovation, and impeded product improvements as a result of PMA requirements. Meanwhile, at least twelve companies are on the market in the EU and the resulting competitive market has lowered costs and provided increased technological choice. A cultural change with new leadership occurred in the early 90's at FDA. The historical culture at the Center for Devices and Radiological Health of collaboration and education gave way to one characterized by a lack of reliance on outside scientific expertise, tolerance of decision making by unqualified reviewers, and an emphasis on enforcement and punishment. Digital mammography fell victim to this cultural change and as a result major innovations like breast CT and computer aided detection technologies are also withheld from the market. The medical device law, currently under review by the Institute of Medicine, should be amended by the Congress so that new technologies can be appropriately classified in accordance with the risk based assessment classification system detailed in Chapter V of the Federal Food, Drug, and Cosmetic Act. A panel of scientific experts chartered by the NIH or IOM should determine the classification appropriate for new technologies that have no historical regulatory framework. This would be binding on FDA. Unless the law is changed we will likely again experience

  20. Insulin-like growth factor binding protein-3 is required for the regulation of rat oval cell proliferation and differentiation in the 2AAF/PHX model

    Directory of Open Access Journals (Sweden)

    Nicole C Steiger-Luther

    2010-02-01

    RNA (siRNA treated animals determined that expression of TGFβ family members, including TGF-βRII and Smads 2–4, were significantly downregulated compared to animals at day 9 post-PHx alone or animals that received negative control siRNA. In conclusion, IGFBP-3 may function as a potent chemoattractant of oval cells during specific types of liver regeneration and may be involved in regulating oval cell proliferation and differentiation in vivo via the TGF-β pathway.Keywords: hepatic stem cells, transforming growth factor-beta, N-2-acetylaminofluorene (2AAF, partial hepatectomy (PHx

  1. 纺织品上限用APEO需要国家标准与法规%Requirement for national standard and regulation on APEO limits in textiles

    Institute of Scientific and Technical Information of China (English)

    陈荣圻

    2012-01-01

    国际绿色和平组织于2011年7~8月发布了2份报告.第一个“时尚之毒”报告,涉及宁波雅戈尔纺织工业城和广东中山国泰染整有限公司2家企业,但报告所列名称和数据较为粗糙,没有说服力.第二个“毒隐于衣”从13个国家采集了15个知名品牌78个服装样品,中国占了28个样品,报告重点是NPEO.从NPEO的急性毒性、水生物毒性、生物降解率及其代谢物的毒性以及应用领域进行了评述.目前国内在生产和应用NPEO上确实存在双重标准,亟待与国际接轨,适时出台纺织品上NPEO的监控标准和法规.%Two reports on textiles were released by Green Peace International during July and August 2011. In the first report named "Dirty Laundry", the detected toxic and hazardous substances in effluents discharged by Youngor Textile Industry City in Ningbo and Well Dyeing Factory Limited in Zhongshan were roughly listed and somewhat lacked of persuasion. In the second report named "Toxin Hide in the Clothes", NPEO residue in 78 pieces of clothing items bearing the label of 15 international brands from 13 countries, including 28 samples from China,are given as a major index. The key of report is NPEO, the acute toxicity, aquatic toxici-ty, biodegradation rate, the metabolite toxicity and its application were introduced. It is pointed out that there exists double standard on NPEO's production and application in home market. Therefore national standards and regulations on NPEO monitoring of textiles in China are suggested to be published in order to conform with the international market.

  2. ATP regulation of type-1 inositol 1,4,5-trisphosphate receptor activity does not require walker A-type ATP-binding motifs.

    Science.gov (United States)

    Betzenhauser, Matthew J; Wagner, Larry E; Park, Hyung Seo; Yule, David I

    2009-06-12

    ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.

  3. Prelimbic cortex extracellular signal-regulated kinase 1/2 activation is required for memory retrieval of long-term inhibitory avoidance.

    Science.gov (United States)

    Luo, Fei; Zheng, Jian; Sun, Xuan; Deng, Wei-Ke; Li, Bao Ming; Liu, Fang

    2017-04-15

    Neural mechanism underlying memory retrieval has been extensively studied in the hippocampus and amygdala. However, little is known about the role of medial prefrontal cortex in long-term memory retrieval. We evaluate this issue in one-trial step-through inhibitory avoidance (IA) paradigm. Our results showed that, 1) inactivation of mPFC by local infusion of GABAA-receptor agonist muscimol caused severe deficits in retrieval of 1-day and 7-day but had no effects on 2-h inhibitory avoidance memory; 2) the protein level of phosphorylated-ERK1/2 in mPFC were significantly increased following retrieval of 1-day and 7-day IA memory, so did the numbers of phosphorylated-ERK (pERK) and phosphorylated-CREB (pCREB) labeled neurons; 3) intra-mPFC infusion of ERK kinase inhibitor PD98095 significantly reduced phosphorylated ERK1/2 levels and phosphorylated-ERK1/2 and phosphorylated-CREB labeled cells, and severely impaired retrieval of 7-day IA memory when the drugs were administrated 30min prior to test. The present study provides evidence that retrieval of long-lasting memory for inhibitory avoidance requires mPFC and involves the ERK-CREB signaling cascade. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Regulation of sterile α- and armadillo motif (SARM) containing protein expression in Pam2CSK4- and Pam3CSK4-activated mouse macrophage cell line (RAW264.7) requires TLR9.

    Science.gov (United States)

    Pudla, Matsayapan; Kulsantiwong, Panthong; Srisaowakarn, Chanya; Utaisincharoen, Pongsak

    2017-09-09

    We aimed to investigate the involvement of surface TLRs and endosomal TLRs in the regulation of SARM expression by TLR2 ligands (Pam2CSK4 and Pam3CSK4). Mouse macrophage cell line (RAW264.7) was treated with either Pam2CSK4 or Pam3CSK4 (TLR2 ligands) at a concentration of 100 ng/ml. At indicated time points, the treated cells were lysed. The gene and protein expression of SARM were determined by RT-PCR and immunoblotting, respectively. For silencing of TLR9 function, the cells were transfected with TLR9 siRNAs before stimulation by these two TLR2 ligands RESULTS: The SARM expression was upregulated at both transcriptional and translational levels in time-dependent manner during activation of Pam2CSK4 and Pam3CSK4 in mouse macrophages. Blocking of ligand internalization by cytochalasin D showed interference effect with SARM expression. Moreover, our results also demonstrated that endosomal acidification and TLR9 were required for SARM expression suggesting the essential role of endosomal compartment acidification and TLR9 in regulating SARM expression. Our findings suggested the collaboration of TLR2-TLR9 at least in the regulation of SARM expression. However, the underlying mechanism that participated in these two TLRs cooperation is underinvestigated.

  5. 78 FR 31551 - Federal Acquisition Regulation; Submission for OMB Review; Commerce Patent Regulations

    Science.gov (United States)

    2013-05-24

    ... Regulation; Submission for OMB Review; Commerce Patent Regulations AGENCIES: Department of Defense (DOD... approved information collection requirement concerning Department of Commerce patent regulations. A notice... Collection 9000- 0095, Commerce Patent Regulations, by any of the following methods: Regulations.gov :...

  6. The TACAN4TGCA motif upstream from the -35 region in the sigma70-sigmaS-dependent Pm promoter of the TOL plasmid is the minimum DNA segment required for transcription stimulation by XylS regulators.

    Science.gov (United States)

    Gallegos, M T; Marqués, S; Ramos, J L

    1996-11-01

    Transcription from the TOL plasmid meta-cleavage pathway operon promoter Pm is dependent on the XylS regulator activated by benzoate effectors or after XylS overproduction. We have generated 5' deletions in Pm and have analyzed expression from wild-type and mutant promoters with the wild-type XylS regulator and XylS mutant regulators that stimulated transcription constitutively. We have found that the motifs T(C or A)CAN4TGCA located between -46 and -57 and -67 and -78 with respect to the main transcription initiation point are required for maximal stimulation of transcription from Pm with effector-activated wild-type XylS. Deletion of the farthest TCCA submotif decreased but did not abolish transcription mediated by the pair XylS with 3-methylbenzoate; however, removal of the motif between -67 and -78 resulted in the loss of stimulation by the wild-type regulator. XylSG44S and XylSS229I stimulated high levels of transcription in the absence of effectors from the wild-type promoter and from a mutant promoter exhibiting only the -46 to -57 motif only when an effector was present. The point mutation Pm5U (with C-47 replaced by G [C-47-->G]) and Pm4 (C-68-->G), located in each 3' TGCA submotif of each motif, resulted in a 90% decrease in transcription stimulation with wild-type XylS; however, the mutant XylSS229I stimulated high levels of transcription from the point mutation promoters both in the presence and in the absence of effectors, while mutant XylSG44S suppressed the two point mutations only with 3-methylbenzoate. Overexpression of XylS and XylSG44S allowed the two regulators to stimulate high levels of transcription from the wild-type promoter, the point mutation Pm4 and Pm5U promoters, and deltaPm promoters exhibiting at least the -46 to -57 motif. Therefore the TACAN4TGCA motif between -46 and -57 represents the minimal DNA segment required for stimulation of transcription from Pm.

  7. Activation of extracellular signal-regulated kinase but not of p38 mitogen-activated protein kinase pathways in lymphocytes requires allosteric activation of SOS.

    Science.gov (United States)

    Jun, Jesse E; Yang, Ming; Chen, Hang; Chakraborty, Arup K; Roose, Jeroen P

    2013-06-01

    Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation.

  8. Changes of Requirements of IVD Reagents Clinical Trial under the New Regulation System%新法规体系下体外诊断试剂类产品临床试验要求的变化

    Institute of Scientific and Technical Information of China (English)

    吕允凤

    2014-01-01

    《体外诊断试剂注册管理办法》(总局令第5号)已经发布实施,相应的体外诊断试剂临床试验技术指导原则也已配套发布实施,本文结合相关法规和指导原则中对临床试验的要求,阐述新法规体系下体外诊断试剂类产品临床试验的具体要求和变化。%Provision on IVD registration(CFDA Decree No.5) had been issued and published by CFDA, fol owing, the guidance for clinical trial of IVD had been posted on CFDA’s website, this paper study the requirements from regulation and guidance, elaborate the speciifc requirements and the change of requirements for clinical trial of IVD.

  9. The Sinorhizobium fredii HH103 MucR1 Global Regulator Is Connected With the nod Regulon and Is Required for Efficient Symbiosis With Lotus burttii and Glycine max cv. Williams.

    Science.gov (United States)

    Acosta-Jurado, Sebastián; Alias-Villegas, Cynthia; Navarro-Gómez, Pilar; Zehner, Susanne; Murdoch, Piedad Del Socorro; Rodríguez-Carvajal, Miguel A; Soto, María J; Ollero, Francisco-Javier; Ruiz-Sainz, José E; Göttfert, Michael; Vinardell, José-María

    2016-09-01

    Sinorhizobium fredii HH103 is a rhizobial strain showing a broad host range of nodulation. In addition to the induction of bacterial nodulation genes, transition from a free-living to a symbiotic state requires complex genetic expression changes with the participation of global regulators. We have analyzed the role of the zinc-finger transcriptional regulator MucR1 from S. fredii HH103 under both free-living conditions and symbiosis with two HH103 host plants, Glycine max and Lotus burttii. Inactivation of HH103 mucR1 led to a severe decrease in exopolysaccharide (EPS) biosynthesis but enhanced production of external cyclic glucans (CG). This mutant also showed increased cell aggregation capacity as well as a drastic reduction in nitrogen-fixation capacity with G. max and L. burttii. However, in these two legumes, the number of nodules induced by the mucR1 mutant was significantly increased and decreased, respectively, with respect to the wild-type strain, indicating that MucR1 can differently affect nodulation depending on the host plant. RNA-Seq analysis carried out in the absence and the presence of flavonoids showed that MucR1 controls the expression of hundreds of genes (including some related to EPS production and CG transport), some of them being related to the nod regulon.

  10. SUMO: regulating the regulator

    Directory of Open Access Journals (Sweden)

    Bossis Guillaume

    2006-06-01

    Full Text Available Abstract Post-translational modifiers of the SUMO (Small Ubiquitin-related Modifier family have emerged as key regulators of protein function and fate. While the past few years have seen an enormous increase in knowledge on SUMO enzymes, substrates, and consequences of modification, regulation of SUMO conjugation is far from being understood. This brief review will provide an overview on recent advances concerning (i the interplay between sumoylation and other post-translational modifications at the level of individual targets and (ii global regulation of SUMO conjugation and deconjugation.

  11. T-cell-receptor engagement and tumor ICAM-1 up-regulation are required to by-pass low susceptibility of melanoma cells to autologous CTL-mediated lysis.

    Science.gov (United States)

    Anichini, A; Mortarini, R; Alberti, S; Mantovani, A; Parmiani, G

    1993-04-01

    Tumor-specific and non-specific CD3+, TcR alpha beta+, CD8+ cytotoxic T-cell (CTL) clones, isolated from tumor-infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL) of a melanoma patient and allogeneic LAK cells, were used to investigate the requirements for bypassing the low lysability of some melanoma clones derived from an s.c. metastasis from which highly lysable clones were also obtained. Cytofluorimetric analysis showed that all melanoma clones expressed ICAM-1, although to different extents, reaching a 10-fold difference in fluorescence units, while HLA class-I antigens were similarly expressed. The differences in expression of ICAM-1 among tumor clones correlated with differences in lysability, by both specific and non-specific CTL, but were not large enough to affect lymphocyte-tumor conjugate formation. Cytokine- or gene-transfer-mediated up-regulation of ICAM-1 did not induce de novo lysis of ICAM-1low tumor cells; however, it markedly enhanced a low level of killing of the same cells by tumor-specific, TcR-dependent and HLA-restricted CTL clones but not by non-specific, TcR-independent effectors. In addition, lysis of melanoma clones by any effector was similarly inhibited by anti-ICAM-1 and anti-LFA-1 antibodies. This indicates that by-pass of low lysability of ICAM-1low melanoma clones by CTL clones, after ICAM-1 up-regulation, is possible only if simultaneous LFA-1 and TcR engagement takes place. In addition, these results suggest that the constitutive high level of expression of ICAM-1 on the subset of ICAM-1high melanoma cells must be only one of the factors contributing to the high lysability of these cells by any effector.

  12. 77 FR 43082 - Federal Acquisition Regulation; Information Collection; Commerce Patent Regulations

    Science.gov (United States)

    2012-07-23

    ... Regulation; Information Collection; Commerce Patent Regulations AGENCIES: Department of Defense (DOD... approved information collection requirement concerning Department of Commerce patent regulations. Public...: Submit comments identified by Information Collection 9000- 0095, Commerce Patent Regulations, by any...

  13. 17 CFR 242.204 - Close-out requirement.

    Science.gov (United States)

    2010-04-01

    ... 600(b)(64) of Regulation NMS (17 CFR 242.600(b)(64)). Regulation ATS—Alternative Trading Systems...) REGULATIONS M, SHO, ATS, AC, AND NMS AND CUSTOMER MARGIN REQUIREMENTS FOR SECURITY FUTURES Regulation...

  14. Requirement of cognate CD4+ T-cell recognition for the regulation of allospecific CTL by human CD4+ CD127- CD25+ FOXP3+ cells generated in MLR.

    Directory of Open Access Journals (Sweden)

    Yuming Yu

    Full Text Available Although immunoregulation of alloreactive human CTLs has been described, the direct influence of CD4(+ Tregs on CD8(+ cytotoxicity and the interactive mechanisms have not been well clarified. Therefore, human CD4(+CD127(-CD25(+FOXP3(+ Tregs were generated in MLR, immunoselected and their allospecific regulatory functions and associated mechanisms were then tested using modified (51Chromium release assays (Micro-CML, MLRs and CFSE-based multi-fluorochrome flow cytometry proliferation assays. It was observed that increased numbers of CD4(+CD127(-CD25(+FOXP3(+ cells were generated after a 7 day MLR. After immunoselection for CD4(+CD127(-CD25(+ cells, they were designated as MLR-Tregs. When added as third component modulators, MLR-Tregs inhibited the alloreactive proliferation of autologous PBMC in a concentration dependent manner. The inhibition was quasi-antigen specific, in that the inhibition was non-specific at higher MLR-Treg modulator doses, but non-specificity disappeared with lower numbers at which specific inhibition was still significant. When tested in micro-CML assays CTL inhibition occurred with PBMC and purified CD8(+ responders. However, antigen specificity of CTL inhibition was observed only with unpurified PBMC responders and not with purified CD8(+ responders or even with CD8(+ responders plus Non-T "APC". However, allospecificity of CTL regulation was restored when autologous purified CD4(+ T cells were added to the CD8(+ responders. Proliferation of CD8(+ cells was suppressed by MLR-Tregs in the presence or absence of IL-2. Inhibition by MLR-Tregs was mediated through down-regulation of intracellular perforin, granzyme B and membrane-bound CD25 molecules on the responding CD8(+ cells. Therefore, it was concluded that human CD4(+CD127(-CD25(+FOXP3(+ MLR-Tregs down-regulate alloreactive cytotoxic responses. Regulatory allospecificity, however, requires the presence of cognate responding CD4(+ T cells. CD8(+ CTL regulatory

  15. Software requirements

    CERN Document Server

    Wiegers, Karl E

    2003-01-01

    Without formal, verifiable software requirements-and an effective system for managing them-the programs that developers think they've agreed to build often will not be the same products their customers are expecting. In SOFTWARE REQUIREMENTS, Second Edition, requirements engineering authority Karl Wiegers amplifies the best practices presented in his original award-winning text?now a mainstay for anyone participating in the software development process. In this book, you'll discover effective techniques for managing the requirements engineering process all the way through the development cy

  16. In the Staphylococcus aureus two-component system sae, the response regulator SaeR binds to a direct repeat sequence and DNA binding requires phosphorylation by the sensor kinase SaeS.

    Science.gov (United States)

    Sun, Fei; Li, Chunling; Jeong, Dowon; Sohn, Changmo; He, Chuan; Bae, Taeok

    2010-04-01

    Staphylococcus aureus uses the SaeRS two-component system to control the expression of many virulence factors such as alpha-hemolysin and coagulase; however, the molecular mechanism of this signaling has not yet been elucidated. Here, using the P1 promoter of the sae operon as a model target DNA, we demonstrated that the unphosphorylated response regulator SaeR does not bind to the P1 promoter DNA, while its C-terminal DNA binding domain alone does. The DNA binding activity of full-length SaeR could be restored by sensor kinase SaeS-induced phosphorylation. Phosphorylated SaeR is more resistant to digestion by trypsin, suggesting conformational changes. DNase I footprinting assays revealed that the SaeR protection region in the P1 promoter contains a direct repeat sequence (GTTAAN(6)GTTAA [where N is any nucleotide]). This sequence is critical to the binding of phosphorylated SaeR. Mutational changes in the repeat sequence greatly reduced both the in vitro binding of SaeR and the in vivo function of the P1 promoter. From these results, we concluded that SaeR recognizes the direct repeat sequence as a binding site and that binding requires phosphorylation by SaeS.

  17. 48 CFR 242.7302 - Requirements.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Requirements. 242.7302 Section 242.7302 Federal Acquisition Regulations System DEFENSE ACQUISITION REGULATIONS SYSTEM, DEPARTMENT.../Pension Review 242.7302 Requirements. Follow the procedures at PGI 242.7302 to determine if a CIPR...

  18. Energy requirements

    NARCIS (Netherlands)

    Hulzebos, Christian V.; Sauer, Pieter J. J.

    2007-01-01

    The determination of the appropriate energy and nutritional requirements of a newborn infant requires a clear goal of the energy and other compounds to be administered, valid methods to measure energy balance and body composition, and knowledge of the neonatal metabolic capacities. Providing an appr

  19. Energy requirements

    NARCIS (Netherlands)

    Hulzebos, Christian V.; Sauer, Pieter J. J.

    The determination of the appropriate energy and nutritional requirements of a newborn infant requires a clear goal of the energy and other compounds to be administered, valid methods to measure energy balance and body composition, and knowledge of the neonatal metabolic capacities. Providing an

  20. Environmental Requirements Management

    Energy Technology Data Exchange (ETDEWEB)

    Cusack, Laura J.; Bramson, Jeffrey E.; Archuleta, Jose A.; Frey, Jeffrey A.

    2015-01-08

    CH2M HILL Plateau Remediation Company (CH2M HILL) is the U.S. Department of Energy (DOE) prime contractor responsible for the environmental cleanup of the Hanford Site Central Plateau. As part of this responsibility, the CH2M HILL is faced with the task of complying with thousands of environmental requirements which originate from over 200 federal, state, and local laws and regulations, DOE Orders, waste management and effluent discharge permits, Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) response and Resource Conservation and Recovery Act (RCRA) corrective action documents, and official regulatory agency correspondence. The challenge is to manage this vast number of requirements to ensure they are appropriately and effectively integrated into CH2M HILL operations. Ensuring compliance with a large number of environmental requirements relies on an organization’s ability to identify, evaluate, communicate, and verify those requirements. To ensure that compliance is maintained, all changes need to be tracked. The CH2M HILL identified that the existing system used to manage environmental requirements was difficult to maintain and that improvements should be made to increase functionality. CH2M HILL established an environmental requirements management procedure and tools to assure that all environmental requirements are effectively and efficiently managed. Having a complete and accurate set of environmental requirements applicable to CH2M HILL operations will promote a more efficient approach to: • Communicating requirements • Planning work • Maintaining work controls • Maintaining compliance

  1. Regulating the private security industry

    CERN Document Server

    Percy, Sarah

    2002-01-01

    The under-regulation of the private security industry has increasingly become a topic of media and academic interest. This Adelphi Paper enters the debate by explaining why the industry requires further regulation, and what is wrong with the current system. It begins by briefly defining the industry and explaining the need for more effective regulation, before analysing three types of regulation: domestic, international and informal (including self-regulation).

  2. Market, Regulation, Market, Regulation

    DEFF Research Database (Denmark)

    Frankel, Christian; Galland, Jean-Pierre

    2015-01-01

    This paper focuses on the European Regulatory system which was settled both for opening the Single Market for products and ensuring the consumers' safety. It claims that the New Approach and Standardization, and the Global Approach to conformity assessment, which suppressed the last technical...... barriers to trade in Europe, realized the free movement of products by organizing progressively several orders of markets and regulation. Based on historical and institutional documents, on technical publications, and on interviews, this article relates how the European Commission and the Member States had...... alternatively recourse to markets and to regulations, at the three main levels of the New Approach Directives implementation. The article focuses also more specifically on the Medical Devices sector, not only because this New Approach sector has long been controversial in Europe, and has recently been concerned...

  3. Market, Regulation, Market, Regulation

    DEFF Research Database (Denmark)

    Frankel, Christian; Galland, Jean-Pierre

    2015-01-01

    This paper focuses on the European Regulatory system which was settled both for opening the Single Market for products and ensuring the consumers' safety. It claims that the New Approach and Standardization, and the Global Approach to conformity assessment, which suppressed the last technical...... barriers to trade in Europe, realized the free movement of products by organizing progressively several orders of markets and regulation. Based on historical and institutional documents, on technical publications, and on interviews, this article relates how the European Commission and the Member States had...... alternatively recourse to markets and to regulations, at the three main levels of the New Approach Directives implementation. The article focuses also more specifically on the Medical Devices sector, not only because this New Approach sector has long been controversial in Europe, and has recently been concerned...

  4. Requirements dilemma

    OpenAIRE

    2006-01-01

    This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University. Knowing ‘what’ to build is an integral part of an Information System Development, and it is generally understood that this, which is known as Requirements, is achievable through a process of understanding, communication and management. It is currently maintained by the Requirements theorists that successful system design clarifies the interrelations between information and its representations...

  5. A novel sterol regulatory element-binding protein gene (sreA identified in penicillium digitatum is required for prochloraz resistance, full virulence and erg11 (cyp51 regulation.

    Directory of Open Access Journals (Sweden)

    Jing Liu

    Full Text Available Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014 at its carboxyl terminus and a helix-loop-helix (HLH leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA in a prochloraz-resistant strain (PdHS-F6 by Agrobacterium tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA. A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression.

  6. A novel sterol regulatory element-binding protein gene (sreA) identified in penicillium digitatum is required for prochloraz resistance, full virulence and erg11 (cyp51) regulation.

    Science.gov (United States)

    Liu, Jing; Yuan, Yongze; Wu, Zhi; Li, Na; Chen, Yuanlei; Qin, Tingting; Geng, Hui; Xiong, Li; Liu, Deli

    2015-01-01

    Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014) at its carboxyl terminus and a helix-loop-helix (HLH) leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA) in a prochloraz-resistant strain (PdHS-F6) by Agrobacterium tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA). A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression.

  7. Identification of a Classical Mutant in the Industrial Host Aspergillus niger by Systems Genetics: LaeA Is Required for Citric Acid Production and Regulates the Formation of Some Secondary Metabolites.

    Science.gov (United States)

    Niu, Jing; Arentshorst, Mark; Nair, P Deepa S; Dai, Ziyu; Baker, Scott E; Frisvad, Jens C; Nielsen, Kristian F; Punt, Peter J; Ram, Arthur F J

    2015-11-13

    The asexual filamentous fungus Aspergillus niger is an important industrial cell factory for citric acid production. In this study, we genetically characterized a UV-generated A. niger mutant that was originally isolated as a nonacidifying mutant, which is a desirable trait for industrial enzyme production. Physiological analysis showed that this mutant did not secrete large amounts of citric acid and oxalic acid, thus explaining the nonacidifying phenotype. As traditional complementation approaches to characterize the mutant genotype were unsuccessful, we used bulk segregant analysis in combination with high-throughput genome sequencing to identify the mutation responsible for the nonacidifying phenotype. Since A. niger has no sexual cycle, parasexual genetics was used to generate haploid segregants derived from diploids by loss of whole chromosomes. We found that the nonacidifying phenotype was caused by a point mutation in the laeA gene. LaeA encodes a putative methyltransferase-domain protein, which we show here to be required for citric acid production in an A. niger lab strain (N402) and in other citric acid production strains. The unexpected link between LaeA and citric acid production could provide new insights into the transcriptional control mechanisms related to citric acid production in A. niger. Interestingly, the secondary metabolite profile of a ΔlaeA strain differed from the wild-type strain, showing both decreased and increased metabolite levels, indicating that LaeA is also involved in regulating the production of secondary metabolites. Finally, we show that our systems genetics approach is a powerful tool to identify trait mutations. Copyright © 2016 Niu et al.

  8. Identification of a Classical Mutant in the Industrial Host Aspergillus niger by Systems Genetics: LaeA Is Required for Citric Acid Production and Regulates the Formation of Some Secondary Metabolites

    Directory of Open Access Journals (Sweden)

    Jing Niu

    2016-01-01

    Full Text Available The asexual filamentous fungus Aspergillus niger is an important industrial cell factory for citric acid production. In this study, we genetically characterized a UV-generated A. niger mutant that was originally isolated as a nonacidifying mutant, which is a desirable trait for industrial enzyme production. Physiological analysis showed that this mutant did not secrete large amounts of citric acid and oxalic acid, thus explaining the nonacidifying phenotype. As traditional complementation approaches to characterize the mutant genotype were unsuccessful, we used bulk segregant analysis in combination with high-throughput genome sequencing to identify the mutation responsible for the nonacidifying phenotype. Since A. niger has no sexual cycle, parasexual genetics was used to generate haploid segregants derived from diploids by loss of whole chromosomes. We found that the nonacidifying phenotype was caused by a point mutation in the laeA gene. LaeA encodes a putative methyltransferase-domain protein, which we show here to be required for citric acid production in an A. niger lab strain (N402 and in other citric acid production strains. The unexpected link between LaeA and citric acid production could provide new insights into the transcriptional control mechanisms related to citric acid production in A. niger. Interestingly, the secondary metabolite profile of a ΔlaeA strain differed from the wild-type strain, showing both decreased and increased metabolite levels, indicating that LaeA is also involved in regulating the production of secondary metabolites. Finally, we show that our systems genetics approach is a powerful tool to identify trait mutations.

  9. Benchmarking and Regulation

    DEFF Research Database (Denmark)

    Agrell, Per J.; Bogetoft, Peter

    nchmarking methods, and in particular Data Envelopment Analysis (DEA), have become well-established and informative tools for economic regulation. DEA is now routinely used by European regulators to set reasonable revenue caps for energy transmission and distribution system operators....... The application of benchmarking in regulation, however, requires specific steps in terms of data validation, model specification and outlier detection that are not systematically documented in open publications, leading to discussions about regulatory stability and economic feasibility of these techniques....... In this paper, we review the modern foundations for frontier-based regulation and we discuss its actual use in several jurisdictions....

  10. Rab7 Regulates CDH1 Endocytosis, Circular Dorsal Ruffles Genesis, and Thyroglobulin Internalization in a Thyroid Cell Line.

    Science.gov (United States)

    Mascia, Anna; Gentile, Flaviana; Izzo, Antonella; Mollo, Nunzia; De Luca, Maria; Bucci, Cecilia; Nitsch, Lucio; Calì, Gaetano

    2016-08-01

    Rab7 regulates the biogenesis of late endosomes, lysosomes, and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over-expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over-expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over-expressing cells, Rab7 and LC3B-II co-localized and co-localization in large circular structures occurred only in Rab7 over-expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co-localization of Rab7 with cortactin and f-actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over-expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5, or Rab8, was inducible by cAMP and that cAMP-stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over-expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. J. Cell. Physiol. 231: 1695-1708, 2016. © 2015 Wiley Periodicals, Inc.

  11. Attenuation of PAMP-triggered immunity in maize requires down-regulation of the key β-1,6-glucan synthesis genes KRE5 and KRE6 in biotrophic hyphae of Colletotrichum graminicola.

    Science.gov (United States)

    Oliveira-Garcia, Ely; Deising, Holger B

    2016-08-01

    In plants, pathogen defense is initiated by recognition of pathogen-associated molecular patterns (PAMPs) via plasma membrane-localized pattern-recognition receptors (PRRs). Fungal structural cell wall polymers such as branched β-glucans are essential for infection structure rigidity and pathogenicity, but at the same time represent PAMPs. Kre5 and Kre6 are key enzymes in β-1,6-glucan synthesis and formation of branch points of the β-glucan network. In spite of the importance of branched β-glucan for hyphal rigidity and plant-fungus interactions, neither the role of KRE5 and KRE6 in pathogenesis nor mechanisms allowing circumventing branched β-glucan-triggered immune responses are known. We functionally characterized KRE5 and KRE6 of the ascomycete Colletotrichum graminicola, a hemibiotroph that infects maize (Zea mays). After appressorial plant invasion, this fungus sequentially differentiates biotrophic and highly destructive necrotrophic hyphae. RNAi-mediated reduction of KRE5 and KRE6 transcript abundance caused appressoria to burst and swelling of necrotrophic hyphae, indicating that β-1,6-glucosidic bonds are essential in these cells. Live cell imaging employing KRE5:mCherry and KRE6:mCherry knock-in strains and probing of infection structures with a YFP-conjugated β-1,6-glucan-binding protein showed expression of these genes and exposure of β-1,6-glucan in conidia, appressoria and necrotrophic, but not in biotrophic hyphae. Overexpression of KRE5 and KRE6 in biotrophic hyphae led to activation of broad-spectrum plant defense responses, including papilla and H2 O2 formation, as well as transcriptional activation of several defense-related genes. Collectively, our results strongly suggest that down-regulation of synthesis and avoidance of exposure of branched β-1,3-β-1,6-glucan in biotrophic hyphae is required for attenuation of plant immune responses. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  12. Regulation of Vif mRNA splicing by human immunodeficiency virus type 1 requires 5' splice site D2 and an exonic splicing enhancer to counteract cellular restriction factor APOBEC3G.

    Science.gov (United States)

    Mandal, Dibyakanti; Exline, Colin M; Feng, Zehua; Stoltzfus, C Martin

    2009-06-01

    The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5' splice site (5'ss) D1, to the first splice acceptor, 3'ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5'ss D2, which is 50 nucleotides downstream of 3'ss A1; a GGGG silencer motif proximal to 5'ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5'ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3'ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5'ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5'ss D2 and ESEVif are required for production of

  13. Hepcidin: regulation of the master iron regulator

    Science.gov (United States)

    Rishi, Gautam; Wallace, Daniel F.; Subramaniam, V. Nathan

    2015-01-01

    Iron, an essential nutrient, is required for many diverse biological processes. The absence of a defined pathway to excrete excess iron makes it essential for the body to regulate the amount of iron absorbed; a deficiency could lead to iron deficiency and an excess to iron overload and associated disorders such as anaemia and haemochromatosis respectively. This regulation is mediated by the iron-regulatory hormone hepcidin. Hepcidin binds to the only known iron export protein, ferroportin (FPN), inducing its internalization and degradation, thus limiting the amount of iron released into the blood. The major factors that are implicated in hepcidin regulation include iron stores, hypoxia, inflammation and erythropoiesis. The present review summarizes our present knowledge about the molecular mechanisms and signalling pathways contributing to hepcidin regulation by these factors. PMID:26182354

  14. 40 CFR 141.80 - General requirements.

    Science.gov (United States)

    2010-07-01

    ... exceeding the lead or copper action level shall implement all applicable source water treatment requirements...) NATIONAL PRIMARY DRINKING WATER REGULATIONS Control of Lead and Copper § 141.80 General requirements. (a... drinking water regulations for lead and copper. Unless otherwise indicated, each of the provisions of...

  15. 10 CFR 70.3 - License requirements.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false License requirements. 70.3 Section 70.3 Energy NUCLEAR... License requirements. No person subject to the regulations in this part shall receive title to, own... license issued by the Commission pursuant to these regulations....

  16. Federal Gasoline Regulations

    Science.gov (United States)

    The Clean Air Act requires EPA to regulate fuels and fuel additives for use in mobile sources if such fuel, fuel additive or any emission products causes or contributes to air or water pollution that may endanger the public health or welfare.

  17. Regulating Transplants

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Legislation to determine brain death is viewed as essential in controlling the organ transplant industry Organ transplant represents a very sensitive and complicated issue. Experts say the temporary administrative regulations recently promulgated by the Central Government are an important step, but relevant laws and regulations must follow. Among these, the

  18. KdmB, a Jumonji Histone H3 Demethylase, Regulates Genome-Wide H3K4 Trimethylation and Is Required for Normal Induction of Secondary Metabolism in Aspergillus nidulans.

    Directory of Open Access Journals (Sweden)

    Agnieszka Gacek-Matthews

    2016-08-01

    Full Text Available Histone posttranslational modifications (HPTMs are involved in chromatin-based regulation of fungal secondary metabolite biosynthesis (SMB in which the corresponding genes-usually physically linked in co-regulated clusters-are silenced under optimal physiological conditions (nutrient-rich but are activated when nutrients are limiting. The exact molecular mechanisms by which HPTMs influence silencing and activation, however, are still to be better understood. Here we show by a combined approach of quantitative mass spectrometry (LC-MS/MS, genome-wide chromatin immunoprecipitation (ChIP-seq and transcriptional network analysis (RNA-seq that the core regions of silent A. nidulans SM clusters generally carry low levels of all tested chromatin modifications and that heterochromatic marks flank most of these SM clusters. During secondary metabolism, histone marks typically associated with transcriptional activity such as H3 trimethylated at lysine-4 (H3K4me3 are established in some, but not all gene clusters even upon full activation. KdmB, a Jarid1-family histone H3 lysine demethylase predicted to comprise a BRIGHT domain, a zinc-finger and two PHD domains in addition to the catalytic Jumonji domain, targets and demethylates H3K4me3 in vivo and mediates transcriptional downregulation. Deletion of kdmB leads to increased transcription of about ~1750 genes across nutrient-rich (primary metabolism and nutrient-limiting (secondary metabolism conditions. Unexpectedly, an equally high number of genes exhibited reduced expression in the kdmB deletion strain and notably, this group was significantly enriched for genes with known or predicted functions in secondary metabolite biosynthesis. Taken together, this study extends our general knowledge about multi-domain KDM5 histone demethylases and provides new details on the chromatin-level regulation of fungal secondary metabolite production.

  19. In the Staphylococcus aureus Two-Component System sae, the Response Regulator SaeR Binds to a Direct Repeat Sequence and DNA Binding Requires Phosphorylation by the Sensor Kinase SaeS ▿

    OpenAIRE

    Sun, Fei; Li, Chunling; Jeong, Dowon; Sohn, Changmo; He, Chuan; Bae, Taeok

    2010-01-01

    Staphylococcus aureus uses the SaeRS two-component system to control the expression of many virulence factors such as alpha-hemolysin and coagulase; however, the molecular mechanism of this signaling has not yet been elucidated. Here, using the P1 promoter of the sae operon as a model target DNA, we demonstrated that the unphosphorylated response regulator SaeR does not bind to the P1 promoter DNA, while its C-terminal DNA binding domain alone does. The DNA binding activity of full-length Sae...

  20. 7 CFR 1942.117 - General requirements.

    Science.gov (United States)

    2010-01-01

    ... § 1942.117 General requirements. (a) Reserve requirements. Loans under this subpart are subject to the... 7 Agriculture 13 2010-01-01 2009-01-01 true General requirements. 1942.117 Section 1942.117 Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, RURAL BUSINESS...

  1. 16 CFR 1505.4 - Manufacturing requirements.

    Science.gov (United States)

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Manufacturing requirements. 1505.4 Section... USE BY CHILDREN Regulations § 1505.4 Manufacturing requirements. (a) General. (1) Only materials safe...-equipped manufacturing establishment. Each component of a toy shall comply with the requirements set...

  2. 24 CFR 891.765 - Lease requirements.

    Science.gov (United States)

    2010-04-01

    ... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Lease requirements. 891.765 Section 891.765 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued... Individuals-Section 162 Assistance § 891.765 Lease requirements. The lease requirements are provided in §...

  3. 24 CFR 891.625 - Lease requirements.

    Science.gov (United States)

    2010-04-01

    ... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Lease requirements. 891.625 Section 891.625 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued... Assistance § 891.625 Lease requirements. The lease requirements are provided in § 891.425....

  4. Transcriptional Elongation Regulator 1 Affects Transcription and Splicing of Genes Associated with Cellular Morphology and Cytoskeleton Dynamics and Is Required for Neurite Outgrowth in Neuroblastoma Cells and Primary Neuronal Cultures

    DEFF Research Database (Denmark)

    Muñoz-Cobo, Juan Pablo; Sánchez-Hernández, Noemí; Gutiérrez-Enríquez, Sara

    2017-01-01

    ) pathogenesis. At present, the molecular mechanism underlying TCERG1-mediated neuronal effects is unknown. Here, we show that TCERG1 depletion led to widespread alterations in mRNA processing that affected different types of alternative transcriptional or splicing events, indicating that TCERG1 plays a broad......TCERG1 is a highly conserved human protein implicated in interactions with the transcriptional and splicing machinery that is associated with neurodegenerative disorders. Biochemical, neuropathological, and genetic evidence suggests an important role for TCERG1 in Huntington’s disease (HD...... role in the regulation of alternative splicing. We observed considerable changes in the transcription and alternative splicing patterns of genes involved in cytoskeleton dynamics and neurite outgrowth. Accordingly, TCERG1 depletion in the neuroblastoma SH-SY5Y cell line and primary mouse neurons...

  5. Albania’s challenge for access and competitiveness in EU Market: When good will requires more than legal regulation. An inside of Albania’s efforts to increase the presence of its domestic product in EU markets.

    Directory of Open Access Journals (Sweden)

    Edi Spaho

    2012-06-01

    Full Text Available Through the presentation of two case studies this paper aims to engage the theoretical debate on the persistence of public space in the contemporary city, and focuses the attention on the way people practice these spaces and on the policies which are regulating their uses. Starting from the description of different ways in which some urban spaces in Rome (Italy are used by two different communities of people, one mainly composed by immigrants and the other by young city users, and the diverse ways in which their different practices are seen and tolerated, the aim of this paper is to reflect on public spaces. Observing how city users practice public spaces, and analysing the way in which these practices are considered, are particularly exciting perspectives that can offer an interesting vision of the spatial and social reality of the city and of hegemonic relations which govern it.

  6. Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice.

    NARCIS (Netherlands)

    Rossum, A.G. van; Bragt, M.P. van; Schuuring-Scholtes, E.; Ploeg, J.C. van der; Krieken, J.H.J.M. van; Kluin, P.M.; Schuuring, E.

    2006-01-01

    BACKGROUND: In human breast cancers, amplification of chromosome 11q13 correlates with lymph node metastasis and increased mortality. To date, two genes located within this amplicon, CCND1 and EMS1, were considered to act as oncogenes, because overexpression of both proteins, respectively cyclin D1

  7. 48 CFR 3452.228-70 - Required insurance.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Required insurance. 3452.228-70 Section 3452.228-70 Federal Acquisition Regulations System DEPARTMENT OF EDUCATION ACQUISITION REGULATION CLAUSES AND FORMS SOLICITATION PROVISIONS AND CONTRACT CLAUSES Texts of Provisions and Clauses 3452.228-70 Required insurance. As...

  8. Restriction of Legionella pneumophila Replication in Macrophages Requires Concerted Action of the Transcriptional Regulators Irf1 and Irf8 and Nod-Like Receptors Naip5 and Nlrc4▿ †

    Science.gov (United States)

    Fortier, Anne; Doiron, Karine; Saleh, Maya; Grinstein, Sergio; Gros, Philippe

    2009-01-01

    The unique permissiveness of A/J mouse macrophages for replication of Legionella pneumophila is caused by a deficiency in the Nod-like receptor (NLR) protein and intracellular sensor for L. pneumophila flagellin (Naip5). The signaling pathways and proteins activated by Naip5 sensing in macrophages were investigated. Transcript profiling of macrophages from susceptible A/J mice and from resistant A/J mice harboring a transgenic wild-type copy of Naip5 at 4 h following L. pneumophila infection suggested that two members of the Irf transcriptional regulator family, Irf1 and Irf8, are regulated in response to Naip5 sensing of L. pneumophila. We show that macrophages having defective alleles of either Irf1 (Irf1−/−) or its heterodimerization partner gene Irf8 (Irf8R294C) become permissive for L. pneumophila replication, indicating that both the Irf1 and Irf8 proteins are essential for macrophage defense against L. pneumophila. Moreover, macrophages doubly heterozygous (Naip5AJ/WT Irf8R294C/WT or Nlrc4−/+ Irf8R294C/WT) for combined loss-of-function mutations in Irf8 and in either Naip5 or Nlrc4 are highly susceptible to L. pneumophila, indicating that there is a strong genetic interaction between Irf8 and the NLR protein family in the macrophage response to L. pneumophila. Legionella-containing phagosomes (LCPs) formed in permissive Irf1−/− or Irf8R294C macrophages behave like LCPs formed in Naip5-insufficient and Nlrc4-deficient macrophages which fail to acidify. These results suggest that, in addition to Naip5 and Nlrc4, Irf1 and Irf8 play a critical role in the early response of macrophages to infection with L. pneumophila, including antagonizing the ability of L. pneumophila to block phagosome acidification. They also suggest that flagellin sensing by the NLR proteins Naip5 and Nlrc4 may be coupled to Irf1-Irf8-mediated transcriptional activation of key effector genes essential for macrophage resistance to L. pneumophila infection. PMID:19720760

  9. Restriction of Legionella pneumophila replication in macrophages requires concerted action of the transcriptional regulators Irf1 and Irf8 and nod-like receptors Naip5 and Nlrc4.

    Science.gov (United States)

    Fortier, Anne; Doiron, Karine; Saleh, Maya; Grinstein, Sergio; Gros, Philippe

    2009-11-01

    The unique permissiveness of A/J mouse macrophages for replication of Legionella pneumophila is caused by a deficiency in the Nod-like receptor (NLR) protein and intracellular sensor for L. pneumophila flagellin (Naip5). The signaling pathways and proteins activated by Naip5 sensing in macrophages were investigated. Transcript profiling of macrophages from susceptible A/J mice and from resistant A/J mice harboring a transgenic wild-type copy of Naip5 at 4 h following L. pneumophila infection suggested that two members of the Irf transcriptional regulator family, Irf1 and Irf8, are regulated in response to Naip5 sensing of L. pneumophila. We show that macrophages having defective alleles of either Irf1 (Irf1-/-) or its heterodimerization partner gene Irf8 (Irf8R294C) become permissive for L. pneumophila replication, indicating that both the Irf1 and Irf8 proteins are essential for macrophage defense against L. pneumophila. Moreover, macrophages doubly heterozygous (Naip5AJ/WT Irf8R294C/WT or Nlrc4-/+ Irf8R294C/WT) for combined loss-of-function mutations in Irf8 and in either Naip5 or Nlrc4 are highly susceptible to L. pneumophila, indicating that there is a strong genetic interaction between Irf8 and the NLR protein family in the macrophage response to L. pneumophila. Legionella-containing phagosomes (LCPs) formed in permissive Irf1-/- or Irf8R294C macrophages behave like LCPs formed in Naip5-insufficient and Nlrc4-deficient macrophages which fail to acidify. These results suggest that, in addition to Naip5 and Nlrc4, Irf1 and Irf8 play a critical role in the early response of macrophages to infection with L. pneumophila, including antagonizing the ability of L. pneumophila to block phagosome acidification. They also suggest that flagellin sensing by the NLR proteins Naip5 and Nlrc4 may be coupled to Irf1-Irf8-mediated transcriptional activation of key effector genes essential for macrophage resistance to L. pneumophila infection.

  10. Type IV(B) pili are required for invasion but not for adhesion of Salmonella enterica serovar Typhi into BHK epithelial cells in a cystic fibrosis transmembrane conductance regulator-independent manner.

    Science.gov (United States)

    Bravo, Denisse; Blondel, Carlos J; Hoare, Anilei; Leyton, Lisette; Valvano, Miguel A; Contreras, Inés

    2011-11-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed as an epithelial cell receptor for the entry of Salmonella Typhi but not Salmonella Typhimurium. The bacterial ligand recognized by CFTR is thought to reside either in the S. Typhi lipopolysaccharide core region or in the type IV pili. Here, we assessed the ability of virulent strains of S. Typhi and S. Typhimurium to adhere to and invade BHK epithelial cells expressing either the wild-type CFTR protein or the ∆F508 CFTR mutant. Both S. Typhi and S. Typhimurium invaded the epithelial cells in a CFTR-independent fashion. Furthermore and also in a CFTR-independent manner, a S. Typhi pilS mutant adhered normally to BHK cells but displayed a 50% reduction in invasion as compared to wild-type bacteria. Immunofluorescence microscopy revealed that bacteria and CFTR do not colocalize at the epithelial cell surface. Together, our results strongly argue against the established dogma that CFTR is a receptor for entry of Salmonella to epithelial cells.

  11. Tumor necrosis factor (TNF) receptor-associated factor 7 is required for TNFα-induced Jun NH2-terminal kinase activation and promotes cell death by regulating polyubiquitination and lysosomal degradation of c-FLIP protein.

    Science.gov (United States)

    Scudiero, Ivan; Zotti, Tiziana; Ferravante, Angela; Vessichelli, Mariangela; Reale, Carla; Masone, Maria C; Leonardi, Antonio; Vito, Pasquale; Stilo, Romania

    2012-02-17

    The pro-inflammatory cytokine tumor necrosis factor (TNF) α signals both cell survival and death. The biological outcome of TNFα treatment is determined by the balance between survival factors and Jun NH(2)-terminal kinase (JNK) signaling, which promotes cell death. Here, we show that TRAF7, the most recently identified member of the TNF receptor-associated factors (TRAFs) family of proteins, is essential for activation of JNK following TNFα stimulation. We also show that TRAF6 and TRAF7 promote unconventional polyubiquitination of the anti-apoptotic protein c-FLIP(L) and demonstrate that degradation of c-FLIP(L) also occurs through a lysosomal pathway. RNA interference-mediated depletion of TRAF7 correlates with increased c-FLIP(L) expression level, which, in turn, results in resistance to TNFα cytotoxicity. Collectively, our results indicate an important role for TRAF7 in the activation of JNK following TNFα stimulation and clearly point to an involvement of this protein in regulating the turnover of c-FLIP and, consequently, cell death.

  12. Tumor Necrosis Factor (TNF) Receptor-associated Factor 7 Is Required for TNFα-induced Jun NH2-terminal Kinase Activation and Promotes Cell Death by Regulating Polyubiquitination and Lysosomal Degradation of c-FLIP Protein*

    Science.gov (United States)

    Scudiero, Ivan; Zotti, Tiziana; Ferravante, Angela; Vessichelli, Mariangela; Reale, Carla; Masone, Maria C.; Leonardi, Antonio; Vito, Pasquale; Stilo, Romania

    2012-01-01

    The pro-inflammatory cytokine tumor necrosis factor (TNF) α signals both cell survival and death. The biological outcome of TNFα treatment is determined by the balance between survival factors and Jun NH2-terminal kinase (JNK) signaling, which promotes cell death. Here, we show that TRAF7, the most recently identified member of the TNF receptor-associated factors (TRAFs) family of proteins, is essential for activation of JNK following TNFα stimulation. We also show that TRAF6 and TRAF7 promote unconventional polyubiquitination of the anti-apoptotic protein c-FLIPL and demonstrate that degradation of c-FLIPL also occurs through a lysosomal pathway. RNA interference-mediated depletion of TRAF7 correlates with increased c-FLIPL expression level, which, in turn, results in resistance to TNFα cytotoxicity. Collectively, our results indicate an important role for TRAF7 in the activation of JNK following TNFα stimulation and clearly point to an involvement of this protein in regulating the turnover of c-FLIP and, consequently, cell death. PMID:22219201

  13. 7 CFR 205.202 - Land requirements.

    Science.gov (United States)

    2010-01-01

    ... Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing Practices), DEPARTMENT OF AGRICULTURE (CONTINUED) ORGANIC FOODS PRODUCTION ACT PROVISIONS NATIONAL ORGANIC PROGRAM Organic Production and Handling Requirements § 205.202 Land...

  14. 75 FR 12803 - Fingerprint Submission Requirements Rule

    Science.gov (United States)

    2010-03-17

    ...Notice of the Compact Council's establishment of a process for initiating noncriminal justice criminal history record checks during times of emergencies and disasters under the authority of the Fingerprint Submission Requirements Rule, title 28 Code of Federal Regulations (CFR), part...

  15. 7 CFR 926.17 - Reporting requirements.

    Science.gov (United States)

    2010-01-01

    ... Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE DATA COLLECTION, REPORTING AND RECORDKEEPING REQUIREMENTS APPLICABLE TO CRANBERRIES NOT SUBJECT TO THE CRANBERRY MARKETING ORDER §...

  16. Clinical comparability and European biosimilar regulations

    NARCIS (Netherlands)

    Schellekens, H.; Moors, Ellen

    2010-01-01

    Clinical trials required by European regulators to compare biosimilar products with corresponding biologic brands are surplus to requirements and may even be a barrier for the development of biosimilars of more complicated biologics.

  17. Identification of a Classical Mutant in the Industrial Host Aspergillus niger by Systems Genetics: LaeA Is Required for Citric Acid Production and Regulates the Formation of Some Secondary Metabolites

    DEFF Research Database (Denmark)

    Niu, Jing; Arentshorst, Mark; Nair, P. Deepa S.

    2015-01-01

    The asexual filamentous fungus Aspergillus niger is an important industrial cell factory for citric acid production. In this study, we genetically characterized a UV-generated A. niger mutant that was originally isolated as a nonacidifying mutant, which is a desirable trait for industrial enzyme...... production. Physiological analysis showed that this mutant did not secrete large amounts of citric acid and oxalic acid, thus explaining the nonacidifying phenotype. As traditional complementation approaches to characterize the mutant genotype were unsuccessful, we used bulk segregant analysis in combination...... was caused by a point mutation in the laeA gene. LaeA encodes a putative methyltransferase-domain protein, which we show here to be required for citric acid production in an A. niger lab strain (N402) and in other citric acid production strains. The unexpected link between LaeA and citric acid production...

  18. Limited portability of G-patch domains in regulators of the Prp43 RNA helicase required for pre-mRNA splicing and ribosomal RNA maturation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Banerjee, Daipayan; McDaniel, Peter M; Rymond, Brian C

    2015-05-01

    The Prp43 DExD/H-box protein is required for progression of the biochemically distinct pre-messenger RNA and ribosomal RNA (rRNA) maturation pathways. In Saccharomyces cerevisiae, the Spp382/Ntr1, Sqs1/Pfa1, and Pxr1/Gno1 proteins are implicated as cofactors necessary for Prp43 helicase activation during spliceosome dissociation (Spp382) and rRNA processing (Sqs1 and Pxr1). While otherwise dissimilar in primary sequence, these Prp43-binding proteins each contain a short glycine-rich G-patch motif required for function and thought to act in protein or nucleic acid recognition. Here yeast two-hybrid, domain-swap, and site-directed mutagenesis approaches are used to investigate G-patch domain activity and portability. Our results reveal that the Spp382, Sqs1, and Pxr1 G-patches differ in Prp43 two-hybrid response and in the ability to reconstitute the Spp382 and Pxr1 RNA processing factors. G-patch protein reconstitution did not correlate with the apparent strength of the Prp43 two-hybrid response, suggesting that this domain has function beyond that of a Prp43 tether. Indeed, while critical for Pxr1 activity, the Pxr1 G-patch appears to contribute little to the yeast two-hybrid interaction. Conversely, deletion of the primary Prp43 binding site within Pxr1 (amino acids 102-149) does not impede rRNA processing but affects small nucleolar RNA (snoRNA) biogenesis, resulting in the accumulation of slightly extended forms of select snoRNAs, a phenotype unexpectedly shared by the prp43 loss-of-function mutant. These and related observations reveal differences in how the Spp382, Sqs1, and Pxr1 proteins interact with Prp43 and provide evidence linking G-patch identity with pathway-specific DExD/H-box helicase activity.

  19. NORM regulations

    Energy Technology Data Exchange (ETDEWEB)

    Gray, P. [ed.

    1997-02-01

    The author reviews the question of regulation for naturally occuring radioactive material (NORM), and the factors that have made this a more prominent concern today. Past practices have been very relaxed, and have often involved very poor records, the involvment of contractors, and the disposition of contaminated equipment back into commercial service. The rationale behind the establishment of regulations is to provide worker protection, to exempt low risk materials, to aid in scrap recycling, to provide direction for remediation and to examine disposal options. The author reviews existing regulations at federal and state levels, impending legislation, and touches on the issue of site remediation and potential liabilities affecting the release of sites contaminated by NORM.

  20. 29 CFR 552.110 - Recordkeeping requirements.

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 3 2010-07-01 2010-07-01 false Recordkeeping requirements. 552.110 Section 552.110 Labor Regulations Relating to Labor (Continued) WAGE AND HOUR DIVISION, DEPARTMENT OF LABOR REGULATIONS APPLICATION OF THE FAIR LABOR STANDARDS ACT TO DOMESTIC SERVICE Interpretations § 552.110 Recordkeeping...

  1. 31 CFR 306.101 - Evidence required.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 2 2010-07-01 2010-07-01 false Evidence required. 306.101 Section 306.101 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) FISCAL SERVICE, DEPARTMENT OF THE TREASURY BUREAU OF THE PUBLIC DEBT GENERAL REGULATIONS GOVERNING...

  2. 31 CFR 306.125 - Additional requirements.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 2 2010-07-01 2010-07-01 false Additional requirements. 306.125 Section 306.125 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) FISCAL SERVICE, DEPARTMENT OF THE TREASURY BUREAU OF THE PUBLIC DEBT GENERAL REGULATIONS GOVERNING...

  3. 7 CFR 33.10 - Minimum requirements.

    Science.gov (United States)

    2010-01-01

    ... Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... ISSUED UNDER AUTHORITY OF THE EXPORT APPLE ACT Regulations § 33.10 Minimum requirements. No person shall... Early: Provided, That apples for export to Pacific ports of Russia shall grade at least U.S. Utility...

  4. Standard types of regulation loops; Chaines de regulation types

    Energy Technology Data Exchange (ETDEWEB)

    Bertrand, M. [ENSAM, Centre d`Enseignement et de Recherche de Lille, 59 - Lille (France)

    1997-12-01

    The aim of this paper is to give help in the analysis of industrial regulation problems using different types of real installations. The increasing complexity of industrial systems requires the use of a decomposition-recomposition procedure using a scheme with different blocs. Examples are given to help the non-specialist users in the mastery of essential choices and in the distinction between operational and material separations. The examples concern: the heating loop of a central heating installation, the sensors and actuators of industrial systems (the temperature regulation of a tubular furnace, the electro-hydraulic positioning systems used in machine tools, forming, aeronautics etc.., the regulation of a mixing system for hot and cold fluids, and the regulation of a fluidizing system. The usual types of regulation loops are presented with the different steps of the resolution of a regulation problem. (J.S.) 7 refs.

  5. 48 CFR 2146.270 - FEGLI Program quality assurance requirements.

    Science.gov (United States)

    2010-10-01

    ... ASSURANCE Contract Quality Requirements 2146.270 FEGLI Program quality assurance requirements. (a) The... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true FEGLI Program quality assurance requirements. 2146.270 Section 2146.270 Federal Acquisition Regulations System OFFICE OF PERSONNEL...

  6. 48 CFR 2152.246-70 - Quality assurance requirements.

    Science.gov (United States)

    2010-10-01

    ... requirements. As prescribed by 2146.270-1 insert the following clause: Quality Assurance Requirements (OCT 2005... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Quality assurance requirements. 2152.246-70 Section 2152.246-70 Federal Acquisition Regulations System OFFICE OF PERSONNEL...

  7. 16 CFR 1512.10 - Requirements for tires.

    Science.gov (United States)

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Requirements for tires. 1512.10 Section 1512... REGULATIONS REQUIREMENTS FOR BICYCLES Regulations § 1512.10 Requirements for tires. The manufacturer's recommended inflation pressure shall be molded into or onto the sidewall of the tire in lettering no less...

  8. 7 CFR 981.51 - Requirements for reserve.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Requirements for reserve. 981.51 Section 981.51... Regulating Handling Volume Regulation § 981.51 Requirements for reserve. Each handler may satisfy his reserve... include grade requirements for reserve almonds delivered to human consumption outlets....

  9. 29 CFR 547.1 - Essential requirements for qualifications.

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 3 2010-07-01 2010-07-01 false Essential requirements for qualifications. 547.1 Section 547.1 Labor Regulations Relating to Labor (Continued) WAGE AND HOUR DIVISION, DEPARTMENT OF LABOR REGULATIONS REQUIREMENTS OF A âBONA FIDE THRIFT OR SAVINGS PLANâ § 547.1 Essential requirements for...

  10. 14 CFR 249.10 - Waiver of requirements.

    Science.gov (United States)

    2010-01-01

    ...) ECONOMIC REGULATIONS PRESERVATION OF AIR CARRIER RECORDS General Instructions § 249.10 Waiver of requirements. A waiver from any provision of this regulation may be made by the Director, Office of Airline...

  11. Nanomaterials: Regulation and Risk Assessment

    DEFF Research Database (Denmark)

    Hansen, Steffen Foss; Grieger, Khara Deanne; Baun, Anders

    2013-01-01

    The topics of regulation and risk assessment of nanomaterials have never been more relevant and controversial in Europe than they are at this point in time. In this entry, we present and discuss a number of major pieces of legislation relevant for the regulation of nanomaterials, including REACH...... Regulation. Chemical risk assessment provides a fundamental element in support of existing legislation. Risk assessment is normally said to consist of four elements, i.e., hazard identification, dose–response assessment, exposure assessment, and risk characterization. Each of these four elements hold......, the Water Framework Directive, pharmaceuticals regulation, and the Novel Foods Regulation. Current regulation of nanomaterials entail three overall challenges: 1) limitations in regard to terminology and definitions of key terms such as a “substance,” “novel food,” etc.; 2) safety assessment requirements...

  12. Capital Requirements and Banks' Leniency

    DEFF Research Database (Denmark)

    Dietrich, J. Kimball; Wihlborg, Clas

    2003-01-01

    We investigate the effect of changes in capital regulation on the strictness(leniency) of loan terms using a simple model of bank capital requirements andasset quality examinations. Banks offer different levels of `leniency' in the senseof willingness to offer automatic extensions of loans...... in the presence of temporarypayment difficulties of borrowers. Banks offering lenient (less strict) loan termsmust have higher initial levels of capital and charge higher loan rates. Whencapital requirements are increased, both strict and lenient banks hold higher levelsof initial capital and they raise loan...... rates. As capital requirements increase thedifference between initial capital levels and between interest rates of strict andlenient banks decrease. Thus, higher capital requirements in recessions tend toreduce the interest rate premium paid for leniency. If a recession is interpreted asan increase...

  13. Safety Requirement for Nuclear Logging in Logging While Drilling as an Application of Nuclear Energy in Industrial Facilities; an Overview for the Improvement of Nuclear Energy Regulating Process in Indonesia

    Energy Technology Data Exchange (ETDEWEB)

    Setianingsih, Lilis Susanti [Korea Advanced Institute of Science and Technology, Daejeon (Korea, Republic of); Choi, Kwang Sik [Korea Institute of Nuclear Safety, Daejeon (Korea, Republic of)

    2012-05-15

    Data transmission processes in LWD are basically performed in two ways: data transmission within the downhole assembly and data transmission to surface. Tools readings can either be stored in downhole memory or transmitted to surface using mud pulse telemetry. Whenever required the two methods can be combined by storing some data in memory and transmitting some in real time. Yet in the case of data supply exceeding data transmission capacity, only key data is transmitted uphole whilst the rest of some sensors output shall be stored downhole. Another way of transmitting data for real time reading is by taking a limited data sample, for example one in every four readings to be sent uphole. The remaining of data readings will be stored to be downloaded to computer once the memory-pack within the tool is brought to surface. In general, analog data from LWD are converted to binary form downhole. Data are transmitted by using a flow-restricting mechanism in the drilling-fluid flow stream and produce positive or negative pressure pulses which are then transmitted through the mud column inside the drill pipe, read at the surface by pressure sensors and later on recorded and processed. Data transmission can also be performed by using rotary valve pressure-pulse generators, which alternately restrict and and open the drilling-fluid flow, causing varying pressure waves generated in the drilling-fluid at carrier frequency which is proportional to the rate of interruption. Downhole sensor-response data are transmitted to the surface by modulating this acoustic carrier frequency

  14. 7 CFR 983.50 - Aflatoxin regulations.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Aflatoxin regulations. 983.50 Section 983.50..., ARIZONA, AND NEW MEXICO Regulations § 983.50 Aflatoxin regulations. The committee shall establish, with the approval of the Secretary, such aflatoxin sampling, analysis, and inspection requirements...

  15. Identification of a classical mutant in the industrial host Aspergillus niger by systems genetics: LaeA is required for citric acid production and regulates the formation of some secondary metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jing; Arentshorst, Mark; Nair, Deepa; Dai, Ziyu; Baker, Scott E.; Frisvad, Jens; Nielsen, Kristian F.; Punt, Peter J.; Ram, Arthur F.

    2016-01-11

    Rapid acidification of the culture medium by the production of organic acids and the production of acid-induced proteases are key characteristics of the filamentous fungus Aspergillus niger. The D15 mutant of A. niger is non-acidifying mutant and used often for the expression of recombinant proteins in A. niger, because of its reduced production of extracellular proteases under non-acidic conditions. In this study, the D15 mutant is characterized in detail. Strongly reduced levels of citric and oxalic acid were observed in the D15 mutant both in shake flask cultures and in controlled batch cultivations. To identify the mutation in the D15 mutant, we successfully combined high-throughput sequencing (Illumina) with bulk segregant analysis. Because of the lack of a sexual cycle for A. niger, the parasexual cycle was used to generate a pool of segregants. From the 52 single nucleotide polymorphisms (SNPs) between the parental strains, three SNPs were homozygous in the genomic DNA of pool of segregants. These three SNPs mapped to all the right arm of chromosome II, indicating that this region contains the genetic locus affecting the phenotype related to acid production. Of the three SNPs, one mutation resulted in a missense mutation in the gene encoding the A. niger homologue of the A. nidulans methyltransferase gene laeA. Complementation analysis of the original mutant with the laeA gene and targeted disruption of laeA further confirmed that LaeA is involved in citric acid production in A. niger lab (N402) and citric acid production strains (ATCC 11414). Analysis of the secondary metabolite (SM) profile of the laeA mutants indicate that LaeA is required for the production of several SMs (asperrubrol, atromentin and JBIR86), but deletion of laeA also resulted in the presence of SMs (aspernigrin A/B and BMS-192548) that were not detected in the wild-type strain. The levels of ten other SMs were not strongly affected as a result of laeA deletion indicating that only a

  16. Groupware requirements evolution patterns

    NARCIS (Netherlands)

    Pumareja, Dulce Trinidad

    2013-01-01

    Requirements evolution is a generally known problem in software development. Requirements are known to change all throughout a system's lifecycle. Nevertheless, requirements evolution is a poorly understood phenomenon. Most studies on requirements evolution focus on changes to written specifications

  17. Legal requirements governing proxy voting in Denmark

    DEFF Research Database (Denmark)

    Werlauff, Erik

    2008-01-01

    The requirements in Danish company law concerning proxy voting in companies whose shares have been accepted for listing on a regulated market have been successively tightened in recent years, and corporate governance principles have also led to the introduction of several requirements concerning...

  18. Regulatory capital requirements and bail in mechanisms

    NARCIS (Netherlands)

    Joosen, B.P.M.; Haentjens, M.; Wessels, B.

    2015-01-01

    With the introduction of the Capital Requirements Regulation (CRR) in the European Union, the qualitative requirements for bank regulatory capital have changed. These changes aim at implementing in Europe the Basel III principles for better bank capital that is able to absorb losses of banks,

  19. 7 CFR 160.11 - Quality requirements.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Quality requirements. 160.11 Section 160.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... STANDARDS FOR NAVAL STORES General § 160.11 Quality requirements. The several standards for spirits of...

  20. 7 CFR 3560.3 - Environmental requirements.

    Science.gov (United States)

    2010-01-01

    ... Environmental requirements. RHS will consider environmental impacts of proposed housing as equal with economic... 7 Agriculture 15 2010-01-01 2010-01-01 false Environmental requirements. 3560.3 Section 3560.3 Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, DEPARTMENT...

  1. 12 CFR 725.19 - Collateral requirements.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Collateral requirements. 725.19 Section 725.19 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING CREDIT UNIONS NATIONAL CREDIT UNION ADMINISTRATION CENTRAL LIQUIDITY FACILITY § 725.19 Collateral requirements. (a)...

  2. 12 CFR 370.9 - Recordkeeping requirements.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 4 2010-01-01 2010-01-01 false Recordkeeping requirements. 370.9 Section 370.9 Banks and Banking FEDERAL DEPOSIT INSURANCE CORPORATION REGULATIONS AND STATEMENTS OF GENERAL POLICY TEMPORARY LIQUIDITY GUARANTEE PROGRAM § 370.9 Recordkeeping requirements. The FDIC will establish...

  3. 49 CFR 130.21 - Packaging requirements.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Packaging requirements. 130.21 Section 130.21 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Packaging requirements. Each packaging used for the transportation of oil subject to this part must...

  4. 10 CFR 40.3 - License requirements.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false License requirements. 40.3 Section 40.3 Energy NUCLEAR REGULATORY COMMISSION DOMESTIC LICENSING OF SOURCE MATERIAL General Provisions § 40.3 License requirements. A..., unless authorized in a specific or general license issued by the Commission under the regulations in...

  5. 31 CFR 103.28 - Identification required.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Identification required. 103.28 Section 103.28 Money and Finance: Treasury Regulations Relating to Money and Finance FINANCIAL RECORDKEEPING AND REPORTING OF CURRENCY AND FOREIGN TRANSACTIONS Reports Required To Be Made §...

  6. 18 CFR 430.15 - Conservation requirements.

    Science.gov (United States)

    2010-04-01

    ... 18 Conservation of Power and Water Resources 2 2010-04-01 2010-04-01 false Conservation requirements. 430.15 Section 430.15 Conservation of Power and Water Resources DELAWARE RIVER BASIN COMMISSION SPECIAL REGULATIONS GROUND WATER PROTECTION AREA: PENNSYLVANIA § 430.15 Conservation requirements....

  7. Regulatory capital requirements and bail in mechanisms

    NARCIS (Netherlands)

    Joosen, B.P.M.; Haentjens, M.; Wessels, B.

    2015-01-01

    With the introduction of the Capital Requirements Regulation (CRR) in the European Union, the qualitative requirements for bank regulatory capital have changed. These changes aim at implementing in Europe the Basel III principles for better bank capital that is able to absorb losses of banks, withou

  8. 40 CFR 141.130 - General requirements.

    Science.gov (United States)

    2010-07-01

    ... treatment technique requirements for disinfection byproduct precursors in § 141.135. (2) The regulations in... established MCLs for TTHM and HAA5 and treatment technique requirements for disinfection byproduct precursors..., distribution line breaks, storm run-off events, source water contamination events, or cross-connection events....

  9. Feed tank transfer requirements

    Energy Technology Data Exchange (ETDEWEB)

    Freeman-Pollard, J.R.

    1998-09-16

    This document presents a definition of tank turnover; DOE responsibilities; TWRS DST permitting requirements; TWRS Authorization Basis (AB) requirements; TWRS AP Tank Farm operational requirements; unreviewed safety question (USQ) requirements; records and reporting requirements, and documentation which will require revision in support of transferring a DST in AP Tank Farm to a privatization contractor for use during Phase 1B.

  10. VLSI Hybrid DC-DC Regulator

    OpenAIRE

    Cosp Vilella, Jordi; Martínez García, Herminio

    2015-01-01

    Hybrid DC-DC regulators are structures that combine both a linear voltage regulator and a switching DC-DC converter. The main objective of this hybrid topology is to converge, in a single circuit topology, the best of both alternatives: a small voltage output ripple, which is a common characteristic of linear regulator circuits, and good energy efficiency, as in switching alternatives. While the linear regulator fixes the required output voltage to a fixed value with negligible steady-state r...

  11. Internationalization of regulatory requirements.

    Science.gov (United States)

    Juillet, Y

    2003-02-01

    The aim of harmonisation of medicines regulatory requirements is to allow the patient quicker access to new drugs and to avoid animal and human duplications. Harmonisation in the European Union (EU) is now completed, and has led to the submission of one dossier in one language study leading to European marketing authorizations, thanks in particular to efficacy guidelines published at the European level. With the benefit of the European experience since 1989, more than 40 guidelines have been harmonised amongst the EU, Japan and the USA through the International Conference on Harmonisation (ICH). ICH is a unique process gathering regulators and industry experts from the three regions. Its activity is built on expertise and trust. The Common Technical Document (CTD), an agreed common format for application in the three regions, is a logical follow-up to the ICH first phase harmonising the content of the dossier. The CTD final implementation in July 2003 will have considerable influence on the review process and on the exchange of information in the three regions.

  12. 48 CFR 246.202 - Types of contract quality requirements.

    Science.gov (United States)

    2010-10-01

    ... SYSTEM, DEPARTMENT OF DEFENSE CONTRACT MANAGEMENT QUALITY ASSURANCE Contract Quality Requirements 246.202 Types of contract quality requirements. ... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Types of contract quality...

  13. 24 CFR 280.25 - Other Federal requirements.

    Science.gov (United States)

    2010-04-01

    ... housing marketing requirements at 24 CFR part 200, subpart M, and the implementing regulations at 24 CFR part 108. (2) Racial and ethnic data collection requirements. Recipients must maintain current data...

  14. Regulated Gene Therapy.

    Science.gov (United States)

    Breger, Ludivine; Wettergren, Erika Elgstrand; Quintino, Luis; Lundberg, Cecilia

    2016-01-01

    Gene therapy represents a promising approach for the treatment of monogenic and multifactorial neurological disorders. It can be used to replace a missing gene and mutated gene or downregulate a causal gene. Despite the versatility of gene therapy, one of the main limitations lies in the irreversibility of the process: once delivered to target cells, the gene of interest is constitutively expressed and cannot be removed. Therefore, efficient, safe and long-term gene modification requires a system allowing fine control of transgene expression.Different systems have been developed over the past decades to regulate transgene expression after in vivo delivery, either at transcriptional or post-translational levels. The purpose of this chapter is to give an overview on current regulatory system used in the context of gene therapy for neurological disorders. Systems using external regulation of transgenes using antibiotics are commonly used to control either gene expression using tetracycline-controlled transcription or protein levels using destabilizing domain technology. Alternatively, specific promoters of genes that are regulated by disease mechanisms, increasing expression as the disease progresses or decreasing expression as disease regresses, are also examined. Overall, this chapter discusses advantages and drawbacks of current molecular methods for regulated gene therapy in the central nervous system.

  15. Precision Adjustable Liquid Regulator (ALR)

    Science.gov (United States)

    Meinhold, R.; Parker, M.

    2004-10-01

    A passive mechanical regulator has been developed for the control of fuel or oxidizer flow to a 450N class bipropellant engine for use on commercial and interplanetary spacecraft. There are several potential benefits to the propulsion system, depending on mission requirements and spacecraft design. This system design enables more precise control of main engine mixture ratio and inlet pressure, and simplifies the pressurization system by transferring the function of main engine flow rate control from the pressurization/propellant tank assemblies, to a single component, the ALR. This design can also reduce the thermal control requirements on the propellant tanks, avoid costly Qualification testing of biprop engines for missions with more stringent requirements, and reduce the overall propulsion system mass and power usage. In order to realize these benefits, the ALR must meet stringent design requirements. The main advantage of this regulator over other units available in the market is that it can regulate about its nominal set point to within +/-0.85%, and change its regulation set point in flight +/-4% about that nominal point. The set point change is handled actively via a stepper motor driven actuator, which converts rotary into linear motion to affect the spring preload acting on the regulator. Once adjusted to a particular set point, the actuator remains in its final position unpowered, and the regulator passively maintains outlet pressure. The very precise outlet regulation pressure is possible due to new technology developed by Moog, Inc. which reduces typical regulator mechanical hysteresis to near zero. The ALR requirements specified an outlet pressure set point range from 225 to 255 psi, and equivalent water flow rates required were in the 0.17 lb/sec range. The regulation output pressure is maintained at +/-2 psi about the set point from a P (delta or differential pressure) of 20 to over 100 psid. Maximum upstream system pressure was specified at 320 psi

  16. 76 FR 81408 - Contractor Legal Management Requirements; Acquisition Regulations

    Science.gov (United States)

    2011-12-28

    ... principles and contract clauses. Consequently, the Department has an ongoing obligation to monitor, supervise... covered by this part? 719.4 Are law firms that are retained by contract by the department covered by this... contract the Department awards directly to retained legal counsel exceeding $100,000. Sec. 719.4 Are law...

  17. Haematopoietic stem cells require a highly regulated protein synthesis rate.

    Science.gov (United States)

    Signer, Robert A J; Magee, Jeffrey A; Salic, Adrian; Morrison, Sean J

    2014-05-01

    Many aspects of cellular physiology remain unstudied in somatic stem cells, for example, there are almost no data on protein synthesis in any somatic stem cell. Here we set out to compare protein synthesis in haematopoietic stem cells (HSCs) and restricted haematopoietic progenitors. We found that the amount of protein synthesized per hour in HSCs in vivo was lower than in most other haematopoietic cells, even if we controlled for differences in cell cycle status or forced HSCs to undergo self-renewing divisions. Reduced ribosome function in Rpl24(Bst/+) mice further reduced protein synthesis in HSCs and impaired HSC function. Pten deletion increased protein synthesis in HSCs but also reduced HSC function. Rpl24(Bst/+) cell-autonomously rescued the effects of Pten deletion in HSCs; blocking the increase in protein synthesis, restoring HSC function, and delaying leukaemogenesis. Pten deficiency thus depletes HSCs and promotes leukaemia partly by increasing protein synthesis. Either increased or decreased protein synthesis impairs HSC function.

  18. 78 FR 80369 - Federal Acquisition Regulation; Service Contracts Reporting Requirements

    Science.gov (United States)

    2013-12-31

    ..., on a bilateral basis in accordance with FAR 1.108(d)(3), within six months of the effective date of... to 41 U.S.C. Sec. 1906 and states that the law applies to acquisitions of commercial items; or 3. Is... stipulation ``for services'' was added at FAR 4.1703(a)(1) and 4.1703(a)(3) to clarify that...

  19. 78 FR 25795 - Contractor Legal Management Requirements; Acquisition Regulations

    Science.gov (United States)

    2013-05-03

    ... unallowable costs for reimbursement, and therefore it adds no value. Response 29: The Department agrees that..., litigation against contractors often directly affects the reputation of the Department and, directly or...

  20. A better regulation is required in viral hepatitis smartphone applications

    Directory of Open Access Journals (Sweden)

    M.ª R. Cantudo-Cuenca

    2014-03-01

    Full Text Available Aim. To describe the characteristics and content of the available viral hepatitis mobile applications, as well as assess the level of participation of medical professionals in their development. Methods. A descriptive observational study was carried out in September 2013. We searched smartphone apps specifically relating to the viral hepatitis for using a keyword search with the following terms; «hepatitis», «hepatology», «hbv» and «hcv» in the Google Play Store (Android and the Apple App Store (iOS. Data recorded included: name, platform, category, cost, user star rating, number of downloads, date the app was updated by the developer and target audience. We analysed the content of the applications, and these were then categorised based on the viral hepatitis type into three groups. We conducted an analysis in which we specifically examined the authorship in order to assess the prevalence of health professional participation in their development. Results. A total of 33 apps were included (from 232 that were identified, among which there were 10 duplicates. Most of these apps were uploaded under the medical category. Three had ratings less than 3.9 stars (out of 5. Only 6 apps had exceeded 1000 downloads. A total of 12 apps were aimed at health professionals, while 4 focused on patients (7 for both of them. The participation of health professionals in the development of apps was 56.6%. Conclusions. Viral hepatitis apps are available for both professionals and patients; however, much of the information contained within them is often not validated. They should be certificated.

  1. A better regulation is required in viral hepatitis smartphone applications.

    Science.gov (United States)

    Cantudo-Cuenca, Ma R; Robustillo-Cortés, Ma A; Cantudo-Cuenca, Ma D; Morillo-Verdugo, R

    2014-04-01

    To describe the characteristics and content of the available viral hepatitis mobile applications, as well as assess the level of participation of medical professionals in their development. A descriptive observational study was carried out in September 2013. We searched smartphone apps specifically relating to the viral hepatitis for using a keyword search with the following terms; «hepatitis», «hepatology», «hbv» and «hcv» in the Google Play Store (Android) and the Apple App Store (iOS). Data recorded included: name, platform, category, cost, user star rating, number of downloads, date the app was updated by the developer and target audience. We analysed the content of the applications, and these were then categorised based on the viral hepatitis type into three groups. We conducted an analysis in which we specifically examined the authorship in order to assess the prevalence of health professional participation in their development. A total of 33 apps were included (from 232 that were identified), among which there were 10 duplicates. Most of these apps were uploaded under the medical category. Three had ratings less than 3.9 stars (out of 5). Only 6 apps had exceeded 1000 downloads. A total of 12 apps were aimed at health professionals, while 4 focused on patients (7 for both of them). The participation of health professionals in the development of apps was 56.6%. Viral hepatitis apps are available for both professionals and patients; however, much of the information contained within them is often not validated. They should be certificated. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

  2. Wiring regulations in brief

    CERN Document Server

    Tricker, Ray

    2012-01-01

    Tired of trawling through the Wiring Regs?Perplexed by Part P?Confused by cables, conductors and circuits?Then look no further! This handy guide provides an on-the-job reference source for Electricians, Designers, Service Engineers, Inspectors, Builders, Students, DIY enthusiastsTopic-based chapters link areas of working practice - such as cables, installations, testing and inspection, special locations - with the specifics of the Regulations themselves. This allows quick and easy identification of the official requirements relating to the situati

  3. Feed tank transfer requirements

    Energy Technology Data Exchange (ETDEWEB)

    Freeman-Pollard, J.R.

    1998-09-16

    This document presents a definition of tank turnover. Also, DOE and PC responsibilities; TWRS DST permitting requirements; TWRS Authorization Basis (AB) requirements; TWRS AP Tank Farm operational requirements; unreviewed safety question (USQ) requirements are presented for two cases (i.e., tank modifications occurring before tank turnover and tank modification occurring after tank turnover). Finally, records and reporting requirements, and documentation which will require revision in support of transferring a DST in AP Tank Farm to a privatization contractor are presented.

  4. 47 CFR 76.925 - Costs of franchise requirements.

    Science.gov (United States)

    2010-10-01

    ... 47 Telecommunication 4 2010-10-01 2010-10-01 false Costs of franchise requirements. 76.925 Section... MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE Cable Rate Regulation § 76.925 Costs of franchise requirements. (a) Franchise requirement costs may include cost increases required by the franchising authority...

  5. 12 CFR 204.4 - Computation of required reserves.

    Science.gov (United States)

    2010-01-01

    ... RESERVE REQUIREMENTS OF DEPOSITORY INSTITUTIONS (REGULATION D) § 204.4 Computation of required reserves. (a) In determining the reserve requirement under this part, the amount of cash items in process of... reserves are computed by applying the reserve requirement ratios below to net transaction...

  6. 12 CFR 204.9 - Emergency reserve requirement.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 2 2010-01-01 2010-01-01 false Emergency reserve requirement. 204.9 Section... RESERVE REQUIREMENTS OF DEPOSITORY INSTITUTIONS (REGULATION D) § 204.9 Emergency reserve requirement. (a..., additional reserve requirements on depository institutions at any ratio on any liability upon a finding by...

  7. 16 CFR 303.4 - English language requirement.

    Science.gov (United States)

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false English language requirement. 303.4 Section... AND REGULATIONS UNDER THE TEXTILE FIBER PRODUCTS IDENTIFICATION ACT § 303.4 English language requirement. All required information shall be set out in the English language. If the required...

  8. Cyclin G2 Promotes Hypoxia- Driven Local Invasion of Glioblastoma by Orchestrating Cytoskeletal Dynamics

    Directory of Open Access Journals (Sweden)

    Atsushi Fujimura

    2013-11-01

    Full Text Available Microenvironmental conditions such as hypoxia potentiate the local invasion of malignant tumors including glioblastomas by modulating signal transduction and protein modification, yet the mechanism by which hypoxia controls cytoskeletal dynamics to promote the local invasion is not well defined. Here, we show that cyclin G2 plays pivotal roles in the cytoskeletal dynamics in hypoxia-driven invasion by glioblastoma cells. Cyclin G2 is a hypoxia-induced and cytoskeleton-associated protein and is required for glioblastoma expansion. Mechanistically, cyclin G2 recruits cortactin to the juxtamembrane through its SH3 domain-binding motif and consequently promotes the restricted tyrosine phosphorylation of cortactin in concert with src. Moreover, cyclin G2 interacts with filamentous actin to facilitate the formation of membrane ruffles. In primary glioblastoma, cyclin G2 is abundantly expressed in severely hypoxic regions such as pseudopalisades, which consist of actively migrating glioma cells. Furthermore, we show the effectiveness of dasatinib against hypoxia-driven, cyclin G2-involved invasion in vitro and in vivo. Our findings elucidate the mechanism of cytoskeletal regulation by which severe hypoxia promotes the local invasion and may provide a therapeutic target in glioblastoma.

  9. 48 CFR 52.204-2 - Security Requirements.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 2 2010-10-01 2010-10-01 false Security Requirements. 52.204-2 Section 52.204-2 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION... Contractor agrees to insert terms that conform substantially to the language of this clause, including...

  10. 48 CFR 801.602-70 - General review requirements.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false General review requirements. 801.602-70 Section 801.602-70 Federal Acquisition Regulations System DEPARTMENT OF VETERANS AFFAIRS GENERAL DEPARTMENT OF VETERANS AFFAIRS ACQUISITION REGULATION SYSTEM Career Development, Contracting Authority, and Responsibilities 801.602-70...

  11. 77 FR 73911 - Flightcrew Member Duty and Rest Requirements

    Science.gov (United States)

    2012-12-12

    ... and Rest Requirements AGENCY: Federal Aviation Administration (FAA), DOT. ACTION: Availability of... Regulatory Impact Analysis of its final rule amending its existing flight, duty and rest regulations... Register as Flight Crew Member Duty and Rest Requirements on January 4, 2012. 77 FR 330. The regulations...

  12. 49 CFR 376.11 - General leasing requirements.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 5 2010-10-01 2010-10-01 false General leasing requirements. 376.11 Section 376.11 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL MOTOR CARRIER... INTERCHANGE OF VEHICLES Leasing Regulations § 376.11 General leasing requirements. Other than through...

  13. 48 CFR 311.7000 - Defining electronic information technology requirements.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Defining electronic information technology requirements. 311.7000 Section 311.7000 Federal Acquisition Regulations System HEALTH... Accessibility Standards 311.7000 Defining electronic information technology requirements. HHS staff that...

  14. 7 CFR 735.300 - Warehouse receipt requirements.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 7 2010-01-01 2010-01-01 false Warehouse receipt requirements. 735.300 Section 735..., DEPARTMENT OF AGRICULTURE REGULATIONS FOR WAREHOUSES REGULATIONS FOR THE UNITED STATES WAREHOUSE ACT Warehouse Receipts § 735.300 Warehouse receipt requirements. (a) Warehouse receipts may be: (1)...

  15. 24 CFR 200.36 - Financial reporting requirements.

    Science.gov (United States)

    2010-04-01

    ... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Financial reporting requirements. 200.36 Section 200.36 Housing and Urban Development Regulations Relating to Housing and Urban... Regulations § 200.36 Financial reporting requirements. The mortgagor must comply with the financial...

  16. 7 CFR 800.25 - Required elevator and merchandising records.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 7 2010-01-01 2010-01-01 false Required elevator and merchandising records. 800.25 Section 800.25 Agriculture Regulations of the Department of Agriculture (Continued) GRAIN INSPECTION... REGULATIONS Recordkeeping and Access to Facilities § 800.25 Required elevator and merchandising records. (a...

  17. 48 CFR 1337.110-70 - Personnel security processing requirements.

    Science.gov (United States)

    2010-10-01

    ... where foreign national access to any DOC facility or DOC IT system is required. The language of the... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Personnel security processing requirements. 1337.110-70 Section 1337.110-70 Federal Acquisition Regulations System DEPARTMENT...

  18. 48 CFR 26.303 - Data collection and reporting requirements.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Data collection and reporting requirements. 26.303 Section 26.303 Federal Acquisition Regulations System FEDERAL ACQUISITION... and Minority Institutions 26.303 Data collection and reporting requirements. Executive Order...

  19. 15 CFR 30.17 - Customs and Border Protection regulations.

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Customs and Border Protection regulations. 30.17 Section 30.17 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade... Requirements § 30.17 Customs and Border Protection regulations. Refer to the DHS's CBP regulations, 19 CFR...

  20. Discovering system requirements

    Energy Technology Data Exchange (ETDEWEB)

    Bahill, A.T.; Bentz, B. [Univ. of Arizona, Tucson, AZ (United States). Systems and Industrial Engineering; Dean, F.F. [Sandia National Labs., Albuquerque, NM (United States)

    1996-07-01

    Cost and schedule overruns are often caused by poor requirements that are produced by people who do not understand the requirements process. This report provides a high-level overview of the system requirements process, explaining types, sources, and characteristics of good requirements. System requirements, however, are seldom stated by the customer. Therefore, this report shows ways to help you work with your customer to discover the system requirements. It also explains terminology commonly used in the requirements development field, such as verification, validation, technical performance measures, and the various design reviews.

  1. Bed Bug Laws and Regulations

    Science.gov (United States)

    21 states have some level of regulation with regard to bed bugs. Most of these requirements focus on hotels and landlords or other property managers. The Department of Housing and Urban Development has guidance on controlling bed bugs in public housing.

  2. Digital Convergence and Content Regulation

    Directory of Open Access Journals (Sweden)

    Michael John Starks

    2014-12-01

    Full Text Available Broadcasting, Press and Internet journalism systems of distribution are converging: the same infrastructure can deliver all three historically separate services. Reception devices mirror this: the Connected TV, the tablet and the smart phone overlap in their functionality. Service overlaps are evident too, with broadcasters providing online and on-demand services and newspapers developing electronic versions. Does this mean that media regulation policies must converge too?My argument is that they should, though only where historically different communications are now fulfilling a similar function, e.g. broadcaster online services and electronic versions of newspapers. Convergence requires a degree of harmonisation and, to this end, I advocate a review of UK broadcasting's 'due impartiality' requirement and of the UK's application of the public service concept. I also argue for independent self-regulation (rather than state-based regulation of non-public-service broadcasting journalism.

  3. Improving CS regulations.

    Energy Technology Data Exchange (ETDEWEB)

    Nesse, R.J.; Scheer, R.M.; Marasco, A.L.; Furey, R.

    1980-10-01

    President Carter issued Executive Order 12044 (3/28/78) that required all Federal agencies to distinguish between significant and insignificant regulations, and to determine whether a regulation will result in major impacts. This study gathered information on the impact of the order and the guidelines on the Office of Conservation and Solar Energy (CS) regulatory practices, investigated problems encountered by the CS staff when implementing the order and guidelines, and recommended solutions to resolve these problems. Major tasks accomplished and discussed are: (1) legislation, Executive Orders, and DOE Memoranda concerning Federal administrative procedures relevant to the development and analysis of regulations within CS reviewed; (2) relevant DOE Orders and Memoranda analyzed and key DOE and CS staff interviewed in order to accurately describe the current CS regulatory process; (3) DOE staff from the Office of the General Counsel, the Office of Policy and Evaluation, the Office of the Environment, and the Office of the Secretary interviewed to explore issues and problems encountered with current CS regulatory practices; (4) the regulatory processes at five other Federal agencies reviewed in order to see how other agencies have approached the regulatory process, dealt with specific regulatory problems, and responded to the Executive Order; and (5) based on the results of the preceding four tasks, recommendations for potential solutions to the CS regulatory problems developed. (MCW)

  4. Ontology Requirements Specification

    OpenAIRE

    Suárez-Figueroa, Mari Carmen; Gómez-Pérez, A.

    2012-01-01

    The goal of the ontology requirements specification activity is to state why the ontology is being built, what its intended uses are, who the end users are, and which requirements the ontology should fulfill. This chapter presents detailed methodological guidelines for specifying ontology requirements efficiently. These guidelines will help ontology engineers to capture ontology requirements and produce the ontology requirements specification document (ORSD). The ORSD will play a key role dur...

  5. Olympic Legislation Requirements

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Management of the number of visitors to the city during the Olympic Games Rules related to free transportation services on railways, highways and city buses Regulations on traffic control and special traffic routes Regulations involving dogs, such as seeing-eye dogs, in public places

  6. Medical device regulation for manufacturers.

    Science.gov (United States)

    McAllister, P; Jeswiet, J

    2003-01-01

    Manufacturers of medical devices are held to a higher standard than manufacturers of many other products due to the potential severity of the consequences of introducing inferior or unsafe products to the market-place. In Canada, the medical device industry is regulated by Health Canada under the Medical Device Regulations of the Food and Drug Act. The Medical Device Regulations define requirements of medical device design, development and manufacture to ensure that products reaching the public are safe and effective. Health Canada also requires that medical device manufacturers maintain distribution records to ensure that devices can be traced to the source and consumers can be contacted successfully in the event that a device is recalled. Medical devices exported from Canada must be compliant with the regulations of the country of import. The Canadian Medical Device Regulations were based on the Medical Device Directives of the European Union thus facilitating approval of Canadian devices for the European market. The United States Food and Drug Administration has separate and distinct requirements for safety and quality of medical devices. While effort has been made to facilitate approval and trade of Canadian medical devices in the United States and the European Union, obtaining approval from multiple regulatory bodies can result in increased device development time and cost. The Global Harmonization Task Force is an organization composed of members from Japanese, Australian, European, Canadian and American medical device regulatory bodies. This organization was formed with the objective of harmonizing medical device regulations in an effort to facilitate international trade and standardize the quality of medical devices available to all countries. This paper discusses the requirements that must be met by manufacturers when designing and manufacturing medical devices.

  7. Ways of standardizing mining regulations

    Energy Technology Data Exchange (ETDEWEB)

    Macionga, R.

    1990-07-01

    Deals with ways of improving and standardizing mining regulations in Poland. Shortcomings of existing mining regulations and legal rules are discussed, their main disadvantage being their great multitude and variety. Apart from institutional faults there are also constitutional failures, e.g. exaggerated safety requirements. Another weakness of the existing regulations is the terminology used that is not compatible with standardized mining terminology. Conversion of all regulations, directions and legal acts into standards is recommended as the best means of improvement and the best methods of standarization are suggested. The standards should be issued by only one institution which should be the Higher Mining Office. Establishing Higher and District Mining Offices as the only control structures and a standarized mining law is postulated.

  8. Assessing Requirements Quality through Requirements Coverage

    Science.gov (United States)

    Rajan, Ajitha; Heimdahl, Mats; Woodham, Kurt

    2008-01-01

    In model-based development, the development effort is centered around a formal description of the proposed software system the model. This model is derived from some high-level requirements describing the expected behavior of the software. For validation and verification purposes, this model can then be subjected to various types of analysis, for example, completeness and consistency analysis [6], model checking [3], theorem proving [1], and test-case generation [4, 7]. This development paradigm is making rapid inroads in certain industries, e.g., automotive, avionics, space applications, and medical technology. This shift towards model-based development naturally leads to changes in the verification and validation (V&V) process. The model validation problem determining that the model accurately captures the customer's high-level requirements has received little attention and the sufficiency of the validation activities has been largely determined through ad-hoc methods. Since the model serves as the central artifact, its correctness with respect to the users needs is absolutely crucial. In our investigation, we attempt to answer the following two questions with respect to validation (1) Are the requirements sufficiently defined for the system? and (2) How well does the model implement the behaviors specified by the requirements? The second question can be addressed using formal verification. Nevertheless, the size and complexity of many industrial systems make formal verification infeasible even if we have a formal model and formalized requirements. Thus, presently, there is no objective way of answering these two questions. To this end, we propose an approach based on testing that, when given a set of formal requirements, explores the relationship between requirements-based structural test-adequacy coverage and model-based structural test-adequacy coverage. The proposed technique uses requirements coverage metrics defined in [9] on formal high-level software

  9. Emotion regulation choice : A conceptual framework and supporting evidence

    NARCIS (Netherlands)

    Sheppes, Gal; Scheibe, Susanne; Suri, Gaurav; Radu, Peter; Blechert, Jens; Gross, James J.

    2014-01-01

    Choice behavior is considered the fundamental means by which individuals exert control over their environments. One important choice domain that remains virtually unexplored is that of emotion regulation. This is surprising given that healthy adaptation requires flexibly choosing between regulation

  10. 34 CFR 350.4 - What regulations apply?

    Science.gov (United States)

    2010-07-01

    ... (IRB) reviews research that purposefully requires inclusion of children with disabilities or... REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION DISABILITY AND REHABILITATION RESEARCH PROJECTS AND CENTERS PROGRAM General § 350.4 What regulations apply? The following regulations apply to the Disability...

  11. Shank-cortactin interactions control actin dynamics to maintain flexibility of neuronal spines and synapses

    NARCIS (Netherlands)

    Mac Gillavry, H.D.; Kerr, JM; Kassner, J; Frost, NA; Blanpied, TA

    2016-01-01

    The family of Shank scaffolding molecules (comprising Shank1, 2 and 3) are core components of the postsynaptic density (PSD) in neuronal synapses. Shanks link surface receptors to other scaffolding molecules within the PSD, as well as to the actin cytoskeleton. However, determining the function of S

  12. Margin requirements, margin loans, and margin rates: practice and principles

    OpenAIRE

    Peter Fortune

    2000-01-01

    The Board of Governors of the Federal Reserve System establishes initial margin requirements under Regulations T, U, and X. Recent margin loan increases, both in aggregate value and relative to market capitalization, have rekindled the debate about using margin requirements as an instrument to affect the prices of common stocks. Proponents of a more active margin requirement policy see the regulations as instruments for affecting the level and volatility of stock prices by influencing investo...

  13. WT1 regulates angiogenesis in Ewing Sarcoma

    National Research Council Canada - National Science Library

    Katuri, Varalakshmi; Gerber, Stephanie; Qiu, Xiaofei; McCarty, Gregory; Goldstein, Seth D; Hammers, Hans; Montgomery, Elizabeth; Chen, Allen R; Loeb, David M

    2014-01-01

    Angiogenesis is required for tumor growth. WT1, a protein that affects both mRNA transcription and splicing, has recently been shown to regulate expression of vascular endothelial growth factor (VEGF...

  14. Characterization of signal transduction pathways regulating myelopoiesis

    NARCIS (Netherlands)

    Geest, C.R.

    2009-01-01

    Studies have demonstrated that hematopoiesis requires coordinated expression of many genes that may directly or indirectly govern HSC and progenitor cell maintenance, lineage commitment, differentiation and mature blood cell function. Although it is evident that correct regulation of proliferation,

  15. Medical devices regulations, standards and practices

    CERN Document Server

    Ramakrishna, Seeram; Wang, Charlene

    2015-01-01

    Medical Devices and Regulations: Standards and Practices will shed light on the importance of regulations and standards among all stakeholders, bioengineering designers, biomaterial scientists and researchers to enable development of future medical devices. Based on the authors' practical experience, this book provides a concise, practical guide on key issues and processes in developing new medical devices to meet international regulatory requirements and standards. Provides readers with a global perspective on medical device regulationsConcise and comprehensive information on how to desig

  16. Postmarket Requirements and Commitments

    Data.gov (United States)

    U.S. Department of Health & Human Services — Provides information to the public on postmarket requirements and commitments. The phrase postmarket requirements and commitments refers to studies and clinical...

  17. Requirements for existing buildings

    DEFF Research Database (Denmark)

    Thomsen, Kirsten Engelund; Wittchen, Kim Bjarne

    2012-01-01

    This report collects energy performance requirements for existing buildings in European member states by June 2012.......This report collects energy performance requirements for existing buildings in European member states by June 2012....

  18. Replacing reserve requirements

    OpenAIRE

    Edward J. Stevens

    1993-01-01

    An examination of the fading significance of the Federal Reserve System's reserve requirements and the recent flowering of required clearing balances, a rapidly growing feature of Reserve Bank operations.

  19. Requirements for existing buildings

    DEFF Research Database (Denmark)

    Thomsen, Kirsten Engelund; Wittchen, Kim Bjarne

    2012-01-01

    This report collects energy performance requirements for existing buildings in European member states by June 2012.......This report collects energy performance requirements for existing buildings in European member states by June 2012....

  20. Replacing reserve requirements

    OpenAIRE

    Edward J. Stevens

    1993-01-01

    An examination of the fading significance of the Federal Reserve System's reserve requirements and the recent flowering of required clearing balances, a rapidly growing feature of Reserve Bank operations.