Science.gov

Sample records for regulator rcac require

  1. Photoelectronic trajectory anatomy of RCA-C-73435 image converter and its performance

    SciTech Connect

    Lai, C.C.; Olk, L.B.; Lear, R.D.

    1985-06-07

    A two-dimensional electron-optics code, which fully incorporates space-charge and self-magnetic effects, was used to compute the photoelectronic trajectories in a RCA-C73435 image-converter streak tube. The code reveals the beam-trace formations in graphic detail as well as provides numerical data on static and dynamic imaging parameters, including focused-field profile, spatial resolution, linear distortion, transit-time dispersion and chromatic aberration. These computational results correlate qualitatively with the experimental ones obtained under corresponding conditions. The study thus establishes a set of computer-simulation estimates on tube performance as a function of the electron-emitting characteristics at the photocathode and as a function of the electrode voltages. 3 refs., 7 figs.

  2. Informational Requirements for Transcriptional Regulation

    PubMed Central

    O'Neill, Patrick K.; Forder, Robert

    2014-01-01

    Abstract Transcription factors (TFs) regulate transcription by binding to specific sites in promoter regions. Information theory provides a useful mathematical framework to analyze the binding motifs associated with TFs but imposes several assumptions that limit their applicability to specific regulatory scenarios. Explicit simulations of the co-evolution of TFs and their binding motifs allow the study of the evolution of regulatory networks with a high degree of realism. In this work we analyze the impact of differential regulatory demands on the information content of TF-binding motifs by means of evolutionary simulations. We generalize a predictive index based on information theory, and we validate its applicability to regulatory scenarios in which the TF binds significantly to the genomic background. Our results show a logarithmic dependence of the evolved information content on the occupancy of target sites and indicate that TFs may actively exploit pseudo-sites to modulate their occupancy of target sites. In regulatory networks with differentially regulated targets, we observe that information content in TF-binding motifs is dictated primarily by the fraction of total probability mass that the TF assigns to its target sites, and we provide a predictive index to estimate the amount of information associated with arbitrarily complex regulatory systems. We observe that complex regulatory patterns can exert additional demands on evolved information content, but, given a total occupancy for target sites, we do not find conclusive evidence that this effect is because of the range of required binding affinities. PMID:24689750

  3. MAPPING GASOLINE REQUIREMENTS, APPLICABLE REGULATIONS AND BANS

    EPA Science Inventory

    Federal and State regulations play an important role in understanding gasoline composition around the United States. Multiple sources of information on these programs were used to develop reliable, up-to-date maps showing gasoline requirements imposed by various regulations. Th...

  4. New HCFA regulations clarify PSO requirements.

    PubMed

    Brock, T H

    1998-06-01

    In March and April of 1998, HCFA promulgated regulations regarding various requirements for provider-sponsored organizations (PSOs). These regulations define what constitutes an affiliated provider to a PSO, identify what percentage of services must be provided directly to beneficiaries by PSO affiliated providers, define what constitutes provider ownership in a PSO, and set minimum capitalization and liquidity standards for PSOs.

  5. Requirements and Regulations for Open Burning and Fire Training

    EPA Pesticide Factsheets

    Intentional burning of facilities is considered demolition under federal asbestos regulations, even if no asbestos is present. Learn about regulations and requirements for open burning and fire training.

  6. 78 FR 29247 - Contractor Legal Management Requirements; Acquisition Regulations; Correction

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-20

    ..., on May 14, 2013. Paul Bosco, Director, Office of Acquisition and Project Management. BILLING CODE... Part 952 RIN 1990-AA37 Contractor Legal Management Requirements; Acquisition Regulations; Correction..., DOE revised existing regulations covering contractor legal management requirements....

  7. 40 CFR 63.312 - Existing regulations and requirements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... PROGRAMS (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS FOR SOURCE CATEGORIES National Emission Standards for Coke Oven Batteries § 63.312 Existing regulations and requirements. (a) The... 40 Protection of Environment 9 2010-07-01 2010-07-01 false Existing regulations and...

  8. Melatonin is required for the circadian regulation of sleep.

    PubMed

    Gandhi, Avni V; Mosser, Eric A; Oikonomou, Grigorios; Prober, David A

    2015-03-18

    Sleep is an evolutionarily conserved behavioral state whose regulation is poorly understood. A classical model posits that sleep is regulated by homeostatic and circadian mechanisms. Several factors have been implicated in mediating the homeostatic regulation of sleep, but molecules underlying the circadian mechanism are unknown. Here we use animals lacking melatonin due to mutation of arylalkylamine N-acetyltransferase 2 (aanat2) to show that melatonin is required for circadian regulation of sleep in zebrafish. Sleep is dramatically reduced at night in aanat2 mutants maintained in light/dark conditions, and the circadian regulation of sleep is abolished in free-running conditions. We find that melatonin promotes sleep downstream of the circadian clock as it is not required to initiate or maintain circadian rhythms. Additionally, we provide evidence that melatonin may induce sleep in part by promoting adenosine signaling, thus potentially linking circadian and homeostatic control of sleep.

  9. General requirements for RCRA regulated hazardous waste tanks

    SciTech Connect

    1995-11-01

    The Resource Conservation and Recovery Act (RCRA), as amended, requires that tanks used for the storage or treatment of hazardous waste (HazW) be permitted, and comply with the requirements contained within the Code of Federal Regulations (CFR) TItle 40 in Subpart J of Part 264/265, unless those tanks have been exempted. Subpart J specifies requirements for the design, construction, installation, operation, inspection, maintenance, repair, release, response, and closure of HazW tanks. Also, the regulations make a distinction between new and existing tanks. Effective December 6, 1995, standards for controlling volatile organic air emissions will apply to non-exempt HazW tanks. HazW tanks will have to be equipped with a cover or floating roof, or be designed to operate as a closed system, to be in compliance with the air emission control requirements. This information brief describes those tanks that are subject to the Subpart J requirements, and will also discuss secondary containment, inspection, restrictions on waste storage, release response, and closure requirements associated with regulated HazW tanks.

  10. 40 CFR 63.312 - Existing regulations and requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... National Emission Standards for Coke Oven Batteries § 63.312 Existing regulations and requirements. (a) The..., topside port lids, coke oven doors, and charging operations in effect on September 15, 1992, or which have... method of monitoring in effect on September 15, 1992, and that ensures coke oven emission...

  11. 40 CFR 63.312 - Existing regulations and requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... National Emission Standards for Coke Oven Batteries § 63.312 Existing regulations and requirements. (a) The..., topside port lids, coke oven doors, and charging operations in effect on September 15, 1992, or which have... method of monitoring in effect on September 15, 1992, and that ensures coke oven emission...

  12. 40 CFR 63.312 - Existing regulations and requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... National Emission Standards for Coke Oven Batteries § 63.312 Existing regulations and requirements. (a) The..., topside port lids, coke oven doors, and charging operations in effect on September 15, 1992, or which have... method of monitoring in effect on September 15, 1992, and that ensures coke oven emission...

  13. 40 CFR 63.312 - Existing regulations and requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... National Emission Standards for Coke Oven Batteries § 63.312 Existing regulations and requirements. (a) The..., topside port lids, coke oven doors, and charging operations in effect on September 15, 1992, or which have... method of monitoring in effect on September 15, 1992, and that ensures coke oven emission...

  14. 78 FR 17186 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-20

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS); Notification Requirements for Critical Safety Items AGENCY: Defense... Acquisition Regulation Supplement (DFARS), Notification Requirements for Critical Safety Items; OMB...

  15. Cellular growth in plants requires regulation of cell wall biochemistry.

    PubMed

    Chebli, Youssef; Geitmann, Anja

    2017-02-01

    Cell and organ morphogenesis in plants are regulated by the chemical structure and mechanical properties of the extracellular matrix, the cell wall. The two primary load bearing components in the plant cell wall, the pectin matrix and the cellulose/xyloglucan network, are constantly remodelled to generate the morphological changes required during plant development. This remodelling is regulated by a plethora of loosening and stiffening agents such as pectin methyl-esterases, calcium ions, expansins, and glucanases. The tight spatio-temporal regulation of the activities of these agents is a sine qua non condition for proper morphogenesis at cell and tissue levels. The pectin matrix and the cellulose-xyloglucan network operate in concert and their behaviour is mutually dependent on their chemical, structural and mechanical modifications. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Review of orders and regulations requiring environmental protection

    SciTech Connect

    Kelly, E.; Cunningham, R.; Michael, D.

    1996-09-01

    With the increased awareness of and interest in potential ecological risks associated with past, current, and future Department of Energy (DOE) activities, DOE`s Defense Programs (DP) Office of Technical and Environmental Support sponsored a study to (1) evaluate the effectiveness of the current compliance-driven environmental protection and assessment efforts relative to ecological concerns; (2) explore the need for a more focused, integrated approach to address ecological impacts; and (3) identify the requirements for an integrated approach. The study explored four questions. (a) Which federal regulations and DOE orders either explicitly require ecological assessments or implicitly require them through environmental protection language? (b) What currently is being done at selected DOE facilities to implement these regulations and orders? (c) What are private sector industries doing in terms of ecological risk assessments and how do industry approaches and issues compare with those of DOE? (d) What, if anything, in addition to current efforts is needed to ensure the protection of ecological resources associated with DOE facilities, to support defensible decision making, and to improve efficiency? The results of this study are presented in a report titled {open_quotes}Integrated, Comprehensive Ecological Impact Assessments In Support of Department of Energy Decision Making{close_quotes}. This report is a companion document to that report. This report provides a more detailed discussion of the document reviews of the relevant environmental protection regulations and current and pending DOE orders. The main goal of the document reviews was to understand existing requirements for ecological data collection and impact assessments.

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

    PubMed

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

    2014-09-08

    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.

  18. 75 FR 20825 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 211...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-21

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 211, Describing Agency Needs AGENCY: Defense Acquisition Regulations System... the message. Fax: 703-602-0350. Mail: Defense Acquisition Regulations System, Attn: Ms....

  19. 75 FR 10790 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-09

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Notification Requirements for Critical Safety Items AGENCY: Defense Acquisition... Supplement (DFARS) Notification Requirements for Critical Safety Items; OMB Control Number 0704-0441....

  20. Pheromone-regulated genes required for yeast mating differentiation.

    PubMed

    Erdman, S; Lin, L; Malczynski, M; Snyder, M

    1998-02-09

    Yeast cells mate by an inducible pathway that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging screen to identify genes whose expression is regulated by mating pheromone. 91,200 transformants containing random lacZ insertions were screened for beta-galactosidase (beta-gal) expression in the presence and absence of alpha factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or had not previously been known to be pheromone regulated were examined for effects on the mating process. Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects. Three of the proteins encoded by these genes, Fig1p, Fig2p, and Fig4p, are dispensible for cell polarization in uniform concentrations of mating pheromone, but are required for normal cell polarization in mating mixtures, conditions that involve cell-cell communication. Fig1p and Fig2p are also important for cell fusion and conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p and Fig2p are likely to act at the cell surface as Fig1:: beta-gal and Fig2::beta-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p. Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.

  1. Neuroendocrine regulation of Drosophila metamorphosis requires TGFβ/Activin signaling

    PubMed Central

    Gibbens, Ying Y.; Warren, James T.; Gilbert, Lawrence I.; O'Connor, Michael B.

    2011-01-01

    In insects, initiation of metamorphosis requires a surge in the production of the steroid hormone 20-hydroxyecdysone from the prothoracic gland, the primary endocrine organ of juvenile larvae. Here, we show that blocking TGFβ/Activin signaling, specifically in the Drosophila prothoracic gland, results in developmental arrest prior to metamorphosis. The terminal, giant third instar larval phenotype results from a failure to induce the large rise in ecdysteroid titer that triggers metamorphosis. We further demonstrate that activin signaling regulates competence of the prothoracic gland to receive PTTH and insulin signals, and that these two pathways act at the mRNA and post-transcriptional levels, respectively, to control ecdysone biosynthetic enzyme expression. This dual regulatory circuitry may provide a cross-check mechanism to ensure that both developmental and nutritional inputs are synchronized before initiating the final genetic program leading to reproductive adult development. As steroid hormone production in C. elegans and mammals is also influenced by TGFβ/Activin signaling, this family of secreted factors may play a general role in regulating developmental transitions across phyla. PMID:21613324

  2. 77 FR 58817 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-24

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF DEFENSE Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS); Contract Financing AGENCY: Defense Acquisition Regulations System, Department...

  3. 75 FR 9114 - Defense Federal Acquisition Regulation Supplement; Additional Requirements Applicable to...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-01

    ... Defense Acquisition Regulations System 48 CFR Part 217 Defense Federal Acquisition Regulation Supplement; Additional Requirements Applicable to Multiyear Contracts (DFARS Case 2008-D023) AGENCY: Defense Acquisition... is issuing this interim rule amending the Defense Federal Acquisition Regulation Supplement (DFARS...

  4. 78 FR 68829 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Contract...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-15

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF DEFENSE Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Contract Financing AGENCY: Defense Acquisition Regulations System, Department...

  5. 75 FR 41161 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 251...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-15

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 251, Contractor Use of Government Supply Sources AGENCY: Defense Acquisition... extension of an approved information collection requirement. SUMMARY: In compliance with Section 3506(c)(2...

  6. translin is required for metabolic regulation of sleep

    PubMed Central

    Stahl, Bethany A.; Masek, Pavel; Mehta, Aradhana; Heidker, Rebecca; Bollinger, Wesley; Gingras, Robert M.; Kim, Young-Joon; Ja, William W.; Suter, Beat; DiAngelo, Justin R.; Keene, Alex C.

    2016-01-01

    Summary Dysregulation of sleep or feeding has enormous health consequences. In humans, acute sleep loss is associated with increased appetite and insulin insensitivity, while chronically sleep-deprived individuals are more likely to develop obesity, metabolic syndrome, type II diabetes, and cardiovascular disease. Conversely, metabolic state potently modulates sleep and circadian behavior; yet, the molecular basis for sleep-metabolism interactions remains poorly understood. Here, we describe the identification of translin (trsn), a highly conserved RNA/DNA binding protein, as essential for starvation-induced sleep suppression. Strikingly, trsn does not appear to regulate energy stores, free glucose levels, or feeding behavior suggesting the sleep phenotype of trsn mutant flies is not a consequence of general metabolic dysfunction or blunted response to starvation. While broadly expressed in all neurons, trsn is transcriptionally upregulated in the heads of flies in response to starvation. Spatially restricted rescue or targeted knockdown localizes trsn function to neurons that produce the tachykinin-family neuropeptide Leucokinin. Manipulation of neural activity in Leucokinin neurons revealed these neurons to be required for starvation-induced sleep suppression. Taken together, these findings establish trsn as an essential integrator of sleep and metabolic state, with implications for understanding the neural mechanism underlying sleep disruption in response to environmental perturbation. PMID:27020744

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

    PubMed

    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.

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

    PubMed Central

    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

    2012-01-01

    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. PMID:23257356

  9. Army Regulation 71-9, Materiel Requirements, 30 April 1997

    DTIC Science & Technology

    1998-08-01

    provides the policy for streamlining requirements through horizontal technology integration and the Warfighting Rapid Acquisition Program; and (6) updates policies for preparing requirements documents and conducting supporting analyses.

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

    PubMed Central

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

    2013-01-01

    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. PMID:23437009

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

    SciTech Connect

    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.

  12. 76 FR 72915 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DoD...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ... appropriate frequency allocation has not been made. Ynette R. Shelkin, Editor, Defense Acquisition Regulations... Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DoD Acquisition Process... following methods: Regulations.gov : http://www.regulations.gov . Submit comments via the Federal...

  13. 78 FR 63462 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-24

    ... organizational conflicts of interest arising in a systems engineering and technical assistance contract for an... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition...: Defense Acquisition Regulations System, Department of Defense (DoD). ACTION: Notice and request for...

  14. 49 CFR 171.26 - Additional requirements for the use of the IAEA Regulations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... International Transport Standards and Regulations § 171.26 Additional requirements for the use of the IAEA... may be offered for transportation or transported in accordance with the IAEA Regulations (IBR, see...

  15. 76 FR 35424 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-17

    ... Regulation Supplement; Acquisition of Information Technology AGENCY: Defense Acquisition Regulations System... technology. The Office of Management and Budget (OMB) has approved this information collection requirement... Supplement (DFARS) Part 239, Acquisition of Information Technology, and the associated clauses at DFARS...

  16. 77 FR 12754 - Contractor Legal Management Requirements; Acquisition Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-02

    ... Part 719 48 Parts 931, 952 and 970 RIN 1990-AA37 Contractor Legal Management Requirements; Acquisition... period. SUMMARY: This document announces a reopening of the time period for submitting comments on the... covering contractor legal management requirements and make conforming amendments to the Department of...

  17. 78 FR 80369 - Federal Acquisition Regulation; Service Contracts Reporting Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-31

    ...), and National Aeronautics and Space Administration (NASA). ACTION: Final rule. SUMMARY: DoD, GSA, and NASA are issuing a final rule amending the Federal Acquisition Regulation (FAR) to implement a section..., GSA, and NASA published a proposed rule in the Federal Register at 76 FR 22070 on April 20, 2011, to...

  18. 49 CFR 171.26 - Additional requirements for the use of the IAEA Regulations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... GENERAL INFORMATION, REGULATIONS, AND DEFINITIONS Authorization and Requirements for the Use of... passing through the United States in the course of being shipped between places outside the United...

  19. The regulation of gene expression required for C4 photosynthesis.

    PubMed

    Hibberd, Julian M; Covshoff, Sarah

    2010-01-01

    C(4) photosynthesis is normally associated with the compartmentation of photosynthesis between mesophyll (M) and bundle sheath (BS) cells. The mechanisms regulating the differential accumulation of photosynthesis proteins in these specialized cells are fundamental to our understanding of how C(4) photosynthesis operates. Cell-specific accumulation of proteins in M or BS can be mediated by posttranscriptional processes and translational efficiency as well as by differences in transcription. Individual genes are likely regulated at multiple levels. Although cis-elements have been associated with cell-specific expression in C(4) leaves, there has been little progress in identifying trans-factors. When C(4) photosynthesis genes from C(4) species are placed in closely related C(3) species, they are often expressed in a manner faithful to the C(4) cycle. Next-generation sequencing and comprehensive analysis of the extent to which genes from C(4) species are expressed in M or BS cells of C(3) plants should provide insight into how the C(4) pathway is regulated and evolved.

  20. Compliance with 504 regulations need not require costly renovation.

    PubMed

    Rees, F W; Gary, J G

    1978-05-16

    By June 3, 1980, all hospitals receiving federal funds must have completed plans to make facilities accessible to the handicapped. In many instances, doing so will require hospitals to renovate existing facilities. However, through careful interpretation of the law and creative planning, hospitals can avoid making expensive, unnecessary building modifications.

  1. 49 CFR 171.26 - Additional requirements for the use of the IAEA Regulations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... may be offered for transportation or transported in accordance with the IAEA Regulations (IBR, see... 49 Transportation 2 2013-10-01 2013-10-01 false Additional requirements for the use of the IAEA Regulations. 171.26 Section 171.26 Transportation Other Regulations Relating to Transportation PIPELINE...

  2. 49 CFR 171.26 - Additional requirements for the use of the IAEA Regulations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... may be offered for transportation or transported in accordance with the IAEA Regulations (IBR, see... 49 Transportation 2 2012-10-01 2012-10-01 false Additional requirements for the use of the IAEA Regulations. 171.26 Section 171.26 Transportation Other Regulations Relating to Transportation PIPELINE...

  3. 49 CFR 192.13 - What general requirements apply to pipelines regulated under this part?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... regulated under this part? 192.13 Section 192.13 Transportation Other Regulations Relating to Transportation... General § 192.13 What general requirements apply to pipelines regulated under this part? (a) No person may... second column, unless: (1) The pipeline has been designed, installed, constructed, initially inspected...

  4. 75 FR 60263 - Federal Acquisition Regulation; Offering a Construction Requirement-8(a) Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    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), to...

  5. Prediction of Regulation Reserve Requirements in California ISO Control Area based on BAAL Standard

    SciTech Connect

    Etingov, Pavel V.; Makarov, Yuri V.; Samaan, Nader A.; Ma, Jian; Loutan, Clyde

    2013-07-21

    This paper presents new methodologies developed at Pacific Northwest National Laboratory (PNNL) to estimate regulation capacity requirements in the California ISO control area. Two approaches have been developed: (1) an approach based on statistical analysis of actual historical area control error (ACE) and regulation data, and (2) an approach based on balancing authority ACE limit control performance standard. The approaches predict regulation reserve requirements on a day-ahead basis including upward and downward requirements, for each operating hour of a day. California ISO data has been used to test the performance of the proposed algorithms. Results show that software tool allows saving up to 30% on the regulation procurements cost .

  6. 75 FR 68330 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-05

    ... occupational safety. DoD contracting officers use this information to-- Verify compliance with requirements for... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Occupational Safety and Drug-Free Work Force (OMB Control Number 0704-0272)...

  7. 49 CFR 390.11 - Motor carrier to require observance of driver regulations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...) FEDERAL MOTOR CARRIER SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION FEDERAL MOTOR CARRIER SAFETY REGULATIONS FEDERAL MOTOR CARRIER SAFETY REGULATIONS; GENERAL General Requirements and Information § 390.11... 49 Transportation 5 2010-10-01 2010-10-01 false Motor carrier to require observance of driver...

  8. [Legal framework of data protection : current requirements in Germany and requirements in planned European Union regulations].

    PubMed

    Schütze, B

    2013-05-01

    The federal system in Germany necessitates that in addition to federal laws, country and church-specific legislations must also be considered during the evaluation of relevant legal stipulations concerning data protection. Furthermore, there are also special legal regulations for hospitals in almost every federal state which are governed by the principle of subsidiarity: special legal regulations are to be preferentially used, so that findings from one federal state are difficult to transfer to another federal state.Patient data may only be used and processed without legal regulations with informed consent of the patient. The use of patient data for purposes of quality assurance, research and further education of students and doctors is possible under the present laws according to a positive weighting of interests. Patient data can also be exchanged via online services for the purposes of patient care; however, informed consent of the patient for medical online services is almost always unavoidable.

  9. GRIP1 is required for homeostatic regulation of AMPAR trafficking

    PubMed Central

    Tan, Han L.; Queenan, Bridget N.; Huganir, Richard L.

    2015-01-01

    Homeostatic plasticity is a negative feedback mechanism that stabilizes neurons during periods of perturbed activity. The best-studied form of homeostatic plasticity in the central nervous system is the scaling of excitatory synapses. Postsynaptic AMPA-type glutamate receptors (AMPARs) can be inserted into synapses to compensate for neuronal inactivity or removed to compensate for hyperactivity. However, the molecular mechanisms underlying the homeostatic regulation of AMPARs remain elusive. Here, we show that the expression of GRIP1, a multi-PDZ (postsynaptic density 95/discs large/zona occludens) domain AMPAR-binding protein, is bidirectionally altered by neuronal activity. Furthermore, we observe a subcellular redistribution of GRIP1 and a change in the binding of GRIP1 to GluA2 during synaptic scaling. Using a combination of biochemical, genetic, and electrophysiological methods, we find that loss of GRIP1 blocks the accumulation of surface AMPARs and the scaling up of synaptic strength that occur in response to chronic activity blockade. Collectively, our data point to an essential role of GRIP1-mediated AMPAR trafficking during inactivity-induced synaptic scaling. PMID:26216979

  10. 77 FR 66464 - Federal Acquisition Regulation; Submission for OMB Review; Value Engineering Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-05

    ... Regulation; Submission for OMB Review; Value Engineering Requirements AGENCIES: Department of Defense (DOD... the Office of Management and Budget (OMB) a request to review and approve an extension of a previously approved information collection requirement concerning Value Engineering Requirements. A notice...

  11. 42 CFR 417.478 - Requirements of other laws and regulations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Requirements of other laws and regulations. 417.478 Section 417.478 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN... HEALTH CARE PREPAYMENT PLANS Medicare Contract Requirements § 417.478 Requirements of other laws...

  12. [On meeting sanitary legal requirements in technical regulation of medical equipment safety].

    PubMed

    Kravchenko, O K; Prokopenko, L V

    2007-01-01

    The article covered observance of sanitary legal requirements in special technical regulations "On requirements to medical equipment and medical products safety". The authors discussed problems of applied terminology, classification of medical products, occupational risk, control over observance of safety requirements on all stages of medical products circulation--design, production, usage.

  13. 78 FR 32547 - Loan Originator Compensation Requirements Under the Truth in Lending Act (Regulation Z...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-31

    ... PROTECTION 12 CFR Part 1026 RIN 3170-AA37 Loan Originator Compensation Requirements Under the Truth in... Compensation Requirements under the Truth in Lending Act (Regulation Z) Final Rule, issued on January 20, 2013...\\ One of these final rules was the Loan Originator Compensation Requirements Under the Truth in...

  14. 7 CFR 318.13-3 - General requirements for all regulated articles.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE STATE OF HAWAII AND TERRITORIES QUARANTINE NOTICES Regulated Articles From Hawaii and the Territories § 318.13-3 General requirements for all...

  15. 7 CFR 318.13-3 - General requirements for all regulated articles.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE STATE OF HAWAII AND TERRITORIES QUARANTINE NOTICES Regulated Articles From Hawaii and the Territories § 318.13-3 General requirements for all...

  16. 7 CFR 318.13-3 - General requirements for all regulated articles.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE STATE OF HAWAII AND TERRITORIES QUARANTINE NOTICES Regulated Articles From Hawaii and the Territories § 318.13-3 General requirements for all...

  17. 49 CFR 1013.3 - Review and reporting requirements for regulated carriers.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... THE PROPER USE OF VOTING TRUSTS § 1013.3 Review and reporting requirements for regulated carriers. (a... applicant shows, by clear and convincing evidence, and the Board finds, that the failure to comply...

  18. 76 FR 14562 - Federal Acquisition Regulation; Additional Requirements for Market Research

    Federal Register 2010, 2011, 2012, 2013, 2014

    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 15...

  19. 7 CFR 330.200 - Movement of plant pests regulated; permits required.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 5 2012-01-01 2012-01-01 false Movement of plant pests regulated; permits required... AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE FEDERAL PLANT PEST REGULATIONS; GENERAL; PLANT PESTS; SOIL, STONE, AND QUARRY PRODUCTS; GARBAGE Movement of Plant Pests § 330.200 Movement of...

  20. 7 CFR 330.200 - Movement of plant pests regulated; permits required.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 5 2013-01-01 2013-01-01 false Movement of plant pests regulated; permits required... AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE FEDERAL PLANT PEST REGULATIONS; GENERAL; PLANT PESTS; SOIL, STONE, AND QUARRY PRODUCTS; GARBAGE Movement of Plant Pests § 330.200 Movement of...

  1. 7 CFR 330.200 - Movement of plant pests regulated; permits required.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 5 2014-01-01 2014-01-01 false Movement of plant pests regulated; permits required... AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE FEDERAL PLANT PEST REGULATIONS; GENERAL; PLANT PESTS; SOIL, STONE, AND QUARRY PRODUCTS; GARBAGE Movement of Plant Pests § 330.200 Movement of...

  2. 7 CFR 318.13-3 - General requirements for all regulated articles.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 5 2010-01-01 2010-01-01 false General requirements for all regulated articles. 318.13-3 Section 318.13-3 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND... consignment is of such a nature that no danger of infestation or infection is involved. (i) Persons...

  3. 7 CFR 330.200 - Movement of plant pests regulated; permits required.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... on arrival in the United States to confirm the nature of the material and freedom from risk of plant... 7 Agriculture 5 2011-01-01 2011-01-01 false Movement of plant pests regulated; permits required... AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE FEDERAL PLANT PEST REGULATIONS; GENERAL...

  4. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... material. 150.31 Section 150.31 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXEMPTIONS AND CONTINUED... Requirements for Agreement State regulation of byproduct material. (a) Prior to November 8, 1981, in the licensing and regulation of byproduct material, as defined in § 150.3(c)(2) of this part, or of any activity...

  5. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... material. 150.31 Section 150.31 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXEMPTIONS AND CONTINUED... Requirements for Agreement State regulation of byproduct material. (a) Prior to November 8, 1981, in the licensing and regulation of byproduct material, as defined in § 150.3(c)(2) of this part, or of any activity...

  6. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... material. 150.31 Section 150.31 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXEMPTIONS AND CONTINUED... Requirements for Agreement State regulation of byproduct material. (a) Prior to November 8, 1981, in the licensing and regulation of byproduct material, as defined in § 150.3(c)(2) of this part, or of any activity...

  7. 76 FR 71558 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Requests...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-18

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition..., implements 10 U.S.C. 2410(a). DoD contracting officers and auditors use this information to evaluate... Acquisition Regulations System, Department of Defense (DoD). ACTION: Notice and request for comments. SUMMARY...

  8. 78 FR 72620 - Federal Acquisition Regulation; Higher-Level Contract Quality Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-03

    ... RIN 9000-AM65 Federal Acquisition Regulation; Higher-Level Contract Quality Requirements AGENCY... Acquisition Regulation (FAR) to clarify when to use higher-level quality standards in solicitations and contracts, and to update the examples of higher-level quality standards by revising obsolete standards and...

  9. 12 CFR 350.12 - Disclosure required by applicable banking or securities law or regulations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 4 2010-01-01 2010-01-01 false Disclosure required by applicable banking or securities law or regulations. 350.12 Section 350.12 Banks and Banking FEDERAL DEPOSIT INSURANCE CORPORATION... STATE NONMEMBER BANKS § 350.12 Disclosure required by applicable banking or securities law or...

  10. 49 CFR 390.11 - Motor carrier to require observance of driver regulations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 5 2013-10-01 2013-10-01 false Motor carrier to require observance of driver... Motor carrier to require observance of driver regulations. Whenever in part 325 of subchapter A or in this subchapter a duty is prescribed for a driver or a prohibition is imposed upon the driver, it...

  11. 49 CFR 390.11 - Motor carrier to require observance of driver regulations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 5 2014-10-01 2014-10-01 false Motor carrier to require observance of driver... Motor carrier to require observance of driver regulations. Whenever in part 325 of subchapter A or in this subchapter a duty is prescribed for a driver or a prohibition is imposed upon the driver, it...

  12. 49 CFR 390.11 - Motor carrier to require observance of driver regulations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 5 2011-10-01 2011-10-01 false Motor carrier to require observance of driver... Motor carrier to require observance of driver regulations. Whenever in part 325 of subchapter A or in this subchapter a duty is prescribed for a driver or a prohibition is imposed upon the driver, it...

  13. 49 CFR 390.11 - Motor carrier to require observance of driver regulations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 5 2012-10-01 2012-10-01 false Motor carrier to require observance of driver... Motor carrier to require observance of driver regulations. Whenever in part 325 of subchapter A or in this subchapter a duty is prescribed for a driver or a prohibition is imposed upon the driver, it...

  14. 78 FR 25 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-02

    ... requirements under the Regulated Navigation Area (RNA) established by 33 CFR 165.830 for barges loaded with... managers responsible for CDC barges in the RNA from their dangerous cargo or vessel arrival and movement... contract and lift the suspension, modify the reporting requirements in the RNA, or repeal the RNA...

  15. 76 FR 2827 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-18

    ... Regulated Navigation Area (RNA) established by 33 CFR 165.921 for barges loaded with certain dangerous... requirements, or repeal the RNA. This suspension of the CDC reporting requirements in no way relieves towing vessel operators and fleeting area managers responsible for CDC barges in the RNA from their dangerous...

  16. Technical guidance document for environmental requirements of commercial OTEC licensing regulations (15 CFR Part 981)

    SciTech Connect

    Not Available

    1981-09-01

    This document provides a potential OTEC applicant with the insights believed needed to satisfy the environmental information requirements of the regulations for licensing commercial OTEC facilities and plantships. This information should be used by applicants to define the site-specific details of the needed environmental assessment, and the details should then form a basis for pre-application consultations on the environmental requirements.

  17. 17 CFR 230.508 - Insignificant deviations from a term, condition or requirement of Regulation D.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 17 Commodity and Securities Exchanges 2 2013-04-01 2013-04-01 false Insignificant deviations from a term, condition or requirement of Regulation D. 230.508 Section 230.508 Commodity and Securities... Securities Act of 1933 § 230.508 Insignificant deviations from a term, condition or requirement of...

  18. 78 FR 65218 - Defense Federal Acquisition Regulation Supplement: Private Sector Notification Requirements of In...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-31

    ... Regulation Supplement: Private Sector Notification Requirements of In-Sourcing Actions DFARS Case 2012-D036... notification of in-sourcing actions. DATES: Effective date: October 31, 2013. Comment date: Comments on the...-sourcing actions. II. Discussion and Analysis Section 938 of the NDAA requires the Secretary of Defense to...

  19. 2 CFR 170.200 - Requirements for program announcements, regulations, and application instructions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 2 Grants and Agreements 1 2014-01-01 2014-01-01 false Requirements for program announcements, regulations, and application instructions. 170.200 Section 170.200 Grants and Agreements Office of Management... Transparency Act reporting requirements; and (2) That either: (i) Is issued on or after the effective date of...

  20. 2 CFR 170.200 - Requirements for program announcements, regulations, and application instructions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 2 Grants and Agreements 1 2012-01-01 2012-01-01 false Requirements for program announcements, regulations, and application instructions. 170.200 Section 170.200 Grants and Agreements Office of Management... are subject to Transparency Act reporting requirements; and (2) That either: (i) Is issued on or after...

  1. 2 CFR 170.200 - Requirements for program announcements, regulations, and application instructions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 2 Grants and Agreements 1 2013-01-01 2013-01-01 false Requirements for program announcements, regulations, and application instructions. 170.200 Section 170.200 Grants and Agreements Office of Management... are subject to Transparency Act reporting requirements; and (2) That either: (i) Is issued on or after...

  2. 2 CFR 170.200 - Requirements for program announcements, regulations, and application instructions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 2 Grants and Agreements 1 2011-01-01 2011-01-01 false Requirements for program announcements, regulations, and application instructions. 170.200 Section 170.200 Grants and Agreements Office of Management... Act reporting requirements; and (2) That either: (i) Is issued on or after the effective date of this...

  3. 78 FR 60898 - Regulation on Definition and Requirements for a Nationally Recognized Testing Laboratory...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-02

    ...: Definition and Requirements of a Nationally Recognized Testing Laboratory (29 CFR 1910.7). OMB Control Number... Recognized Testing Laboratory; Revision of the Office of Management and Budget's (OMB) Approval of..., ``definition and requirements for a nationally recognized testing laboratory'' (The Regulation). The...

  4. 31 CFR 103.120 - Anti-money laundering program requirements for financial institutions regulated by a Federal...

    Code of Federal Regulations, 2010 CFR

    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...

  5. 12 CFR 925.8 - Subject to inspection and regulation requirement.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... required by section 4(a)(1)(B) of the Act (12 U.S.C. 1424(a)(1)(B)) and § 925.6(a)(2) of this part, if, in... 12 Banks and Banking 7 2010-01-01 2010-01-01 false Subject to inspection and regulation requirement. 925.8 Section 925.8 Banks and Banking FEDERAL HOUSING FINANCE BOARD FEDERAL HOME LOAN...

  6. 78 FR 4788 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-23

    ... SECURITY Coast Guard 33 CFR Part 165 [USCG-2013-0019 RIN 1625-AA11 Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous Cargoes, Inland Rivers, Ninth Coast Guard District; Stay (Suspension) AGENCY: Coast Guard, DHS. ACTION: Temporary final rule. SUMMARY: The Commander, Ninth Coast Guard...

  7. 78 FR 61183 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-03

    ... SECURITY Coast Guard 33 CFR Part 165 RIN 1625-AA11 Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous Cargoes, Inland Rivers, Ninth Coast Guard District; Stay (Suspension) AGENCY: Coast Guard, DHS. ACTION: Temporary final rule. SUMMARY: The Commander, Ninth Coast Guard...

  8. 76 FR 1360 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-10

    ... SECURITY Coast Guard 33 CFR Part 165 RIN 1625-AA11 Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous Cargoes, Inland Rivers, Eighth Coast Guard District; Stay (Suspension) AGENCY: Coast Guard, DHS. ACTION: Temporary final rule. SUMMARY: The Commander, Eighth Coast...

  9. Hydrogen monitoring requirements in the global technical regulation on hydrogen and fuel cell vehicles

    DOE PAGES

    Buttner, William; Rivkin, C.; Burgess, R.; ...

    2017-02-04

    Here, the United Nations Economic Commission for Europe 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 (FCEVs). GTR Number 13 has been formally adopted and will serve as the basis for the national regulatory standards for FCEV safety in North America (led by the United States), Japan, Korea, and the European Union. The GTR defines safety requirements for these vehicles, including specifications on the allowable hydrogen levels in vehicle enclosures during in-use and post-crash conditions andmore » 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, to be legally binding, methods to verify compliance with 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 with the hydrogen release requirements as specified in the GTR.« less

  10. 50 CFR 14.254 - What are the requirements contained in these regulations?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 1 2010-10-01 2010-10-01 false What are the requirements contained in these regulations? 14.254 Section 14.254 Wildlife and Fisheries UNITED STATES FISH AND WILDLIFE SERVICE... IMPORTATION OF WILDLIFE AND PLANTS IMPORTATION, EXPORTATION, AND TRANSPORTATION OF WILDLIFE Captive Wildlife...

  11. 40 CFR 152.25 - Exemptions for pesticides of a character not requiring FIFRA regulation.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Exemptions for pesticides of a... PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PESTICIDE REGISTRATION AND CLASSIFICATION PROCEDURES Exemptions § 152.25 Exemptions for pesticides of a character not requiring FIFRA regulation. The pesticides...

  12. 40 CFR 152.25 - Exemptions for pesticides of a character not requiring FIFRA regulation.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Exemptions for pesticides of a... PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PESTICIDE REGISTRATION AND CLASSIFICATION PROCEDURES Exemptions § 152.25 Exemptions for pesticides of a character not requiring FIFRA regulation. The pesticides...

  13. 40 CFR 152.25 - Exemptions for pesticides of a character not requiring FIFRA regulation.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Exemptions for pesticides of a... PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PESTICIDE REGISTRATION AND CLASSIFICATION PROCEDURES Exemptions § 152.25 Exemptions for pesticides of a character not requiring FIFRA regulation. The pesticides...

  14. 40 CFR 152.25 - Exemptions for pesticides of a character not requiring FIFRA regulation.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Exemptions for pesticides of a... PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PESTICIDE REGISTRATION AND CLASSIFICATION PROCEDURES Exemptions § 152.25 Exemptions for pesticides of a character not requiring FIFRA regulation. The pesticides...

  15. 40 CFR 152.25 - Exemptions for pesticides of a character not requiring FIFRA regulation.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Exemptions for pesticides of a... PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PESTICIDE REGISTRATION AND CLASSIFICATION PROCEDURES Exemptions § 152.25 Exemptions for pesticides of a character not requiring FIFRA regulation. The pesticides...

  16. 77 FR 31026 - Requirements for Importing Food and Drug Administration Regulated Products Into the United States

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-24

    ... respect to importing pharmaceutical products, medical devices, food products, as well as technology which... No: 2012-12592] DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2012-N-0001] Requirements for Importing Food and Drug Administration Regulated Products Into the...

  17. 76 FR 78126 - Disclosure Requirements for Depository Institutions Lacking Federal Deposit Insurance (Regulation I)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-16

    ... Administrative Procedure Act (APA) \\11\\ generally requires public notice and an opportunity to comment before promulgation of substantive regulations.\\12\\ The APA provides exceptions to notice-and- comment procedures... Bureau, relate to agency organization, procedure, and practice and are thus exempt from the APA's...

  18. 13 CFR 120.1511 - Certification and other reporting and notification requirements for Other Regulated SBLCs.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Certification and other reporting and notification requirements for Other Regulated SBLCs. 120.1511 Section 120.1511 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION BUSINESS LOANS Risk-Based Lender Oversight Enforcement Actions §...

  19. 42 CFR 417.478 - Requirements of other laws and regulations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Requirements of other laws and regulations. 417.478 Section 417.478 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM (CONTINUED) HEALTH MAINTENANCE ORGANIZATIONS, COMPETITIVE...

  20. 42 CFR 51b.107 - Is participation in preventive health service programs required by these regulations?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Is participation in preventive health service..., DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS PROJECT GRANTS FOR PREVENTIVE HEALTH SERVICES General Provisions § 51b.107 Is participation in preventive health service programs required by these regulations...

  1. 7 CFR 330.200 - Movement of plant pests regulated; permits required.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 5 2010-01-01 2010-01-01 false Movement of plant pests regulated; permits required...; PLANT PESTS; SOIL, STONE, AND QUARRY PRODUCTS; GARBAGE Movement of Plant Pests § 330.200 Movement of... plant pest so moving unless such movement is authorized under permit under this part and is made in...

  2. 75 FR 14448 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Federal Register 2010, 2011, 2012, 2013, 2014

    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 Angeles...

  3. 75 FR 51824 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-23

    ...] [FR Doc No: 2010-20834] DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2010-N-0001] Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good Clinical Practice; Public Workshop AGENCY: Food and Drug Administration, HHS. ACTION...

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

    SciTech Connect

    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. 77 FR 34927 - National Pollutant Discharge Elimination System-Proposed Regulations To Establish Requirements...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-12

    ...--Proposed Regulations To Establish Requirements for Cooling Water Intake Structures at Existing Facilities... standards for cooling water intake structures at all existing power generating, manufacturing, and industrial facilities as part of implementing section 316(b) of the Clean Water Act (CWA). This...

  6. 77 FR 34315 - National Pollutant Discharge Elimination System-Proposed Regulations to Establish Requirements...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-11

    ... operates at a minimum cycles of concentration of 3.0 for freshwater and 1.5 for saltwater or brackish water...--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...

  7. 77 FR 24611 - Removal of Regulations Requiring 3% Withholding by Government Entities

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-25

    ... affects government entities that would have been required to withhold and report tax from payments to... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF THE TREASURY... Government Entities AGENCY: Internal Revenue Service (IRS), Treasury. ACTION: Final regulations....

  8. 78 FR 38234 - Defense Federal Acquisition Regulation Supplement: Requirements for Acquisitions Pursuant to...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-26

    ... convention. Delete 216.501-1. Only ``Multiple-award contract'' was defined and it was only used in 216.505-70... Acquisition Regulation Supplement: Requirements for Acquisitions Pursuant to Multiple Award Contracts (DFARS... pursuant to multiple award contracts. Increasing savings in expenditures through competition is...

  9. 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.

    PubMed

    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

  10. RCRA corrective action permit requirements and modifications under Subpart F regulations. RCRA Information Brief

    SciTech Connect

    Coalgate, J.

    1993-07-01

    The ground water protection requirements under the Resource Conservation and Recovery Act (RCRA), 40 CFR 264, Subpart F, apply to surface impoundments, waste plies, land treatment units, and landfills that received hazardous waste after July 26,1982 (i.e., regulated units). There are three phases to the Subpart F ground water protection requirements: detection monitoring, compliance monitoring, and corrective action. Subpart F corrective action applies to remediation of ground water contamination resulting from releases from regulated units at a treatment, storage, or disposal facility (TSDF). The TSDF owner or operator is responsible for complying with these requirements. This Information Brief provides information on the permit requirements under Subpart F. This Information Brief is one of a series on RCRA corrective action. The first step in the permitting process is for the facility to determine the need for ground-water monitoring. The regulations found in 40 CFR 264 Sections 264.90 to 264.100 (Subpart F) apply to all regulated units. A ``regulated unit`` is defined as a surface impoundment, waste pile, landfill, or land treatment unit that received hazardous waste after July 26, 1982. Such units require a permit under RCRA. Subpart F entails a three-phased program designed to detect, evaluate, and, if necessary, respond to ground water contamination. The ground-water protection standard, including identification of maximum contaminant levels (MCLs) under the Safe Drinking Water Act (SDWA) and alternate concentration limits (ACLs), is established with the permit application. Where MCLs and ACLs cannot be established, the standard may be established at background levels.

  11. Strabismus requires Flamingo and Prickle function to regulate tissue polarity in the Drosophila eye.

    PubMed

    Rawls, Amy S; Wolff, Tanya

    2003-05-01

    Tissue polarity in Drosophila is regulated by a number of genes that are thought to function in a complex, many of which interact genetically and/or physically, co-localize, and require other tissue polarity proteins for their localization. We report the enhancement of the strabismus tissue polarity phenotype by mutations in two other tissue polarity genes, flamingo and prickle. Flamingo is autonomously required for the establishment of ommatidial polarity. Its localization is dynamic throughout ommatidial development and is dependent on Frizzled and Notch. Flamingo and Strabismus co-localize for several rows posterior to the morphogenetic furrow and subsequently diverge. While neither of these proteins is required for the other's localization, Prickle localization is influenced by Strabismus function. Our data suggest that Strabismus, Flamingo and Prickle function together to regulate the establishment of tissue polarity in the Drosophila eye.

  12. Root growth regulation and gravitropism in maize roots does not require the epidermis

    NASA Technical Reports Server (NTRS)

    Bjorkman, T.; Cleland, R. E.

    1991-01-01

    We have earlier published observations showing that endogenous alterations in growth rate during gravitropism in maize roots (Zea mays L.) are unaffected by the orientation of cuts which remove epidermal and cortical tissue in the growing zone (Bjorkman and Cleland, 1988, Planta 176, 513-518). We concluded that the epidermis and cortex are not essential for transporting a growth-regulating signal in gravitropism or straight growth, nor for regulating the rate of tissue expansion. This conclusion has been challenged by Yang et al. (1990, Planta 180, 530-536), who contend that a shallow girdle around the entire perimeter of the root blocks gravitropic curvature and that this inhibition is the result of a requirement for epidermal cells to transport the growth-regulating signal. In this paper we demonstrate that the entire epidermis can be removed without blocking gravitropic curvature and show that the position of narrow girdles does not affect the location of curvature. We therefore conclude that the epidermis is not required for transport of a growth-regulating substance from the root cap to the growing zone, nor does it regulate the growth rate of the elongating zone of roots.

  13. Root growth regulation and gravitropism in maize roots does not require the epidermis

    NASA Technical Reports Server (NTRS)

    Bjorkman, T.; Cleland, R. E.

    1991-01-01

    We have earlier published observations showing that endogenous alterations in growth rate during gravitropism in maize roots (Zea mays L.) are unaffected by the orientation of cuts which remove epidermal and cortical tissue in the growing zone (Bjorkman and Cleland, 1988, Planta 176, 513-518). We concluded that the epidermis and cortex are not essential for transporting a growth-regulating signal in gravitropism or straight growth, nor for regulating the rate of tissue expansion. This conclusion has been challenged by Yang et al. (1990, Planta 180, 530-536), who contend that a shallow girdle around the entire perimeter of the root blocks gravitropic curvature and that this inhibition is the result of a requirement for epidermal cells to transport the growth-regulating signal. In this paper we demonstrate that the entire epidermis can be removed without blocking gravitropic curvature and show that the position of narrow girdles does not affect the location of curvature. We therefore conclude that the epidermis is not required for transport of a growth-regulating substance from the root cap to the growing zone, nor does it regulate the growth rate of the elongating zone of roots.

  14. MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

    PubMed Central

    Bogard, Ryan W.; Davies, Bryan W.; Mekalanos, John J.

    2012-01-01

    ABSTRACT LysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel of V. cholerae El Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed that glyA1 and metJ were also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation of glyA1, indicating that misregulation of glyA1 is likely responsible for the colonization defect observed in the metR mutant. The glyA1 mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor. PMID:23015737

  15. The RNA-binding protein Rbfox1 regulates splicing required for skeletal muscle structure and function

    PubMed Central

    Pedrotti, Simona; Giudice, Jimena; Dagnino-Acosta, Adan; Knoblauch, Mark; Singh, Ravi K.; Hanna, Amy; Mo, Qianxing; Hicks, John; Hamilton, Susan; Cooper, Thomas A.

    2015-01-01

    The Rbfox family of RNA-binding proteins is highly conserved with established roles in alternative splicing (AS) regulation. High-throughput studies aimed at understanding transcriptome remodeling have revealed skeletal muscle as displaying one of the largest number of AS events. This finding is consistent with requirements for tissue-specific protein isoforms needed to sustain muscle-specific functions. Rbfox1 is abundant in vertebrate brain, heart and skeletal muscle. Genome-wide genetic approaches have linked the Rbfox1 gene to autism, and a brain-specific knockout mouse revealed a critical role for this splicing regulator in neuronal function. Moreover, a Caenorhabditis elegans Rbfox1 homolog regulates muscle-specific splicing. To determine the role of Rbfox1 in muscle function, we developed a conditional knockout mouse model to specifically delete Rbfox1 in adult tissue. We show that Rbfox1 is required for muscle function but a >70% loss of Rbfox1 in satellite cells does not disrupt muscle regeneration. Deep sequencing identified aberrant splicing of multiple genes including those encoding myofibrillar and cytoskeletal proteins, and proteins that regulate calcium handling. Ultrastructure analysis of Rbfox1−/− muscle by electron microscopy revealed abundant tubular aggregates. Immunostaining showed mislocalization of the sarcoplasmic reticulum proteins Serca1 and Ryr1 in a pattern indicative of colocalization with the tubular aggregates. Consistent with mislocalization of Serca1 and Ryr1, calcium handling was drastically altered in Rbfox1−/− muscle. Moreover, muscle function was significantly impaired in Rbfox1−/− muscle as indicated by decreased force generation. These results demonstrate that Rbfox1 regulates a network of AS events required to maintain multiple aspects of muscle physiology. PMID:25575511

  16. Cryptococcus neoformans Mediator Protein Ssn8 Negatively Regulates Diverse Physiological Processes and Is Required for Virulence

    PubMed Central

    Wang, Lin-Ing; Lin, Yu-Sheng; Liu, Kung-Hung; Jong, Ambrose Y.; Shen, Wei-Chiang

    2011-01-01

    Cryptococcus neoformans is a ubiquitously distributed human pathogen. It is also a model system for studying fungal virulence, physiology and differentiation. Light is known to inhibit sexual development via the evolutionarily conserved white collar proteins in C. neoformans. To dissect molecular mechanisms regulating this process, we have identified the SSN8 gene whose mutation suppresses the light-dependent CWC1 overexpression phenotype. Characterization of sex-related phenotypes revealed that Ssn8 functions as a negative regulator in both heterothallic a-α mating and same-sex mating processes. In addition, Ssn8 is involved in the suppression of other physiological processes including invasive growth, and production of capsule and melanin. Interestingly, Ssn8 is also required for the maintenance of cell wall integrity and virulence. Our gene expression studies confirmed that deletion of SSN8 results in de-repression of genes involved in sexual development and melanization. Epistatic and yeast two hybrid studies suggest that C. neoformans Ssn8 plays critical roles downstream of the Cpk1 MAPK cascade and Ste12 and possibly resides at one of the major branches downstream of the Cwc complex in the light-mediated sexual development pathway. Taken together, our studies demonstrate that the conserved Mediator protein Ssn8 functions as a global regulator which negatively regulates diverse physiological and developmental processes and is required for virulence in C. neoformans. PMID:21559476

  17. p73 is Required for Multiciliogenesis and Regulates the Foxj1-Associated Gene Network

    PubMed Central

    Marshall, Clayton B; Mays, Deborah J; Beeler, J Scott; Rosenbluth, Jennifer M; Boyd, Kelli L; Guasch, Gabriela L Santos; Shaver, Timothy M; Tang, Lucy J; Liu, Qi; Shyr, Yu; Venters, Bryan J; Magnuson, Mark A; Pietenpol, Jennifer A

    2016-01-01

    Summary We report that p73 is expressed in multiciliated cells (MCCs), is required for MCC differentiation, and directly regulates transcriptional modulators of multiciliogenesis. Loss of ciliary biogenesis provides a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus, hippocampal dysgenesis, sterility and chronic inflammation/infection of lung, middle ear and sinus. Through p73 and p63 ChIP-seq using murine tracheal cells, we identified over 100 putative p73 target genes that regulate MCC differentiation and homeostasis. We validated Foxj1, a transcriptional regulator of multiciliogenesis, and many other cilia-associated genes as direct target genes of p73 and p63. We show p73 and p63 are co-expressed in a subset of basal cells, and suggest that p73 ‘marks’ these cells for MCC differentiation. In sum, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation and, like p63, has an essential role in development of tissues. PMID:26947080

  18. MEDICAL PRIVACY REGULATION: Questions Remain About Implementing the New Consent Requirement

    DTIC Science & Technology

    2001-04-01

    protection of patient confidentiality , views differ as to the best ways in practice to achieve that goal. Pressures are increasing from insurers, providers...protection of patient confidentiality , views differ as to the best ways in practice to achieve that goal. Pressures are increasing from insurers, providers...obligated under the regulation to protect the confidentiality of patient information. Moreover, with or without the consent requirement, patients

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

    PubMed

    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.

  20. putzig Is Required for Cell Proliferation and Regulates Notch Activity in Drosophila

    PubMed Central

    Kugler, Sabrina J.

    2007-01-01

    We have identified the gene putzig (pzg) as a key regulator of cell proliferation and of Notch signaling in Drosophila. pzg encodes a Zn-finger protein that was found earlier within a macromolecular complex, including TATA-binding protein-related factor 2 (TRF2)/DNA replication-related element factor (DREF). This complex is involved in core promoter selection, where DREF functions as a transcriptional activator of replication-related genes. Here, we provide the first in vivo evidence that pzg is required for the expression of cell cycle and replication-related genes, and hence for normal developmental growth. Independent of its role in the TRF2/DREF complex, pzg acts as a positive regulator of Notch signaling that may occur by chromatin activation. Down-regulation of pzg activity inhibits Notch target gene activation, whereas Hedgehog (Hh) signal transduction and growth regulation are unaffected. Our findings uncover different modes of operation of pzg during imaginal development of Drosophila, and they provide a novel mechanism of Notch regulation. PMID:17634285

  1. putzig is required for cell proliferation and regulates notch activity in Drosophila.

    PubMed

    Kugler, Sabrina J; Nagel, Anja C

    2007-10-01

    We have identified the gene putzig (pzg) as a key regulator of cell proliferation and of Notch signaling in Drosophila. pzg encodes a Zn-finger protein that was found earlier within a macromolecular complex, including TATA-binding protein-related factor 2 (TRF2)/DNA replication-related element factor (DREF). This complex is involved in core promoter selection, where DREF functions as a transcriptional activator of replication-related genes. Here, we provide the first in vivo evidence that pzg is required for the expression of cell cycle and replication-related genes, and hence for normal developmental growth. Independent of its role in the TRF2/DREF complex, pzg acts as a positive regulator of Notch signaling that may occur by chromatin activation. Down-regulation of pzg activity inhibits Notch target gene activation, whereas Hedgehog (Hh) signal transduction and growth regulation are unaffected. Our findings uncover different modes of operation of pzg during imaginal development of Drosophila, and they provide a novel mechanism of Notch regulation.

  2. 49 CFR 40.7 - How can you get an exemption from a requirement in this regulation?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 1 2012-10-01 2012-10-01 false How can you get an exemption from a requirement in this regulation? 40.7 Section 40.7 Transportation Office of the Secretary of Transportation PROCEDURES... can you get an exemption from a requirement in this regulation? (a) If you want an exemption from any...

  3. 49 CFR 40.7 - How can you get an exemption from a requirement in this regulation?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 1 2014-10-01 2014-10-01 false How can you get an exemption from a requirement in this regulation? 40.7 Section 40.7 Transportation Office of the Secretary of Transportation PROCEDURES... can you get an exemption from a requirement in this regulation? (a) If you want an exemption from any...

  4. 49 CFR 40.7 - How can you get an exemption from a requirement in this regulation?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false How can you get an exemption from a requirement in this regulation? 40.7 Section 40.7 Transportation Office of the Secretary of Transportation PROCEDURES... can you get an exemption from a requirement in this regulation? (a) If you want an exemption from any...

  5. 49 CFR 40.7 - How can you get an exemption from a requirement in this regulation?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 1 2011-10-01 2011-10-01 false How can you get an exemption from a requirement in this regulation? 40.7 Section 40.7 Transportation Office of the Secretary of Transportation PROCEDURES... can you get an exemption from a requirement in this regulation? (a) If you want an exemption from any...

  6. Using Five-Minute Data to Allocate Load-Following and Regulation Requirements among Individual Customers

    SciTech Connect

    Kirby, B.J.

    2001-03-27

    B. Kirby and E. Hirst, Customer-Specific Metrics for the Regulation and Load-Following 1 Ancillary Services, ORNL/CON-474, Oak Ridge National Laboratory, Oak Ridge, Tenn., January 2000. In an earlier project, we analyzed data on total system load and the loads of eight large industrial customers in terms of system- and customer-specific requirements for the regulation and load-following ancillary services. We conducted these analyses using 12 days of data from February 1 1999 plus 12 days of data from August and September 1999. These analyses were conducted using data provided by the control area at the 30-s level, which we then aggregated to the 2-min level for subsequent analysis. The current project analyzes the feasibility of using 5-min revenue-meter (RM) data to allocate load-following and regulation requirements among retail customers. This project does not use the 5-min data to determine the actual ancillary services requirements for individual loads because these requirements depend strongly on the time-averaging period chosen for the load data. In particular, the amount of regulation required declines as the time-averaging period increases. Our earlier project showed that 2 min was a reasonable time-averaging period for this control area. The reason for examining 5-min data is that supervisory control and data acquisition (SCADA) data, which are available every 4 s, are collected for only eight of this control area's very largest industrial customers. On the other hand, this control area collects 5-min RM data for about 600 customers. Although these customers account for less than 0.2% of the retail customers, they account for 60% of total energy sales and more than 40% of total revenues. Thus, if the allocations obtained with 5-min data for these eight large customers agree well with the allocations obtained with the SCADA data for these customers, the method we developed to allocate regulation costs to individual customers can be applied to many more

  7. Regulated membrane protein entry into flagella is facilitated by cytoplasmic microtubules and does not require IFT.

    PubMed

    Belzile, Olivier; Hernandez-Lara, Carmen I; Wang, Qian; Snell, William J

    2013-08-05

    The membrane protein composition of the primary cilium, a key sensory organelle, is dynamically regulated during cilium-generated signaling [1, 2]. During ciliogenesis, ciliary membrane proteins, along with structural and signaling proteins, are carried through the multicomponent, intensely studied ciliary diffusion barrier at the base of the organelle [3-8] by intraflagellar transport (IFT) [9-18]. A favored model is that signaling-triggered accumulation of previously excluded membrane proteins in fully formed cilia [19-21] also requires IFT, but direct evidence is lacking. Here, in studies of regulated entry of a membrane protein into the flagellum of Chlamydomonas, we show that cells use an IFT-independent mechanism to breach the diffusion barrier at the flagellar base. In resting cells, a flagellar signaling component [22], the integral membrane polypeptide SAG1-C65, is uniformly distributed over the plasma membrane and excluded from the flagellar membrane. Flagellar adhesion-induced signaling triggers rapid, striking redistribution of the protein to the apical ends of the cells concomitantly with entry into the flagella. Protein polarization and flagellar enrichment are facilitated by cytoplasmic microtubules. Using a conditional anterograde IFT mutant, we demonstrate that the IFT machinery is not required for regulated SAG1-C65 entry into flagella. Thus, integral membrane proteins can negotiate passage through the ciliary diffusion barrier without the need for a motor.

  8. 50 years of biological research--from oxidative phosphorylation to energy requiring transport regulation.

    PubMed

    Kalckar, H M

    1991-01-01

    In 1930 adenosine triphosphate appeared in the literature from W. A. Engelhardt's work on avian erythrocytes. This was an early example of oxidative phosphorylation in intact cells, and it required methylene blue and oxygen. Both Belitser and I realized that the use of Warburg manometers for aeration was critical in order to generate oxidative phosphorylation of glucose in tissue preparations. Test tube techniques did not work. In 1956 we were able to describe a human type of diabetes called "galactose diabetes," in which consumption of human or cows' milk provokes mental retardation. Replacement of human or cows' milk products with "vegetable milk" formula in early infancy can prevent retardation. We determined that the disease results from a defect of galactose-one-phosphate uridylyl-transferase, a hereditary enzyme. This type of enzyme defect, if discovered and treated in early infancy, is a benign molecular disease. Regulation of transport systems in mammalian cell cultures are frequently complex energized systems. Perhaps my greatest surprise in this regard was the mere fact that an all-cis "odd" hexose-D-allose turned out to be a highly intense down-regulator of the hexose transport system. Additions of inhibitors of oxidative phosphorylation (such as oligomycin or di-nitrophenol) arrested the allose-mediated down-regulation. We have reason to suspect that the strong down-regulator is a phosphorylated form of D-allose. Thus ends my story about oxidative energized biological phosphorylation systems.

  9. Metamorphic T3-response genes have specific co-regulator requirements

    PubMed Central

    Havis, Emmanuelle; Sachs, Laurent M.; Demeneix, Barbara A.

    2003-01-01

    Thyroid hormone receptors (TRs) have several regulatory functions in vertebrates. In the absence of thyroid hormone (T3; triiodothyronine), apo-TRs associate with co-repressors to repress transcription, whereas in the presence of T3, holo-TRs engage transcriptional coactivators. Although many studies have addressed the molecular mechanisms of T3 action, it is not known how specific physiological responses arise. We used T3-dependent amphibian metamorphosis to analyse how TRs interact with particular co-regulators to differentially regulate gene expression during development. Using chromatin immunoprecipitation to study tissue from pre-metamorphic tad-poles, we found that TRs are physically associated with T3-responsive promoters, whether or not T3 is present. Addition of T3 results in histone H4 acetylation specifically on T3-response genes. Most importantly, we show that individual T3-response genes have distinct co-regulator requirements, the T3-dependent co-repressor-to-coactivator switch being gene-specific for both co-regulator categories. PMID:12947412

  10. Beyond transcription factors: The role of chromatin modifying enzymes in regulating transcription required for memory

    PubMed Central

    Barrett, Ruth M.; Wood, Marcelo A.

    2008-01-01

    One of the alluring aspects of examining chromatin modifications in the role of modulating transcription required for long-term memory processes is that these modifications may provide transient and potentially stable epigenetic marks in the service of activating and/or maintaining transcriptional processes. These, in turn, may ultimately participate in the molecular mechanisms required for neuronal changes subserving long-lasting changes in behavior. As an epigenetic mechanism of transcriptional control, chromatin modification has been shown to participate in maintaining cellular memory (e.g., cell fate) and may underlie the strengthening and maintenance of synaptic connections required for long-term changes in behavior. Epigenetics has become central to several fields of neurobiology, where researchers have found that regulation of chromatin modification has a significant role in epilepsy, drug addiction, depression, neurodegenerative diseases, and memory. In this review, we will discuss the role of chromatin modifying enzymes in memory processes, as well as how recent studies in yeast genetics and cancer biology may impact the way we think about how chromatin modification and chromatin remodeling regulate neuronal function. PMID:18583646

  11. Overestimation of required recovery time during repeated sprint exercise with self-regulated recovery.

    PubMed

    Phillips, Shaun M; Thompson, Richard; Oliver, Jon L

    2014-12-01

    This study investigated the reliability and accuracy of self-regulated recovery time and performance during repeated sprinting. On 4 occasions, 14 men (24.5 ± 5.0 years) completed 10 × 6 seconds cycle sprints against 7.5% body mass, self-regulating (SR) recovery time to maintain performance. Subjects then repeated the test, but with a reduced recovery (RR) of 10% less recovery time. Across the first 4 trials, there were no between-trial differences in peak power output (PPO) or mean power output (MPO), recovery time, or fatigue index (p > 0.05). Random variation in recovery time was reduced across trials 3-4 (coefficient of variation [CV] = 7.5%, 95% confidence limits [CL] = 5.4-12.4%) compared with trials 1-2 (CV = 16.0, 95% CL = 11.4-27.0%) and 2-3 (CV = 10.1%, 95% CL = 7.2-16.7%) but was consistent across trials for PPO and MPO (between-trials CV, ≤3.3%). There were no trial effects for any performance, physiological, or perceptual measures when comparing SR with RR (p > 0.05), although heart rate and perceptual measures increased with subsequent sprint efforts (p ≤ 0.05). After 2 familiarization trials, subjects can reliably self-regulate recovery time to maintain performance during repeated sprints. However, subjects overestimate the amount of recovery time required, as reducing this time by 10% had no effect on performance, perceptual, or physiological parameters. Self-regulated sprinting is potentially a reliable training tool, particularly for sprint training where maintenance of work is desired. However, overestimation of required recovery time means that performance improvements may not be achieved if the goal of training is improvement of repeated sprint performance with incomplete recovery.

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

    EPA Pesticide Factsheets

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

    EPA Pesticide Factsheets

    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. Overview of existing regulations for ventilation requirements of enclosed vehicular parking facilities

    SciTech Connect

    Krarti, M.; Ayari, A.M.

    1999-07-01

    This paper provides an overview of the current standards and regulations regarding the ventilation in enclosed parking facilities in the US and other countries. First, the paper discusses the emission rates of motor vehicle pollutants and their health effects. In particular, typical emission rates for different vehicle and fuel types are presented to highlight the effect of various parameters on the ventilation rate requirements for parking garages. In addition, the paper provides a brief description of some of the common ventilation problems reported in the literature for enclosed parking garages.

  15. Different requirements for conserved post-transcriptional regulators in planarian regeneration and stem cell maintenance.

    PubMed

    Rouhana, Labib; Shibata, Norito; Nishimura, Osamu; Agata, Kiyokazu

    2010-05-15

    Planarian regeneration depends on the presence and precise regulation of pluripotent adult somatic stem cells named neoblasts, which differentiate to replace cells of any missing tissue. A characteristic feature of neoblasts is the presence of large perinuclear nonmembranous organelles named "chromatoid bodies", which are comparable to ribonucleoprotein structures found in germ cells of organisms across different phyla. In order to better understand regulation of gene expression in neoblasts, and potentially the function and composition of chromatoid bodies, we characterized homologues to known germ and soma ribonucleoprotein granule components from other organisms and analyzed their function during regeneration of the planarian Dugesia japonica. Expression in neoblasts was detected for 49 of 55 analyzed genes, highlighting the prevalence of post-transcriptional regulation in planarian stem cells. RNAi-mediated knockdown of two factors [ago-2 and bruli] lead to loss of neoblasts, and consequently loss of regeneration, corroborating with results previously reported for a bruli ortholog in the planarian Schmidtea mediterranea (Guo et al., 2006). Conversely, depletion mRNA turnover factors [edc-4 or upf-1], exoribonucleases [xrn-1 or xrn-2], or DEAD box RNA helicases [Djcbc-1 or vas-1] inhibited planarian regeneration, but did not reduce neoblast proliferation or abundance. We also found that depletion of cap-dependent translation initiation factors eIF-3A or eIF-2A interrupted cell cycle progression outside the M-phase of mitosis. Our results show that a set of post-transcriptional regulators is required to maintain the stem cell identity in neoblasts, while another facilitates proper differentiation. We propose that planarian neoblasts maintain pluripotency by employing mechanisms of post-transcriptional regulation exhibited in germ cells and early development of most metazoans. Copyright 2010 Elsevier Inc. All rights reserved.

  16. 76 FR 35424 - Information Collection Requirements; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-17

    ... Regulation Supplement; Construction and Architect-Engineer Contracts AGENCY: Defense Acquisition Regulations... Acquisition Regulation Supplement (DFARS) Part 236, Construction and Architect-Engineer Contracts, and...

  17. 75 FR 45104 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-02

    ... Regulation Supplement; Administrative Matters AGENCY: Defense Acquisition Regulations System, Department of... Federal Acquisition Regulation Supplement (DFARS) Part 204, Administrative Matters, and related clauses...

  18. Successful emotion regulation requires both conviction and skill: beliefs about the controllability of emotions, reappraisal, and regulation success.

    PubMed

    Gutentag, Tony; Halperin, Eran; Porat, Roni; Bigman, Yochanan E; Tamir, Maya

    2016-08-05

    To succeed in self-regulation, people need to believe that it is possible to change behaviour and they also need to use effective means to enable such a change. We propose that this also applies to emotion regulation. In two studies, we found that people were most successful in emotion regulation, the more they believed emotions can be controlled and the more they used an effective emotion regulation strategy - namely, cognitive reappraisal. Cognitive reappraisal moderated the link between beliefs about the controllability of emotion and success in emotion regulation, when reappraisal was measured as a trait (Study 1) or manipulated (Study 2). Such moderation was found when examining the regulation of disgust elicited by emotion-inducing films (Study 1), and the regulation of anger elicited by real political events (Study 2). We discuss the implications of our findings for research and practice in emotion regulation.

  19. Iron-Regulated Biofilm Formation in Staphylococcus aureus Newman Requires ica and the Secreted Protein Emp▿

    PubMed Central

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

    2008-01-01

    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. PMID:18268030

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

    PubMed

    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.

  1. Plasma membrane H⁺ -ATPase regulation is required for auxin gradient formation preceding phototropic growth.

    PubMed

    Hohm, Tim; Demarsy, Emilie; Quan, Clément; Allenbach Petrolati, Laure; Preuten, Tobias; Vernoux, Teva; Bergmann, Sven; Fankhauser, Christian

    2014-09-26

    Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H⁺ -ATPases that are required to control apoplastic pH. Our results show that H⁺ -ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H⁺ -ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  2. Plasma membrane H+-ATPase regulation is required for auxin gradient formation preceding phototropic growth

    PubMed Central

    Hohm, Tim; Demarsy, Emilie; Quan, Clément; Allenbach Petrolati, Laure; Preuten, Tobias; Vernoux, Teva; Bergmann, Sven; Fankhauser, Christian

    2014-01-01

    Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H+-ATPases that are required to control apoplastic pH. Our results show that H+-ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H+-ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH. PMID:25261457

  3. Computational identification of a p38SAPK regulated transcription factor network required for tumor cell quiescence

    PubMed Central

    Adam, Alejandro P.; George, Ajish; Schewe, Denis; Bragado, Paloma; Iglesias, Bibiana V.; Ranganathan, Aparna C.; Kourtidis, Antonis; Conklin, Douglas S.; Aguirre-Ghiso, Julio A.

    2009-01-01

    The stress activated kinase p38 plays key roles in tumor suppression and induction of tumor cell dormancy. However, the mechanisms behind these functions remain poorly understood. Using computational tools we identified a transcription factor (TF) network regulated by p38α/β and required for human squamous carcinoma cell quiescence in vivo. We found that p38 transcriptionally regulates a core network of 46 genes that includes 16 TFs. Activation of p38 induced the expression of the TFs p53 and BHLHB3, while inhibiting c-Jun and FoxM1 expression. Further, induction of p53 by p38 was dependent on c-Jun downregulation. Accordingly, while RNAi downregulation of BHLHB3 or p53 interrupted tumor cell quiescence; downregulation of c-Jun or FoxM1 or overexpression of BHLHB3 in malignant cells mimicked the onset of quiescence. Our results identify components of the regulatory mechanisms driving p38-induced cancer cell quiescence. These may regulate dormancy of residual disease that usually precedes the onset of metastasis in many cancers. PMID:19584293

  4. SRSF10 regulates alternative splicing and is required for adipocyte differentiation.

    PubMed

    Li, Huang; Cheng, Yuanming; Wu, Wenwu; Liu, Yuguo; Wei, Ning; Feng, Xiaoyan; Xie, Zhiqin; Feng, Ying

    2014-06-01

    During adipocyte differentiation, significant alternative splicing changes occur in association with the adipogenic process. However, little is known about roles played by splicing factors in this process. We observed that mice deficient for the splicing factor SRSF10 exhibit severely impaired development of subcutaneous white adipose tissue (WAT) as a result of defects in adipogenic differentiation. To identify splicing events responsible for this, transcriptome sequencing (RNA-seq) analysis was performed using embryonic fibroblast cells. Several SRSF10-affected splicing events that are implicated in adipogenesis have been identified. Notably, lipin1, known as an important regulator during adipogenesis, was further investigated. While lipin1β is mainly involved in lipogenesis, its alternatively spliced isoform lipin1α, generated through the skipping of exon 7, is primarily required for initial adipocyte differentiation. Skipping of exon 7 is controlled by an SRSF10-regulated cis element located in the constitutive exon 8. The activity of this element depends on the binding of SRSF10 and correlates with the relative abundance of lipin1α mRNA. A series of experiments demonstrated that SRSF10 controls the production of lipin1α and thus promotes adipocyte differentiation. Indeed, lipin1α expression could rescue SRSF10-mediated adipogenic defects. Taken together, our results identify SRSF10 as an essential regulator for adipocyte differentiation and also provide new insights into splicing control by SRSF10 in lipin1 pre-mRNA splicing. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium

    PubMed Central

    Bastide, Pauline; Darido, Charbel; Pannequin, Julie; Kist, Ralf; Robine, Sylvie; Marty-Double, Christiane; Bibeau, Frédéric; Scherer, Gerd; Joubert, Dominique; Hollande, Frédéric; Blache, Philippe; Jay, Philippe

    2007-01-01

    The HMG-box transcription factor Sox9 is expressed in the intestinal epithelium, specifically, in stem/progenitor cells and in Paneth cells. Sox9 expression requires an active β-catenin–Tcf complex, the transcriptional effector of the Wnt pathway. This pathway is critical for numerous aspects of the intestinal epithelium physiopathology, but processes that specify the cell response to such multipotential signals still remain to be identified. We inactivated the Sox9 gene in the intestinal epithelium to analyze its physiological function. Sox9 inactivation affected differentiation throughout the intestinal epithelium, with a disappearance of Paneth cells and a decrease of the goblet cell lineage. Additionally, the morphology of the colon epithelium was severely altered. We detected general hyperplasia and local crypt dysplasia in the intestine, and Wnt pathway target genes were up-regulated. These results highlight the central position of Sox9 as both a transcriptional target and a regulator of the Wnt pathway in the regulation of intestinal epithelium homeostasis. PMID:17698607

  6. Phosphoinositide Regulation of Integrin Trafficking Required for Muscle Attachment and Maintenance

    PubMed Central

    Ribeiro, Inês; Yuan, Lin; Tanentzapf, Guy; Dowling, James J.; Kiger, Amy

    2011-01-01

    Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. We show that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila melanogaster, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease. PMID:21347281

  7. Hic-5 is required for myofibroblast differentiation by regulating mechanically dependent MRTF-A nuclear accumulation

    PubMed Central

    Varney, Scott D.; Betts, Courtney B.; Zheng, Rui; Wu, Lei; Hinz, Boris; Zhou, Jiliang; Van De Water, Livingston

    2016-01-01

    ABSTRACT How mechanical cues from the extracellular environment are translated biochemically to modulate the effects of TGF-β on myofibroblast differentiation remains a crucial area of investigation. We report here that the focal adhesion protein, Hic-5 (also known as TGFB1I1), is required for the mechanically dependent generation of stress fibers in response to TGF-β. Successful generation of stress fibers promotes the nuclear localization of the transcriptional co-factor MRTF-A (also known as MKL1), and this correlates with the mechanically dependent induction of α smooth muscle actin (α-SMA) and Hic-5 in response to TGF-β. As a consequence of regulating stress fiber assembly, Hic-5 is required for the nuclear accumulation of MRTF-A and the induction of α-SMA as well as cellular contractility, suggesting a crucial role for Hic-5 in myofibroblast differentiation. Indeed, the expression of Hic-5 was transient in acute wounds and persistent in pathogenic scars, and Hic-5 colocalized with α-SMA expression in vivo. Taken together, these data suggest that a mechanically dependent feed-forward loop, elaborated by the reciprocal regulation of MRTF-A localization by Hic-5 and Hic-5 expression by MRTF-A, plays a crucial role in myofibroblast differentiation in response to TGF-β. PMID:26759173

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

    PubMed Central

    Matsubayashi, Yutaka; Coulson-Gilmer, Camilla

    2015-01-01

    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. PMID:26216900

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

    PubMed

    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.

  10. Fine regulation of RhoA and Rock is required for skeletal muscle differentiation.

    PubMed

    Castellani, Loriana; Salvati, Erica; Alemà, Stefano; Falcone, Germana

    2006-06-02

    The RhoA GTPase controls a variety of cell functions such as cell motility, cell growth, and gene expression. Previous studies suggested that RhoA mediates signaling inputs that promote skeletal myogenic differentiation. We show here that levels and activity of RhoA protein are down-regulated in both primary avian myoblasts and mouse satellite cells undergoing differentiation, suggesting that a fine regulation of this GTPase is required. In addition, ectopic expression of activated RhoA in primary quail myocytes, but not in mouse myocytes, inhibits accumulation of muscle-specific proteins and cell fusion. By disrupting RhoA signaling with specific inhibitors, we have shown that this GTPase, although required for cell identity in proliferating myoblasts, is not essential for commitment to terminal differentiation and muscle gene expression. Ectopic expression of an activated form of its downstream effector, Rock, impairs differentiation of both avian and mouse myoblasts. Conversely, Rock inhibition with specific inhibitors and small interfering RNA-mediated gene silencing leads to accelerated progression in the lineage and enhanced cell fusion, underscoring a negative regulatory function of Rock in myogenesis. Finally, we have reported that Rock acts independently from RhoA in preventing myoblast exit from the cell cycle and commitment to differentiation and may receive signaling inputs from Raf-1 kinase.

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

    SciTech Connect

    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.

  12. IHF Is Required for the Transcriptional Regulation of the Desulfovibrio vulgaris Hildenborough orp Operons

    PubMed Central

    Fiévet, Anouchka; Cascales, Eric; Valette, Odile; Dolla, Alain; Aubert, Corinne

    2014-01-01

    Transcriptional activation of σ54-dependent promoters is usually tightly regulated in response to environmental cues. The high abundance of potential σ54-dependent promoters in the anaerobe bacteria, Desulfovibrio vulgaris Hildenborough, reflects the high versatility of this bacteria suggesting that σ54 factor is the nexus of a large regulatory network. Understanding the key players of σ54-regulation in this organism is therefore essential to gain insights into the adaptation to anaerobiosis. Recently, the D. vulgaris orp genes, specifically found in anaerobe bacteria, have been shown to be transcribed by the RNA polymerase coupled to the σ54 alternative sigma factor. In this study, using in vitro binding experiments and in vivo reporter fusion assays in the Escherichia coli heterologous host, we showed that the expression of the divergent orp promoters is strongly dependent on the integration host factor IHF. Bioinformatic and mutational analysis coupled to reporter fusion activities and mobility shift assays identified two functional IHF binding site sequences located between the orp1 and orp2 promoters. We further determined that the D. vulgaris DVU0396 (IHFα) and DVU1864 (IHFβ) subunits are required to control the expression of the orp operons suggesting that they form a functionally active IHF heterodimer. Interestingly results obtained from the in vivo inactivation of DVU0396, which is required for orp operons transcription, suggest that several functionally IHF active homodimer or heterodimer are present in D. vulgaris. PMID:24466126

  13. IHF is required for the transcriptional regulation of the Desulfovibrio vulgaris Hildenborough orp operons.

    PubMed

    Fiévet, Anouchka; Cascales, Eric; Valette, Odile; Dolla, Alain; Aubert, Corinne

    2014-01-01

    Transcriptional activation of σ(54)-dependent promoters is usually tightly regulated in response to environmental cues. The high abundance of potential σ(54)-dependent promoters in the anaerobe bacteria, Desulfovibrio vulgaris Hildenborough, reflects the high versatility of this bacteria suggesting that σ(54) factor is the nexus of a large regulatory network. Understanding the key players of σ(54)-regulation in this organism is therefore essential to gain insights into the adaptation to anaerobiosis. Recently, the D. vulgaris orp genes, specifically found in anaerobe bacteria, have been shown to be transcribed by the RNA polymerase coupled to the σ(54) alternative sigma factor. In this study, using in vitro binding experiments and in vivo reporter fusion assays in the Escherichia coli heterologous host, we showed that the expression of the divergent orp promoters is strongly dependent on the integration host factor IHF. Bioinformatic and mutational analysis coupled to reporter fusion activities and mobility shift assays identified two functional IHF binding site sequences located between the orp1 and orp2 promoters. We further determined that the D. vulgaris DVU0396 (IHFα) and DVU1864 (IHFβ) subunits are required to control the expression of the orp operons suggesting that they form a functionally active IHF heterodimer. Interestingly results obtained from the in vivo inactivation of DVU0396, which is required for orp operons transcription, suggest that several functionally IHF active homodimer or heterodimer are present in D. vulgaris.

  14. Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains

    PubMed Central

    Barber, Cynthia F.; Jorquera, Ramon A.; Melom, Jan E.

    2009-01-01

    Ca2+ influx into synaptic compartments during activity is a key mediator of neuronal plasticity. Although the role of presynaptic Ca2+ in triggering vesicle fusion though the Ca2+ sensor synaptotagmin 1 (Syt 1) is established, molecular mechanisms that underlie responses to postsynaptic Ca2+ influx remain unclear. In this study, we demonstrate that fusion-competent Syt 4 vesicles localize postsynaptically at both neuromuscular junctions (NMJs) and central nervous system synapses in Drosophila melanogaster. Syt 4 messenger RNA and protein expression are strongly regulated by neuronal activity, whereas altered levels of postsynaptic Syt 4 modify synaptic growth and presynaptic release properties. Syt 4 is required for known forms of activity-dependent structural plasticity at NMJs. Synaptic proliferation and retrograde signaling mediated by Syt 4 requires functional C2A and C2B Ca2+–binding sites, as well as serine 284, an evolutionarily conserved substitution for a key Ca2+-binding aspartic acid found in other synaptotagmins. These data suggest that Syt 4 regulates activity-dependent release of postsynaptic retrograde signals that promote synaptic plasticity, similar to the role of Syt 1 as a Ca2+ sensor for presynaptic vesicle fusion. PMID:19822673

  15. Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains.

    PubMed

    Barber, Cynthia F; Jorquera, Ramon A; Melom, Jan E; Littleton, J Troy

    2009-10-19

    Ca(2+) influx into synaptic compartments during activity is a key mediator of neuronal plasticity. Although the role of presynaptic Ca(2+) in triggering vesicle fusion though the Ca(2+) sensor synaptotagmin 1 (Syt 1) is established, molecular mechanisms that underlie responses to postsynaptic Ca(2+) influx remain unclear. In this study, we demonstrate that fusion-competent Syt 4 vesicles localize postsynaptically at both neuromuscular junctions (NMJs) and central nervous system synapses in Drosophila melanogaster. Syt 4 messenger RNA and protein expression are strongly regulated by neuronal activity, whereas altered levels of postsynaptic Syt 4 modify synaptic growth and presynaptic release properties. Syt 4 is required for known forms of activity-dependent structural plasticity at NMJs. Synaptic proliferation and retrograde signaling mediated by Syt 4 requires functional C2A and C2B Ca(2+)-binding sites, as well as serine 284, an evolutionarily conserved substitution for a key Ca(2+)-binding aspartic acid found in other synaptotagmins. These data suggest that Syt 4 regulates activity-dependent release of postsynaptic retrograde signals that promote synaptic plasticity, similar to the role of Syt 1 as a Ca(2+) sensor for presynaptic vesicle fusion.

  16. Acinetobacter baumannii Response to Host-Mediated Zinc Limitation Requires the Transcriptional Regulator Zur

    PubMed Central

    Mortensen, Brittany L.; Rathi, Subodh; Chazin, Walter J.

    2014-01-01

    Acinetobacter baumannii is a leading cause of ventilator-associated pneumonia in intensive care units, and the increasing rates of antibiotic resistance make treating these infections challenging. Consequently, there is an urgent need to develop new antimicrobials to treat A. baumannii infections. One potential therapeutic option is to target bacterial systems involved in maintaining appropriate metal homeostasis, processes that are critical for the growth of pathogens within the host. The A. baumannii inner membrane zinc transporter ZnuABC is required for growth under low-zinc conditions and for A. baumannii pathogenesis. The expression of znuABC is regulated by the transcriptional repressor Zur. To investigate the role of Zur during the A. baumannii response to zinc limitation, a zur deletion mutant was generated, and transcriptional changes were analyzed using RNA sequencing. A number of Zur-regulated genes were identified that exhibit increased expression both when zur is absent and under low-zinc conditions, and Zur binds to predicted Zur box sequences of several genes affected by zinc levels or the zur mutation. Furthermore, the zur mutant is impaired for growth in the presence of both high and low zinc levels compared to wild-type A. baumannii. Finally, the zur mutant exhibits a defect in dissemination in a mouse model of A. baumannii pneumonia, establishing zinc sensing as a critical process during A. baumannii infection. These results define Zur-regulated genes within A. baumannii and demonstrate a requirement for Zur in the A. baumannii response to the various zinc levels experienced within the vertebrate host. PMID:24816603

  17. Mycobacterium tuberculosis Requires Phosphate-Responsive Gene Regulation To Resist Host Immunity

    PubMed Central

    Leistikow, Rachel L.; Kirksey, Meghan A.; Voskuil, Martin I.; McKinney, John D.

    2013-01-01

    Mycobacterium tuberculosis persists in the tissues of mammalian hosts despite inducing a robust immune response dominated by the macrophage-activating cytokine gamma interferon (IFN-γ). We identified the M. tuberculosis phosphate-specific transport (Pst) system component PstA1 as a factor required to resist IFN-γ-dependent immunity. A ΔpstA1 mutant was fully virulent in IFN-γ−/− mice but attenuated in wild-type (WT) mice and mice lacking specific IFN-γ-inducible immune mechanisms: nitric oxide synthase (NOS2), phagosome-associated p47 GTPase (Irgm1), or phagocyte oxidase (phox). These phenotypes suggest that ΔpstA1 bacteria are sensitized to an IFN-γ-dependent immune mechanism(s) other than NOS2, Irgm1, or phox. In other species, the Pst system has a secondary role as a negative regulator of phosphate starvation-responsive gene expression through an interaction with a two-component signal transduction system. In M. tuberculosis, we found that ΔpstA1 bacteria exhibited dysregulated gene expression during growth in phosphate-rich medium that was mediated by the two-component sensor kinase/response regulator system SenX3-RegX3. Remarkably, deletion of the regX3 gene suppressed the replication and virulence defects of ΔpstA1 bacteria in NOS2−/− mice, suggesting that M. tuberculosis requires the Pst system to negatively regulate activity of RegX3 in response to available phosphate in vivo. We therefore speculate that inorganic phosphate is readily available during replication in the lung and is an important signal controlling M. tuberculosis gene expression via the Pst-SenX3-RegX3 signal transduction system. Inability to sense this environmental signal, due to Pst deficiency, results in dysregulation of gene expression and sensitization of the bacteria to the host immune response. PMID:23132496

  18. A Subset of Cholinergic Mushroom Body Neurons Requires Go Signaling to Regulate Sleep in Drosophila

    PubMed Central

    Yi, Wei; Zhang, Yunpeng; Tian, Yinjun; Guo, Jing; Li, Yan; Guo, Aike

    2013-01-01

    Study Objectives: Identifying the neurochemistry and neural circuitry of sleep regulation is critical for understanding sleep and various sleep disorders. Fruit flies display sleep-like behavior, sharing essential features with sleep of vertebrate. In the fruit fly's central brain, the mushroom body (MB) has been highlighted as a sleep center; however, its neurochemical nature remains unclear, and whether it promotes sleep or wake is still a topic of controversy. Design: We used a video recording system to accurately monitor the locomotor activity and sleep status. Gene expression was temporally and regionally manipulated by heat induction and the Gal4/UAS system. Measurements and Results: We found that expressing pertussis toxin (PTX) in the MB by c309-Gal4 to block Go activity led to unique sleep defects as dramatic sleep increase in daytime and fragmented sleep in nighttime. We narrowed down the c309-Gal4 expressing brain regions to the MB α/β core neurons that are responsible for the Go-mediated sleep effects. Using genetic tools of neurotransmitter-specific Gal80 and RNA interference approach to suppress acetylcholine signal, we demonstrated that these MB α/β core neurons were cholinergic and sleep-promoting neurons, supporting that Go mediates an inhibitory signal. Interestingly, we found that adjacent MB α/β neurons were also cholinergic but wake-promoting neurons, in which Go signal was also required. Conclusion: Our findings in fruit flies characterized a group of sleep-promoting neurons surrounded by a group of wake-promoting neurons. The two groups of neurons are both cholinergic and use Go inhibitory signal to regulate sleep. Citation: Yi W; Zhang Y; Tian Y; Guo J; Li Y; Guo A. A subset of cholinergic mushroom body neurons requires go signaling to regulate sleep in Drosophila. SLEEP 2013;36(12):1809-1821. PMID:24293755

  19. Two new Sinorhizobium meliloti LysR-type transcriptional regulators required for nodulation.

    PubMed

    Luo, Li; Yao, Shi-Yi; Becker, Anke; Rüberg, Silvia; Yu, Guan-Qiao; Zhu, Jia-Bi; Cheng, Hai-Ping

    2005-07-01

    The establishment of an effective nitrogen-fixing symbiosis between Sinorhizobium meliloti and its legume host alfalfa (Medicago sativa) depends on the timely expression of nodulation genes that are controlled by LysR-type regulators. Ninety putative genes coding for LysR-type transcriptional regulators were identified in the recently sequenced S. meliloti genome. All 90 putative lysR genes were mutagenized using plasmid insertions as a first step toward determining their roles in symbiosis. Two new LysR-type symbiosis regulator genes, lsrA and lsrB, were identified in the screening. Both the lsrA and lsrB genes are expressed in free-living S. meliloti cells, but they are not required for cell growth. An lsrA1 mutant was defective in symbiosis and elicited only white nodules that exhibited no nitrogenase activity. Cells of the lsrA1 mutant were recovered from the white nodules, suggesting that the lsrA1 mutant was blocked early in nodulation. An lsrB1 mutant was deficient in symbiosis and elicited a mixture of pink and white nodules on alfalfa plants. These plants exhibited lower overall nitrogenase activity than plants inoculated with the wild-type strain, which is consistent with the fact that most of the alfalfa plants inoculated with the lsrB1 mutant were short and yellow. Cells of the lsrB1 mutant were recovered from both pink and white nodules, suggesting that lsrB1 mutants could be blocked at multiple points during nodulation. The identification of two new LysR-type symbiosis transcriptional regulators provides two new avenues for understanding the complex S. meliloti-alfalfa interactions which occur during symbiosis.

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

    PubMed Central

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

    2013-01-01

    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. PMID:24344181

  1. Cell cycle-regulated histone acetylation required for expression of the yeast HO gene

    PubMed Central

    Krebs, Jocelyn E.; Kuo, Min-Hao; Allis, C. David; Peterson, Craig L.

    1999-01-01

    Expression of the yeast HO gene in late G1 of the cell cycle requires the SWI/SNF chromatin remodeling complex, the Gcn5p histone acetyltransferase, and two different sequence-specific transcriptional activators, Swi5p and Swi4p/Swi6p. We have used chromatin immunoprecipitation assays to investigate the role of each of these trans-acting factors in establishing a cell cycle-regulated domain of histone acetylation surrounding the HO upstream regulatory region. We detect a ∼1-kb domain of H3 and H4 acetylation that is established in mid-G1, prior to and independent of HO transcription, which then declines with kinetics similar to inactivation of HO. This cell cycle burst of histone acetylation requires Gcn5p, SWI/SNF, and the Swi5p activator, but occurs in the absence of the Swi4p activator. We also find that inactivation of the Sin3p/Rpd3p deacetylase complex leads to a high level of acetylation at the HO locus throughout the cell cycle. We propose a sequential model for activation of HO in which the Swi5p-dependent recruitment of the Gcn5p acetyltransferase requires chromatin remodeling events by the SWI/SNF complex. PMID:10364158

  2. 43 CFR 1821.12 - Are these the only regulations that will apply to my application or other required document?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 43 Public Lands: Interior 2 2014-10-01 2014-10-01 false Are these the only regulations that will apply to my application or other required document? 1821.12 Section 1821.12 Public Lands: Interior Regulations Relating to Public Lands (Continued) BUREAU OF LAND MANAGEMENT, DEPARTMENT OF THE INTERIOR GENERAL MANAGEMENT (1000) APPLICATION PROCEDURES...

  3. 43 CFR 1821.12 - Are these the only regulations that will apply to my application or other required document?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 43 Public Lands: Interior 2 2012-10-01 2012-10-01 false Are these the only regulations that will apply to my application or other required document? 1821.12 Section 1821.12 Public Lands: Interior Regulations Relating to Public Lands (Continued) BUREAU OF LAND MANAGEMENT, DEPARTMENT OF THE INTERIOR GENERAL MANAGEMENT (1000) APPLICATION PROCEDURES...

  4. 43 CFR 2568.40 - Does BLM have the authority to ask me for the information required in these regulations?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 43 Public Lands: Interior 2 2013-10-01 2013-10-01 false Does BLM have the authority to ask me for the information required in these regulations? 2568.40 Section 2568.40 Public Lands: Interior Regulations Relating to Public Lands (Continued) BUREAU OF LAND MANAGEMENT, DEPARTMENT OF THE INTERIOR LAND...

  5. 43 CFR 2568.40 - Does BLM have the authority to ask me for the information required in these regulations?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 43 Public Lands: Interior 2 2011-10-01 2011-10-01 false Does BLM have the authority to ask me for the information required in these regulations? 2568.40 Section 2568.40 Public Lands: Interior Regulations Relating to Public Lands (Continued) BUREAU OF LAND MANAGEMENT, DEPARTMENT OF THE INTERIOR LAND...

  6. 43 CFR 2568.40 - Does BLM have the authority to ask me for the information required in these regulations?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 43 Public Lands: Interior 2 2014-10-01 2014-10-01 false Does BLM have the authority to ask me for the information required in these regulations? 2568.40 Section 2568.40 Public Lands: Interior Regulations Relating to Public Lands (Continued) BUREAU OF LAND MANAGEMENT, DEPARTMENT OF THE INTERIOR LAND...

  7. 43 CFR 2568.40 - Does BLM have the authority to ask me for the information required in these regulations?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 43 Public Lands: Interior 2 2012-10-01 2012-10-01 false Does BLM have the authority to ask me for the information required in these regulations? 2568.40 Section 2568.40 Public Lands: Interior Regulations Relating to Public Lands (Continued) BUREAU OF LAND MANAGEMENT, DEPARTMENT OF THE INTERIOR LAND...

  8. 7 CFR 301.76-4 - Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... produced within an area quarantined for citrus greening. 301.76-4 Section 301.76-4 Agriculture Regulations... OF AGRICULTURE DOMESTIC QUARANTINE NOTICES Citrus Greening and Asian Citrus Psyllid § 301.76-4 Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

  9. 7 CFR 301.76-4 - Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... produced within an area quarantined for citrus greening. 301.76-4 Section 301.76-4 Agriculture Regulations... OF AGRICULTURE DOMESTIC QUARANTINE NOTICES Citrus Greening and Asian Citrus Psyllid § 301.76-4 Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

  10. 7 CFR 301.76-4 - Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... produced within an area quarantined for citrus greening. 301.76-4 Section 301.76-4 Agriculture Regulations... OF AGRICULTURE DOMESTIC QUARANTINE NOTICES Citrus Greening and Asian Citrus Psyllid § 301.76-4 Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

  11. 7 CFR 301.76-4 - Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... produced within an area quarantined for citrus greening. 301.76-4 Section 301.76-4 Agriculture Regulations... OF AGRICULTURE DOMESTIC QUARANTINE NOTICES Citrus Greening and Asian Citrus Psyllid § 301.76-4 Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

  12. [Regulation requirements for new investments to protect the environment against electromagnetic fields].

    PubMed

    Kawicki, Artur

    2006-01-01

    The date of 28 July 2005, when the amendments to the Environmental Protection Act and related acts entered into force, is crucial for the future installations of the majority of electromagnetic fields emitters. Since the amendments let the environmental protection assessment procedures be more "independent", set the decision on environmental conditions for development consent and transposed the rules of managing and protection of Natura 2000 sites, they are considered as a revolution in the environmental impact assessment system. According to new rules, the decision on environmental conditions for development consent is granted for the project that may have a significant impact on the environment listed in the regulation issued by the Council of Ministers on 9 November 2004 on the types of projects that may have significant impact on the environment and detailed criteria for screening of the projects for which the environmental impact report may be required or for other projects that may have significant impact on Natura 2000 sites.

  13. Requirement for the c-Maf transcription factor in crystallin gene regulation and lens development

    PubMed Central

    Kim, James I.; Li, Tiansen; Ho, I.-Cheng; Grusby, Michael J.; Glimcher, Laurie H.

    1999-01-01

    The vertebrate lens is a tissue composed of terminally differentiated fiber cells and anterior lens epithelial cells. The abundant, preferential expression of the soluble proteins called crystallins creates a transparent, refractive index gradient in the lens. Several transcription factors such as Pax6, Sox1, and L-Maf have been shown to regulate lens development. Here we show that mice lacking the transcription factor c-Maf are microphthalmic secondary to defective lens formation, specifically from the failure of posterior lens fiber elongation. The marked impairment of crystallin gene expression observed is likely explained by the ability of c-Maf to transactivate the crystallin gene promoter. Thus, c-Maf is required for the differentiation of the vertebrate lens. PMID:10097114

  14. Centrosome maturation requires YB-1 to regulate dynamic instability of microtubules for nucleus reassembly

    PubMed Central

    Kawaguchi, Atsushi; Asaka, Masamitsu N.; Matsumoto, Ken; Nagata, Kyosuke

    2015-01-01

    Microtubule formation from the centrosome increases dramatically at the onset of mitosis. This process is termed centrosome maturation. However, regulatory mechanisms of microtubule assembly from the centrosome in response to the centrosome maturation are largely unknown. Here we found that YB-1, a cellular cancer susceptibility protein, is required for the centrosome maturation. Phosphorylated YB-1 accumulated in the centrosome at mitotic phase. By YB-1 knockdown, microtubules were found detached from the centrosome at telophase and an abnormal nuclear shape called nuclear lobulation was found due to defective reassembly of nuclear envelope by mis-localization of non-centrosomal microtubules. In conclusion, we propose that YB-1 is important for the assembly of centrosomal microtubule array for temporal and spatial regulation of microtubules. PMID:25740062

  15. gon-4, a cell lineage regulator required for gonadogenesis in Caenorhabditis elegans.

    PubMed

    Friedman, L; Santa Anna-Arriola, S; Hodgkin, J; Kimble, J

    2000-12-15

    The gon-4 gene is required for gonadogenesis in the nematode Caenorhabditis elegans. Normally, two precursor cells, Z1 and Z4, follow a reproducible pattern of cell divisions to generate the mature somatic gonadal structures (e.g., uterus in hermaphrodites, vas deferens in males). In contrast, in gon-4 mutants, the Z1/Z4 cell lineages are variably aborted in both hermaphrodites and males: Z1 and Z4 divide much later than normal and subsequent divisions are either absent or severely delayed. In gon-4 adults, normal somatic gonadal structures are never observed, and germ-line and vulval tissues, which depend on somatic gonadal cues for their development, are also aberrant. In contrast, nongonadal tissues and the timing of other developmental events (e.g., molts) appear to be normal in gon-4 mutants. The gon-4 alleles are predicted to be strong loss-of-function or null alleles by both genetic and molecular criteria. We have cloned gon-4 in an attempt to learn how it regulates gonadogenesis. The gon-4 gene encodes a novel, acidic protein. A GON-4::GFP fusion protein, which rescues a gon-4 mutant to fertility, is expressed in somatic gonadal cells during early gonadal development. Furthermore, this fusion protein is nuclear. We conclude that gon-4 is a regulator of the early lineage of Z1 and Z4 and suggest that it is a part of a genetic program common to the regulation of both hermaphrodite and male gonadogenesis. Copyright 2000 Academic Press.

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

    PubMed

    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.

  17. Regulation of longevity by genes required for the functions of AIY interneuron in nematode Caenorhabditis elegans.

    PubMed

    Shen, Lulu; Hu, Yaou; Cai, Ting; Lin, Xingfeng; Wang, Dayong

    2010-01-01

    In Caenorhabditis elegans, functional ttx-3, sra-11, ceh-10, and ceh-23 genes are required for the functions of AIY interneuron. Compared to wild-type N2, mutations in ttx-3 and ceh-10 significantly decreased lifespan, whereas mutations in sra-11 and ceh-23 did not obviously influence nematode lifespan. Mutations in ttx-3 and ceh-10 were associated closely with lower pumping rates at adult day 8 and caused a more rapid accumulated intestinal autofluorescence than wild-type N2. Mutations in ceh-10 remarkably affected fertility and egg number in the uterus. The regulation of ttx-3 and ceh-10 on longevity was not temperature-dependent, and ttx-3, and ceh-10 mutants all formed very few dauers at 27°C. The shortened lifespan of the ttx-3 or ceh-10 mutants was completely or largely rescued by expression of TTX-3 or CEH-10 in AIY interneurons. Moreover, the long-lived phenotype of the daf-2 mutant could be suppressed by both the ttx-3 and the ceh-10 mutations. Furthermore, ablation of AIY interneurons shortened the longevity of wild-type and the daf-2 mutant. Therefore, ttx-3 and ceh-10 regulate the longevity through influencing the insulin/IGF signaling pathway in C. elegans.

  18. DNA sequence-specific recognition by a transcriptional regulator requires indirect readout of A-tracts

    PubMed Central

    Mendieta, Jesús; Pérez-Lago, Laura; Salas, Margarita; Camacho, Ana

    2007-01-01

    The bacteriophage Ø29 transcriptional regulator p4 binds to promoters of different intrinsic activities. The p4–DNA complex contains two identical protomers that make similar interactions with the target sequence 5′-AACTTTTT-15 bp-AAAATGTT-3′. To define how the various elements in the target sequence contribute to p4's affinity, we studied p4 binding to a series of mutated binding sites. The binding specificity depends critically on base pairs of the target sequence through both direct as well as indirect readout. There is only one specific contact between a base and an amino acid residue; other contacts take place with the phosphate backbone. Alteration of direct amino acid–base contacts, or mutation of non-contacted A·T base pairs at A-tracts abolished binding. We generated three 5 ns molecular dynamics (MD) simulations to investigate the basis for the p4–DNA complex specificity. Recognition is controlled by the protein and depends on DNA dynamic properties. MD results on protein–DNA contacts and the divergence of p4 affinity to modified binding sites reveal an inherent asymmetry, which is required for p4-specific binding and may be crucial for transcription regulation. PMID:17452358

  19. Cytokine-regulated neutrophil recruitment is required for brain but not spinal cord inflammation during EAE

    PubMed Central

    Simmons, Sarah B.; Liggitt, Denny; Goverman, Joan M.

    2014-01-01

    Multiple sclerosis (MS) is an autoimmune disease in which inflammatory lesions lead to tissue injury in the brain and/or spinal cord. The specific sites of tissue injury are strong determinants of clinical outcome in MS, but the pathways that determine whether damage occurs in the brain or spinal cord are not understood. Previous studies in mouse models of MS demonstrated that IFN-γ and IL-17 regulate lesion localization within the brain, however, the mechanisms by which these cytokines mediate their effects have not been identified. Here we show that IL-17 promoted, but IFN-γ inhibited, ELR+ chemokine-mediated neutrophil recruitment to the brain, and that neutrophil infiltration was required for parenchymal tissue damage in the brain. In contrast, IFN-γ promoted ELR+ chemokine expression and neutrophil recruitment to the spinal cord. Surprisingly, tissue injury in the spinal cord did not exhibit the same dependence on neutrophil recruitment that was observed for the brain. Our results demonstrate that the brain and spinal cord exhibit distinct sensitivities to cellular mediators of tissue damage, and that IL-17 and IFN-γ differentially regulate recruitment of these mediators to each microenvironment. These findings suggest an approach toward tailoring therapies for patients with distinct patterns of neuroinflammation. PMID:24913979

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

    PubMed

    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.

  1. Spindly/CCDC99 Is Required for Efficient Chromosome Congression and Mitotic Checkpoint Regulation

    PubMed Central

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

    2010-01-01

    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 (NΔ253), or the conserved Spindly box (ΔSB), 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. PMID:20427577

  2. 75 FR 47561 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Contract...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-06

    ... Regulation Supplement; Contract Financing (OMB Control Number 0704- 0359) AGENCY: Defense Acquisition... and OMB Number: Defense Federal Acquisition Regulation Supplement (DFARS) part 232, Contract Financing...

  3. PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis.

    PubMed Central

    Saporito-Irwin, S M; Birse, C E; Sypherd, P S; Fonzi, W A

    1995-01-01

    Candida albicans, like many fungi, exhibits morphological plasticity, a property which may be related to its biological capacity as an opportunistic pathogen of humans. Morphogenesis and alterations in cell shape require integration of many cellular functions and occur in response to environmental signals, most notably pH and temperature in the case of C. albicans. In the course of our studies of differential gene expression associated with dimorphism of C. albicans, we have isolated a gene, designated PHR1, which is regulated in response to the pH of the culture medium. PHR1 expression was repressed at pH values below 5.5 and induced at more alkaline pH. The predicted amino acid sequence of the PHR1 protein was 56% identical to that of the Saccharomyces cerevisiae Ggp1/Gas1 protein, a highly glycosylated cell surface protein attached to the membrane via glycosylphosphatidylinositol. A homozygous null mutant of PHR1 was constructed and found to exhibit a pH-conditional morphological defect. At alkaline pH, the mutant, unlike the parental type, was unable to conduct apical growth of either yeast or hyphal growth forms. This morphological aberration was not associated with defective cytoskeletal polarization or secretion. The results suggest that PHR1 defines a novel function required for apical cell growth and morphogenesis. PMID:7823929

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

    PubMed Central

    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.

    2013-01-01

    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. PMID:24216482

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

    PubMed

    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.

  6. Nutritional Requirements and Nitrogen-Dependent Regulation of Proteinase Activity of Lactobacillus helveticus CRL 1062

    PubMed Central

    Hebert, Elvira M.; Raya, Raul R.; De Giori, Graciela S.

    2000-01-01

    The nutritional requirements of Lactobacillus helveticus CRL 1062 were determined with a simplified chemically defined medium (SCDM) and compared with those of L. helveticus CRL 974 (ATCC 15009). Both strains were found to be prototrophic for alanine, glycine, asparagine, glutamine, and cysteine. In addition, CRL 1062 also showed prototrophy for lysine and serine. The microorganisms also required riboflavin, calcium pantothenate, pyridoxal, nicotinic acid, and uracil for growth in liquid SCDM. The growth rate and the synthesis of their cell membrane-bound serine proteinases, but not of their intracellular leucyl-aminopeptidases, were influenced by the peptide content of the medium. The highest proteinase levels were found during cell growth in basal SCDM, while the synthesis of this enzyme was inhibited in SCDM supplemented with Casitone, Casamino Acids, or β-casein. Low-molecular-mass peptides (<3,000 Da), extracted from Casitone, and the dipeptide leucylproline (final concentration, 5 mM) play important roles in the medium-dependent regulation of proteinase activity. The addition of the dipeptide leucylproline (5 mM) to SCDM reduced proteinase activity by 25%. PMID:11097908

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

    PubMed

    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.

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

    PubMed

    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.

  9. Guide to NRC reporting and recordkeeping requirements. Compiled from requirements in Title 10 of the US Code of Federal Regulations as codified on December 31, 1991

    SciTech Connect

    Collins, M.; Shelton, B.

    1992-11-01

    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 I, on December 31, 1991. It also includes, in a separate section, any of those requirements that were superseded or discontinued from January through December 1991. Finally, the appendix lists mailing and delivery addresses for NRC Headquarters and Regional Offices mentioned in the compilation.

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

    ERIC Educational Resources Information Center

    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…

  11. 76 FR 14275 - Regulations Issued Under the Export Grape and Plum Act; Revision to the Minimum Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-16

    ... Service 7 CFR Part 35 Regulations Issued Under the Export Grape and Plum Act; Revision to the Minimum... requirements under the Export Grape and Plum Act. This rule changes the minimum bunch weight requirement for... under authority of the Export Grape and Plum Act, as amended (7 U.S.C. 591-599), hereinafter referred...

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

    ERIC Educational Resources Information Center

    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…

  13. 49 CFR 40.7 - How can you get an exemption from a requirement in this regulation?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 1 2013-10-01 2013-10-01 false How can you get an exemption from a requirement in... can you get an exemption from a requirement in this regulation? (a) If you want an exemption from any... this part, that make your compliance with a specific provision of this part impracticable. (c) If...

  14. Hygienic and sanitary requirements of gyms in force in Italy: national issues and analysis of regional regulations.

    PubMed

    Capasso, L; Arpesella, M; Gaeta, M; D'Alessandro, D

    2015-01-01

    In recent years, the awareness of the importance of physical activity for human health is growing, as people practicing it; the number of gyms has also substantially increased. Currently, in Italy there is not a consistent regulation regarding hygienic and sanitary requirements of gyms. Several Italian regions issued laws about this topic. The authors analyse the standards required by the Italian Olympic Committee (CONI) and the regional laws, highlighting the numerous issues, among which the possible location of gyms in semi-basements and basements. They eventually call for a national regulation that should clearly transpose the hygienic and sanitary requirements of gyms into a satisfactory rule of law.

  15. BDNF Activates mTOR to Regulate GluR1 Expression Required for Memory Formation

    PubMed Central

    Katche, Cynthia; Cammarota, Martín; Izquierdo, Iván; Medina, Jorge H.

    2009-01-01

    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 at least two

  16. Requirement for non-regulated, constitutive calcium influx in macrophage survival signaling

    SciTech Connect

    Tano, Jean-Yves; Vazquez, Guillermo

    2011-04-08

    Highlights: {yields} We examine the role of constitutive Ca{sup 2+} influx in macrophage survival. {yields} Survival signaling exhibits a mandatory requirement for constitutive Ca{sup 2+} influx. {yields} CAM/CAMKII couples constitutive Ca{sup 2+} influx to survival signaling. -- Abstract: The phosphatidylinositol-3-kinase (PI3K)/AKT axis and the Nuclear Factor kappa B (NF{kappa}B) pathway play critical roles in macrophage survival. In cells other than macrophages proper operation of those two pathways requires Ca{sup 2+} influx into the cell, but if that is the case in macrophages remains unexplored. In the present work we used THP-1-derived macrophages and a pharmacological approach to examine for the first time the role of constitutive, non-regulated Ca{sup 2+} influx in PI3K/AKT and NF{kappa}B signaling. Blocking constitutive function of Ca{sup 2+}-permeable channels with the organic channel blocker SKF96365 completely prevented phosphorylation of I{kappa}B{alpha}, AKT and its downstream target BAD in TNF{alpha}-treated macrophages. A similar effect was observed upon treating macrophages with the calmodulin (CAM) inhibitor W-7 or the calmodulin-dependent kinase II (CAMKII) inhibitor KN-62. In addition, pre-treating macrophages with SKF96365 significantly enhanced TNF{alpha}-induced apoptosis. Our findings suggest that in THP-1-derived macrophages survival signaling depends, to a significant extent, on constitutive Ca{sup 2+} influx presumably through a mechanism that involves the CAM/CAMKII axis as a coupling component between constitutive Ca{sup 2+} influx and activation of survival signaling.

  17. Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator requires a tyrosine-based signal.

    PubMed

    Prince, L S; Peter, K; Hatton, S R; Zaliauskiene, L; Cotlin, L F; Clancy, J P; Marchase, R B; Collawn, J F

    1999-02-05

    We previously demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) is rapidly endocytosed in epithelial cells (Prince, L. S., Workman, R. B., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5192-5196). To determine the structural features of CFTR required for endocytosis, we prepared chimeric molecules consisting of the amino-terminal (residues 2-78) and carboxyl-terminal tail regions (residues 1391-1476) of CFTR, each fused to the transmembrane and extracellular domains of the transferrin receptor. Functional analysis of the CFTR-(2-78) and CFTR-(1391-1476) indicated that both chimeras were rapidly internalized. Deletion of residues 1440-1476 had no effect on chimera internalization. Mutations of potential internalization signals in both cytoplasmic domains reveal that only one mutation inhibits internalization, Y1424A. Using a surface biotinylation reaction, we also examined internalization rates of wild type and mutant CFTRs expressed in COS-7 cells. We found that both wild type and A1440X CFTR were rapidly internalized, whereas the Y1424A CFTR mutant, like the chimeric protein, had approximately 40% reduced internalization activity. Deletions in the amino-terminal tail region of CFTR resulted in defective trafficking of CFTR out of the endoplasmic reticulum to the cell surface, suggesting that an intact amino terminus is critical for biosynthesis. In summary, our results suggest that both tail regions of CFTR are sufficient to promote rapid internalization of a reporter molecule and that tyrosine 1424 is required for efficient CFTR endocytosis.

  18. 76 FR 36905 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Taxes

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-23

    ... Regulation Supplement; Taxes AGENCY: Defense Acquisition Regulations System, Department of Defense (DoD... Supplement (DFARS) Part 229, Taxes, and related clause at DFARS 252.229-7010; OMB Control Number...

  19. 78 FR 70294 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-25

    ... Regulation Supplement; Administrative Matters AGENCY: Defense Acquisition Regulations System, Department of..., Administrative Matters, and related clauses at DFARS 252.204; DD Form 2051, Request for Assignment of a...

  20. 49 CFR 192.13 - What general requirements apply to pipelines regulated under this part?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    .... Regulated onshore gathering line to which this part did not apply until April 14, 2006 March 15 2007. All... Offshore gathering line July 31, 1977. Regulated onshore gathering line to which this part did not apply...

  1. Germline self-renewal requires cyst stem cells and stat regulates niche adhesion in Drosophila testes.

    PubMed

    Leatherman, Judith L; Dinardo, Stephen

    2010-08-01

    Adults maintain tissue-specific stem cells through niche signals. A model for niche function is the Drosophila melanogaster testis, where a small cluster of cells called the hub produce locally available signals that allow only adjacent cells to self-renew. We show here that the principal signalling pathway implicated in this niche, chemokine activation of STAT, does not primarily regulate self-renewal of germline stem cells (GSCs), but rather governs GSC adhesion to hub cells. In fact, GSC renewal does not require hub cell contact, as GSCs can be renewed solely by contact with the second resident stem cell population, somatic cyst stem cells (CySCs), and this involves BMP signalling. These data suggest a modified paradigm whereby the hub cells function as architects of the stem cell environment, drawing into proximity cellular components necessary for niche function. Self-renewal functions are shared by the hub cells and the CySCs. This work also reconciles key differences in GSC renewal between Drosophila testis and ovary niches, and highlights how a niche can coordinate the production of distinct lineages by having one stem cell type rely on a second.

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

    PubMed

    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.

  3. Retinophilin is a light-regulated phosphoprotein required to suppress photoreceptor dark noise in Drosophila.

    PubMed

    Mecklenburg, Kirk L; Takemori, Nobuaki; Komori, Naoka; Chu, Brian; Hardie, Roger C; Matsumoto, Hiroyuki; O'Tousa, Joseph E

    2010-01-27

    Photoreceptor cells achieve high sensitivity, reliably detecting single photons, while limiting the spontaneous activation events responsible for dark noise. We used proteomic, genetic, and electrophysiological approaches to characterize Retinophilin (RTP) (CG10233) in Drosophila photoreceptors and establish its involvement in dark-noise suppression. RTP possesses membrane occupation and recognition nexus (MORN) motifs, a structure shared with mammalian junctophilins and other membrane-associated proteins found within excitable cells. We show the MORN repeats, and both the N- and C-terminal domains, are required for RTP localization in the microvillar light-gathering organelle, the rhabdomere. RTP exists in multiple phosphorylated isoforms under dark conditions and is dephosphorylated by light exposure. An RTP deletion mutant exhibits a high rate of spontaneous membrane depolarization events in dark conditions but retains the normal kinetics of the light response. Photoreceptors lacking neither inactivation nor afterpotential C (NINAC) myosin III, a motor protein/kinase, also display a similar dark-noise phenotype as the RTP deletion. We show that NINAC mutants are depleted for RTP. These results suggest the increase in dark noise in NINAC mutants is attributable to lack of RTP and, furthermore, defines a novel role for NINAC in the rhabdomere. We propose that RTP is a light-regulated phosphoprotein that organizes rhabdomeric components to suppress random activation of the phototransduction cascade and thus increases the signaling fidelity of dark-adapted photoreceptors.

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

    PubMed

    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.

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

    PubMed Central

    Vithayathil, Joseph; Pucilowska, Joanna; Goodnough, L. Henry; Atit, Radhika P.

    2015-01-01

    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. PMID:25926459

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

    PubMed

    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.

  7. 78 FR 68831 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-15

    ... Regulation Supplement (DFARS) Part 211, Describing Agency Needs AGENCY: Defense Acquisition Regulations... Acquisition Regulation Supplement (DFARS, Part 211, Describing Agency Needs, and the associated clauses at... Military or Federal Specifications and Standards; OMB Control Number 0704-0398. Needs and Uses:...

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

    PubMed

    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

  9. 76 FR 71707 - Revising Underground Storage Tank Regulations-Revisions to Existing Requirements and New...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-18

    ... operation and maintenance requirements for UST systems; removing certain deferrals; adding new release... Containment B. Additional Requirements for Operation and Maintenance 1. Walkthrough Inspections 2. Spill... and Maintenance Requirements for Release Detection Equipment C. Addressing Deferrals 1. Emergency...

  10. NFAT5 regulates the canonical Wnt pathway and is required for cardiomyogenic differentiation

    SciTech Connect

    Adachi, Atsuo; Takahashi, Tomosaburo; Ogata, Takehiro; Imoto-Tsubakimoto, Hiroko; Nakanishi, Naohiko; Ueyama, Tomomi; Matsubara, Hiroaki

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer NFAT5 protein expression is downregulated during cardiomyogenesis. Black-Right-Pointing-Pointer Inhibition of NFAT5 function suppresses canonical Wnt signaling. Black-Right-Pointing-Pointer Inhibition of NFAT5 function attenuates mesodermal induction. Black-Right-Pointing-Pointer NFAT5 function is required for cardiomyogenesis. -- Abstract: While nuclear factor of activated T cells 5 (NFAT5), a transcription factor implicated in osmotic stress response, is suggested to be involved in other processes such as migration and proliferation, its role in cardiomyogenesis is largely unknown. Here, we examined the role of NFAT5 in cardiac differentiation of P19CL6 cells, and observed that it was abundantly expressed in undifferentiated P19CL6 cells, and its protein expression was significantly downregulated by enhanced proteasomal degradation during DMSO-induced cardiomyogenesis. Expression of a dominant negative mutant of NFAT5 markedly attenuated cardiomyogenesis, which was associated with the inhibition of mesodermal differentiation. TOPflash reporter assay revealed that the transcriptional activity of canonical Wnt signaling was activated prior to mesodermal differentiation, and this activation was markedly attenuated by NFAT5 inhibition. Pharmacological activation of canonical Wnt signaling by [2 Prime Z, 3 Prime E]-6-bromoindirubin-3 Prime -oxime (BIO) restored Brachyury expression in NFAT5DN-expressing cells. Inhibition of NFAT5 markedly attenuated Wnt3 and Wnt3a induction. Expression of Dkk1 and Cerberus1, which are secreted Wnt antagonists, was also inhibited by NFAT5 inhibition. Thus, endogenous NFAT5 regulates the coordinated expression of Wnt ligands and antagonists, which are essential for cardiomyogenesis through the canonical Wnt pathway. These results demonstrated a novel role of NFAT5 in cardiac differentiation of stem cells.

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

    PubMed Central

    Wang, Jian; Wang, Wen-Xiong

    2016-01-01

    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. PMID:27216705

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

    PubMed

    Wang, Jian; Wang, Wen-Xiong

    2016-05-24

    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.

  13. 25 CFR 518.7 - If a tribe holds a certificate of self-regulation, is it required to report information to the...

    Code of Federal Regulations, 2010 CFR

    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...

  14. 25 CFR 518.7 - If a tribe holds a certificate of self-regulation, is it required to report information to the...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 2 2013-04-01 2013-04-01 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...

  15. 25 CFR 518.7 - If a tribe holds a certificate of self-regulation, is it required to report information to the...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 2 2012-04-01 2012-04-01 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...

  16. Requirements for Teacher Education and Certification. Part One--Certification Regulations.

    ERIC Educational Resources Information Center

    South Carolina State Dept. of Education, Columbia.

    This document sets forth certification requirements for prospective teachers in South Carolina. Areas covered include: (1) persons required to hold a teaching credential; (2) requirements for certification; (3) acceptable credit; (4) required examinations; (5) student teaching; (6) accredited teacher education programs; (7) application procedures;…

  17. 49 CFR 1013.3 - Review and reporting requirements for regulated carriers.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... acquisition of control of a regulated carrier. (b) Any person who establishes an independent trust for the... securities of another I.C.C. regulated carrier (or the listed shares of a company controlling 10 percent or... acquisition of control, through the acquisition of the voting stock of another carrier, is sought, unless the...

  18. 78 FR 30898 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Rights in...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-23

    ... 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... are associated with rights in technical data and computer software. DoD needs this information...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  20. 33 CFR 165.921 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...) Regulated Navigation Area. The following waters are a regulated navigation area (RNA): the Illinois Waterway... RNA. This section does not apply to towing vessel operators responsible for barges not carrying CDC... one or more CDC barges in the RNA must report all the information items specified in table 165.921(f...

  1. 33 CFR 165.921 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) Regulated Navigation Area. The following waters are a regulated navigation area (RNA): the Illinois Waterway... RNA. This section does not apply to towing vessel operators responsible for barges not carrying CDC... one or more CDC barges in the RNA must report all the information items specified in table 165.921(f...

  2. 33 CFR 165.921 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...) Regulated Navigation Area. The following waters are a regulated navigation area (RNA): the Illinois Waterway... RNA. This section does not apply to towing vessel operators responsible for barges not carrying CDC... one or more CDC barges in the RNA must report all the information items specified in table 165.921(f...

  3. 7 CFR 340.8 - Container requirements for the movement of regulated articles.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... articles. 340.8 Section 340.8 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL... requirements—(1) Plants and plant parts. All plants or plant parts, except seeds, cells, and subcellular... strength. (3) Live microorganisms and/or etiologic agents, cells, or subcellular elements. All regulated...

  4. 7 CFR 340.8 - Container requirements for the movement of regulated articles.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... articles. 340.8 Section 340.8 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL... requirements—(1) Plants and plant parts. All plants or plant parts, except seeds, cells, and subcellular... strength. (3) Live microorganisms and/or etiologic agents, cells, or subcellular elements. All regulated...

  5. 75 FR 44163 - Implementation of Regulations Required Under Title XI of the Food, Conservation and Energy Act of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-28

    ...-AB07 Implementation of Regulations Required Under Title XI of the Food, Conservation and Energy Act of... clarify conditions for industry compliance with the P&S Act and provide for a fairer market place. DATES... fairer market place. We have received comments asking for an extension of the comment period, and...

  6. 77 FR 33635 - Amendment to the Bank Secrecy Act Regulations-Requirement That Clerks of Court Report Certain...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-07

    ... Financial Crimes Enforcement Network 31 CFR Part 1010 RIN 1506-AB17 Amendment to the Bank Secrecy Act Regulations--Requirement That Clerks of Court Report Certain Currency Transactions AGENCY: Financial Crimes... Crimes Enforcement Network. This information shall be reported on the same form as prescribed by...

  7. 77 FR 33638 - Amendment to the Bank Secrecy Act Regulations-Exemption From the Requirement To Report...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-07

    ... Financial Crimes Enforcement Network 31 CFR Part 1020 RIN 1506-AB18 Amendment to the Bank Secrecy Act Regulations--Exemption From the Requirement To Report Transactions in Currency AGENCY: Financial Crimes.... Dated: June 1, 2012. James H. Freis, Jr., Director, Financial Crimes Enforcement Network. BILLING...

  8. Immigration Rules, Regulations, and Requirements and Foreign Student Enrollments. Southeast Florida Education Consortium Workshop (Miami, Florida, February 18, 1981).

    ERIC Educational Resources Information Center

    Matthewson, Douglas E., Jr., Comp.

    Information is presented about a workshop on immigration documents, rules, regulations, and requirements associated with international students and nonimmigrant alien students. Twenty-six members of the Southeast Florida Educational Consortium participated, representing campus admissions and registrars offices, international student programs, and…

  9. 14 CFR 11.61 - May I ask FAA to adopt, amend, or repeal a regulation, or grant relief from the requirements of a...

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false May I ask FAA to adopt, amend, or repeal a regulation, or grant relief from the requirements of a current regulation? 11.61 Section 11.61 Aeronautics... to adopt, amend, or repeal a regulation, or grant relief from the requirements of a current...

  10. 14 CFR 11.61 - May I ask FAA to adopt, amend, or repeal a regulation, or grant relief from the requirements of a...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false May I ask FAA to adopt, amend, or repeal a regulation, or grant relief from the requirements of a current regulation? 11.61 Section 11.61 Aeronautics... to adopt, amend, or repeal a regulation, or grant relief from the requirements of a current...

  11. 78 FR 67133 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-08

    ... of Information, and the clause at DFARS 252.205- 7000 require defense prime contractors awarded... DFARS 252.205-7000 require defense prime contractors awarded contracts over $1,000,000 to provide...

  12. 77 FR 59139 - Prompt Corrective Action, Requirements for Insurance, and Promulgation of NCUA Rules and Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-26

    ... From the Federal Register Online via the Government Publishing Office NATIONAL CREDIT UNION..., and Promulgation of NCUA Rules and Regulations AGENCY: National Credit Union Administration (NCUA... consideration of regulatory relief for more small credit unions in future rulemakings. The amended...

  13. 27 CFR 70.411 - Imposition of taxes, qualification requirements, and regulations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... contains the regulations relating to distilled spirits for fuel use and the production of vinegar by the... unmerchantable taxpaid wine; use of wine for distilling material and manufacture of vinegar; and record...

  14. 75 FR 26165 - Regulation of Fuels and Fuel Additives: Alternative Affirmative Defense Requirements for Ultra...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-11

    ...EPA is issuing a proposed rule to amend the diesel sulfur regulations to allow refiners, importers, distributors, and retailers of highway diesel fuel the option to use an alternative affirmative defense if the Agency finds highway diesel fuel samples above the specified sulfur standard at retail facilities. This rule also proposes to amend the gasoline benzene regulations to allow disqualified small refiners the same opportunity to generate gasoline benzene credits as that afforded to non-small refiners.

  15. The transcriptional regulator lola is required for stem cell maintenance and germ cell differentiation in the Drosophila testis

    PubMed Central

    Davies, Erin L.; Lim, Jaclyn G.Y.; Joo, William J.; Tam, Cheuk Ho; Fuller, Margaret T.

    2014-01-01

    Stem cell behavior is regulated by extrinsic signals from specialized microenvironments, or niches, and intrinsic factors required for execution of context-appropriate responses to niche signals. Here we show that function of the transcriptional regulator longitudinals lacking (lola) is required cell autonomously for germline stem cell and somatic cyst stem cell maintenance in the Drosophila testis. In addition, lola is also required for proper execution of key developmental transitions during male germ cell differentiation, including the switch from transit amplifying progenitor to spermatocyte growth and differentiation, as well as meiotic cell cycle progression and spermiogenesis. Different lola isoforms, each having unique C-termini and zinc finger domains, may control different aspects of proliferation and differentiation in the male germline and somatic cyst stem cell lineages. PMID:23159836

  16. Regulation of G-protein signaling via Gnas is required to regulate proximal tubular growth in the Xenopus pronephros

    PubMed Central

    Zhang, Bo; Romaker, Daniel; Ferrell, Nicholas; Wessely, Oliver

    2013-01-01

    In the kidney, proximal tubules are very important for the reabsorption of water, ions and organic solutes from the primary urine. They are composed of highly specialized epithelial cells that are characterized by an elaborate apical brush border to increase transport efficiency. Using the pronephric kidney of Xenopus laevis we discovered that the G-protein modulator cholera toxin resulted in a dramatic reduction of the proximal tubular size. This phenotype was accompanied by changes in the cytoarchitecture characterized by ectopic expression of the distal tubular marker 4A6 and an impairment of yolk platelet degradation. In addition, cholera toxin caused edema formation. However, this phenotype was not due to kidney defects, but rather due to impaired vasculature development. Based on experiments with antisense morpholino oligomers as well as pharmacological agonists and antagonists, we could show that the complex phenotype of cholera toxin in the pronephric kidney was caused by the hyperactivation of a single G-protein alpha subunit, Gnas. This—in turn—caused elevated cAMP levels, triggered a Rapgef4-dependent signaling cassette and perturbed exo- and endocytosis. This perturbation of the secretory pathway by Ctx was not only observed in Xenopus embryos. Also, in a human proximal tubular cell line, cholera toxin or a Rapgef4-specific agonist increased uptake and decreased secretion of FITC-labeled Albumin. Based on these data we propose that the Gnas/cAMP/Rapgef4 pathway regulates the signals inducing the proliferation of proximal tubules to acquire their final organ size. PMID:23352791

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

    PubMed

    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.

  18. Regulation of Gene33 expression by insulin requires MEK-ERK activation.

    PubMed

    Keeton, Adam B; Xu, Jie; Franklin, J Lee; Messina, Joseph L

    2004-09-17

    Gene33 and its human homologue, mitogen inducible gene-6/receptor-associated late transducer (mig-6, RALT), is a 53-kDa soluble protein that was identified as a hepatic gene regulated by glucocorticoids and insulin. Its mRNA is expressed in numerous tissues in addition to the liver. Mitogen inducibility of Gene33 mRNA has been described in several experimental systems. Recent reports have suggested a role for Gene33 in inhibition of proliferation induced by factors that bind to members of the ErbB family of receptors. In the present work, we examine the regulation of Gene33 protein by insulin in hepatoma cells of rat (H4IIE) and human (HepG2/Hep3B) origin. Inhibition of MEK1 significantly inhibited extracellularly regulated kinase (ERK)1/2 activation and insulin-regulated Gene33 transcription and protein levels in H4IIE cells. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity alone did not significantly alter transcription of Gene33. In Hep3B and HepG2 cells, insulin did not significantly induce either ERK1/2 activation or Gene33 expression. This work suggests that the MEK-ERK, but not the phosphatidylinositol 3-kinase (PI3-K), pathway plays a direct role in insulin regulation of Gene33 transcription and protein expression.

  19. 77 FR 8757 - Revising Underground Storage Tank Regulations-Revisions to Existing Requirements and New...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-15

    ..., Hazardous materials, Petroleum, Reporting and recordkeeping requirements, Underground storage ] tanks, Water pollution control, Water supply. 40 CFR Part 281 Environmental protection, Administrative practice and...

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Collagen I and the fibroblast: high protein expression requires a new paradigm of post-transcriptional, feedback regulation.

    PubMed

    Schwarz, Richard I

    2015-09-01

    Scaling protein production seems like a simple perturbation of transcriptional control. However, when embryonic tendon fibroblasts have to produce >50% procollagen and secrete it from the cell 4 times faster than the average protein, this taxes the cellular machinery and requires a fresh look at how the pathway is controlled. Ascorbate, a reducing agent, can stimulate procollagen production 6-fold. Procollagen mRNA levels goes up 6-fold but requires 3 days for the cell to accomplish this task. Secretion rates, the last cellular step in the process, also goes up 6-fold but this occurs in <1 h. What regulatory scheme is consistent with these properties? This review focuses on fibroblasts that make high levels of procollagen (type I) and how they regulate the collagen pathway. Data from many different labs are relevant to this problem but it is hard to see the bigger picture from a large number of small studies. This review aims to consolidate this data into a coherent model and this requires solutions to some controversies and postulating potential mechanisms where the details are still missing. In high collagen producing cells, the pathway is controlled by post-transcriptional regulation. This requires feedback control between secretion and translation rates that is based on the helical structure of the procollagen molecule and additional tissue-specific modifications. Transcriptional control does not scale well to high protein production with rapid regulation. New paradigms lead to better understanding of collagen diseases and tendon morphogenesis.

  2. A comparison of the requirements of the OSHA 29 CFR 1910.119 regulation and API RP 75

    SciTech Connect

    Tibbetts, O.D.

    1995-12-31

    The OSHA Process Safety Management (PSM) Regulation, 29 CFR 1910.119, went into effect on May 26, 1992. API RP 75, Recommended Practices for Development of a Safety and Environmental Management Program for Outer Continental Shelf Operations and Facilities, was published on May 15, 1993. The OSHA regulation applies to all normally manned facilities, onshore and in state waters, that possess the threshold quantities of highly hazardous chemicals. The regulation identifies the specific ``highly hazardous chemicals`` and states the threshold quantities of each. Meanwhile, API RP 75 applies to all operations and facilities on the Outer Continental Shelf. While the implementation of the practices contained in the RP is voluntary, the MMS has urged operators to comply rather than face the alternative of further governmental regulations. It is possible that some operators do not fully understand the scope of the PSM regulation or RP 75. In addition, they may not realize their exposure as far as these standards are concerned. This paper compares the two standards and attempts to show the obligation and exposure which the regulation and the RP create. It also indicates the benefits which the operator can derive from adopting the practices required by these standards.

  3. 75 FR 81337 - Interim Final Regulation Deferring the Reporting Date for Certain Data Elements Required Under...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-27

    ... Elements Required Under the Mandatory Reporting of Greenhouse Gases Rule; Interim Final Rule #0;#0;Federal... Elements Required Under the Mandatory Reporting of Greenhouse Gases Rule AGENCY: Environmental Protection... equations under the Mandatory Greenhouse Gas Reporting Rule. In response to EPA's July 7, 2010...

  4. 49 CFR 195.11 - What is a regulated rural gathering line and what requirements apply?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...) of an unusually sensitive area as defined in § 195.6; and (3) Operates at a maximum pressure... constructed with steel pipe, a pressure of more than 125 psi (861 kPa) gage. (b) Safety requirements. Each... requirements in subpart B of this part. (5) Establish the maximum operating pressure of the pipeline according...

  5. 76 FR 39236 - Federal Acquisition Regulation; Uniform Suspension and Debarment Requirement

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-05

    ... Year 2010. Section 815 extends the flow down of limitations on subcontracting with entities that have... for Debarment, by flowing down the requirements for the contractor or higher-tier subcontractor to... the requirement down to the first tier, the clause was added to FAR 52.212-5, Contract Terms...

  6. 75 FR 29456 - Acquisition Regulation: Subchapter E-General Contracting Requirements, Subchapter F-Special...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-26

    ...--General Contracting Requirements, Subchapter F--Special Categories of Contracting, and Subchapter G... Contracting Requirements, F--Special Categories of Contracting, and G--Contract Management to make changes to... Act E. Review Under the National Environmental Policy Act F. Review Under Executive Order 13132...

  7. 7 CFR 318.13-3 - General requirements for all regulated articles.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... infestation and infection, or without such inspection when the inspector determines that the lot for consignment is of such a nature that no danger of infestation or infection is involved. (i) Persons intending... treatment. (i) Regulated articles for which treatments are approved under part 305 of this chapter may...

  8. 75 FR 6185 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Rights in...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-08

    ... Regulation Supplement; Rights in Technical Data and Computer Software (OMB Control Number 0704-0369) AGENCY... Subpart 227.72, Rights in Computer Software and Computer Software Documentation, and related provisions... technical data and computer software. DoD needs this information to implement 10 U.S.C. 2320, Rights...

  9. 76 FR 65411 - Specialty Crops; Import Regulations; Proposed Pistachio Import Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-21

    ... regulations for all domestic and imported peanuts marketed in the United States, for which USDA has...--Minimum Quality and Handling Standards for Domestic and Imported Peanuts Marketed in the United States... pistachios and shelled kernels because of the additional weight of the shells for inshell pistachios. The...

  10. 77 FR 51686 - Specialty Crops; Import Regulations; New Pistachio Import Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-27

    ... regulations for all domestic and imported peanuts marketed in the United States, for which USDA has...--Minimum Quality and Handling Standards for Domestic and Imported Peanuts Marketed in the United States... and shelled kernels because of the additional weight of the shells for inshell pistachios. The drawing...

  11. 76 FR 4555 - Authority To Require Supervision and Regulation of Certain Nonbank Financial Companies

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-26

    ... also stressed the need for outreach and coordination with the home regulators of foreign institutions... those least affected by the cyclical nature of the economy are less likely to be systemically important... consultation, coordination and judicial review in connection with a determination. Given the importance of...

  12. 78 FR 27308 - Loan Originator Compensation Requirements Under the Truth In Lending Act (Regulation Z...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-10

    ... implementing regulations by that date.\\2\\ To avoid uncertainty and potential disruption in the national... rules, as needed; (4) publication of readiness guides for the new rules; and (5) education of consumers... disruptions in the provision of credit insurance products to consumers while interpretive questions...

  13. NOVA2-mediated RNA regulation is required for axonal pathfinding during development

    PubMed Central

    Saito, Yuhki; Miranda-Rottmann, Soledad; Ruggiu, Matteo; Park, Christopher Y; Fak, John J; Zhong, Ru; Duncan, Jeremy S; Fabella, Brian A; Junge, Harald J; Chen, Zhe; Araya, Roberto; Fritzsch, Bernd; Hudspeth, A J; Darnell, Robert B

    2016-01-01

    The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo. DOI: http://dx.doi.org/10.7554/eLife.14371.001 PMID:27223325

  14. 78 FR 4032 - Prompt Corrective Action, Requirements for Insurance, and Promulgation of NCUA Rules and Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-18

    ... From the Federal Register Online via the Government Publishing Office NATIONAL CREDIT UNION..., and Promulgation of NCUA Rules and Regulations AGENCY: National Credit Union Administration (NCUA... increases the asset threshold that identifies credit unions to which NCUA will give more robust...

  15. TRICHOME AND ARTEMISININ REGULATOR 1 Is Required for Trichome Development and Artemisinin Biosynthesis in Artemisia annua.

    PubMed

    Tan, Hexin; Xiao, Ling; Gao, Shouhong; Li, Qing; Chen, Junfeng; Xiao, Ying; Ji, Qian; Chen, Ruibing; Chen, Wansheng; Zhang, Lei

    2015-09-01

    Trichomes, small protrusions on the surface of many plant species, can produce and store various secondary metabolic products. Artemisinin, the most famous and potent medicine for malaria, is synthesized, stored, and secreted by Artemisia annua trichomes. However, the molecular basis regulating the biosynthesis of artemisinin and the development of trichomes in A. annua remains poorly understood. Here, we report that an AP2 transcription factor, TRICHOME AND ARTEMISININ REGULATOR 1 (TAR1), plays crucial roles in regulating the development of trichomes and the biosynthesis of artemisinin in A. annua. TAR1, which encodes a protein specially located in the nucleus, is mainly expressed in young leaves, flower buds, and some trichomes. In TAR1-RNAi lines, the morphology of trichomes and the composition of cuticular wax were altered, and the artemisinin content was dramatically reduced, which could be significantly increased by TAR1 oeverexpression. Expression levels of several key genes that are involved in artemisinin biosynthesis were altered when TAR1 was silenced or overexpressed. By the electrophoretic mobility shift, yeast one-hybrid and transient transformation β-glucuronidase assays, we showed that ADS and CYP71AV1, two key genes in the biosynthesis pathway of artemisinin, are likely the direct targets of TAR1. Taken together, our results indicate that TAR1 is a key component of the molecular network regulating trichome development and artemisinin biosynthesis in A. annua. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

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

    PubMed

    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.

  17. Tropomyosin exon 6b is troponin-specific and required for correct acto-myosin regulation.

    PubMed

    Maytum, Robin; Bathe, Friederike; Konrad, Manfred; Geeves, Michael A

    2004-04-30

    The specificity of tropomyosin (Tm) exon 6b for interaction with and functioning of troponin (Tn) has been studied using recombinant fibroblast Tm isoforms 5a and 5b. These isoforms differ internally by exons 6a/6b and possess non-muscle exons 1b/9d at the termini, hence they lack the primary TnT(1)-tropomyosin interaction, allowing study of exon 6 exchange in isolation from this. Using kinetic techniques to measure regulation of myosin S1 binding to actin and fluorescently labeled Tm to directly measure Tn binding, we show that binding of Tn to both isoforms is similar (0.1-0.5 microm) and both produce well regulated systems. Calcium has little effect on Tn binding to the actin.Tm complex and both exons produce a 3-fold reduction in the S1 binding rate to actin.Tm.Tn in its absence. This confirms previous results that show exon 6 has little influence on Tn affinity to actin.Tm or its ability to fully inhibit the acto-myosin interaction. Thin filaments reconstituted with Tn and Tm5a or skeletal Tm (containing exon 6b) show nearly identical calcium dependence of acto-myosin regulation. However, Tm5b produces a dramatic increase in calcium sensitivity, shifting the activation mid-point by almost an order of magnitude. This shows that exon 6 sequence and, hence, Tm structure in this region have a significant effect upon the calcium regulation of Tn. This finding supports evidence that familial hypertrophic cardiomyopathy mutations occurring adjacent to this region can effect calcium regulation.

  18. Drosophila Med6 Is Required for Elevated Expression of a Large but Distinct Set of Developmentally Regulated Genes

    PubMed Central

    Gim, Byung Soo; Park, Jin Mo; Yoon, Jeong Ho; Kang, Changwon; Kim, Young-Joon

    2001-01-01

    Mediator is the evolutionarily conserved coactivator required for the integration and recruitment of diverse regulatory signals to basal transcription machinery. To elucidate the functions of metazoan Mediator, we isolated Drosophila melanogaster Med6 mutants. dMed6 is essential for viability and/or proliferation of most cells. dMed6 mutants failed to pupate and died in the third larval instar with severe proliferation defects in imaginal discs and other larval mitotic cells. cDNA microarray, quantitative reverse transcription-PCR, and in situ expression analyses of developmentally regulated genes in dMed6 mutants showed that transcriptional activation of many, but not all, genes was affected. Among the genes found to be affected were some that play a role in cell proliferation and metabolism. Therefore, dMed6 is required in most cells for transcriptional regulation of many genes important for diverse aspects of Drosophila development. PMID:11438678

  19. 78 FR 63461 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Foreign...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-24

    ... forms regarding products that are eligible for duty-free entry. DFARS 252.225-7018, Photovoltaic Devices--Certificate, as prescribed at 225.7017-4(b), requires offerors to certify that no photovoltaic devices with...

  20. CCAAT/Enhancer Binding Protein–α Regulates the Protease/Antiprotease Balance Required for Bronchiolar Epithelium Regeneration

    PubMed Central

    Sato, Atsuyasu; Xu, Yan; Whitsett, Jeffrey A.

    2012-01-01

    Many transcription factors that regulate lung morphogenesis during development are reactivated to mediate repairs of the injured adult lung. We hypothesized that CCAAT/enhancer binding protein–α (C/EBPα), a transcription factor critical for perinatal lung maturation, regulates genes required for the normal repair of the bronchiolar epithelium after injury. Transgenic CebpαΔ/Δ mice, in which Cebpa was conditionally deleted from Clara cells and Type II cells after birth, were used in this study. Airway injury was induced in mice by the intraperitoneal administration of naphthalene to ablate bronchiolar epithelial cells. Although the deletion of C/EBPα did not influence lung structure and function under unstressed conditions, C/EBPα was required for the normal repair of terminal bronchiolar epithelium after naphthalene injury. To identify cellular processes that are influenced by C/EBPα during repair, mRNA microarray was performed on terminal bronchiolar epithelial cells isolated by laser-capture microdissection. Normal repair of the terminal bronchiolar epithelium was highly associated with the mRNAs regulating antiprotease activities, and their induction required C/EBPα. The defective deposition of fibronectin in CebpαΔ/Δ mice was associated with increased protease activity and delayed differentiation of FoxJ1-expressing ciliated cells. The fibronectin and ciliated cells were restored by the intratracheal treatment of CebpαΔ/Δ mice with the serine protease inhibitor. In conclusion, C/EBPα regulates the expression of serine protease inhibitors that are required for the normal increase of fibronectin and the restoration of ciliated cells after injury. Treatment with serine protease inhibitor may aid in the recovery of injured bronchiolar epithelial cells, and prevent common chronic lung diseases. PMID:22652201

  1. Adrenergic Regulation of HCN4 Channel Requires Protein Association with β2-Adrenergic Receptor*

    PubMed Central

    Greene, Derek; Kang, Seungwoo; Kosenko, Anastasia; Hoshi, Naoto

    2012-01-01

    β1- and β2-adrenergic receptors utilize different signaling mechanisms to control cardiac function. Recent studies demonstrated that β2-adrenergic receptors (β2ARs) colocalize with some ion channels that are critical for proper cardiac function. Here, we demonstrate that β2ARs form protein complexes with the pacemaker HCN4 channel, as well as with other subtypes of HCN channels. The adrenergic receptor-binding site was identified at a proximal region of the N-terminal tail of the HCN4 channel. A synthetic peptide derived from the β2AR-binding domain of the HCN4 channel disrupted interaction between HCN4 and β2AR. In addition, treatment with this peptide prevented adrenergic augmentation of pacemaker currents and spontaneous contraction rates but did not affect adrenergic regulation of voltage-gated calcium currents. These results suggest that the ion channel-receptor complex is a critical mechanism in ion channel regulation. PMID:22613709

  2. Drosophila Fatty Acid Transport Protein Regulates Rhodopsin-1 Metabolism and Is Required for Photoreceptor Neuron Survival

    PubMed Central

    Dourlen, Pierre; Bertin, Benjamin; Chatelain, Gilles; Robin, Marion; Napoletano, Francesco; Roux, Michel J.; Mollereau, Bertrand

    2012-01-01

    Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance. PMID:22844251

  3. Adrenergic regulation of HCN4 channel requires protein association with β2-adrenergic receptor.

    PubMed

    Greene, Derek; Kang, Seungwoo; Kosenko, Anastasia; Hoshi, Naoto

    2012-07-06

    β(1)- and β(2)-adrenergic receptors utilize different signaling mechanisms to control cardiac function. Recent studies demonstrated that β(2)-adrenergic receptors (β(2)ARs) colocalize with some ion channels that are critical for proper cardiac function. Here, we demonstrate that β(2)ARs form protein complexes with the pacemaker HCN4 channel, as well as with other subtypes of HCN channels. The adrenergic receptor-binding site was identified at a proximal region of the N-terminal tail of the HCN4 channel. A synthetic peptide derived from the β(2)AR-binding domain of the HCN4 channel disrupted interaction between HCN4 and β(2)AR. In addition, treatment with this peptide prevented adrenergic augmentation of pacemaker currents and spontaneous contraction rates but did not affect adrenergic regulation of voltage-gated calcium currents. These results suggest that the ion channel-receptor complex is a critical mechanism in ion channel regulation.

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

    PubMed

    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.

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

    PubMed Central

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

    2009-01-01

    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. PMID:19646530

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

    PubMed Central

    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

    2009-01-01

    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-κB 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-κB, 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. PMID:19721007

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

    PubMed

    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.

  8. Up-regulation of glycolytic metabolism is required for HIF1α-driven bone formation.

    PubMed

    Regan, Jenna N; Lim, Joohyun; Shi, Yu; Joeng, Kyu Sang; Arbeit, Jeffrey M; Shohet, Ralph V; Long, Fanxin

    2014-06-10

    The bone marrow environment is among the most hypoxic in the body, but how hypoxia affects bone formation is not known. Because low oxygen tension stabilizes hypoxia-inducible factor alpha (HIFα) proteins, we have investigated the effect of expressing a stabilized form of HIF1α in osteoblast precursors. Brief stabilization of HIF1α in SP7-positive cells in postnatal mice dramatically stimulated cancellous bone formation via marked expansion of the osteoblast population. Remarkably, concomitant deletion of vascular endothelial growth factor A (VEGFA) in the mouse did not diminish bone accrual caused by HIF1α stabilization. Thus, HIF1α-driven bone formation is independent of VEGFA up-regulation and increased angiogenesis. On the other hand, HIF1α stabilization stimulated glycolysis in bone through up-regulation of key glycolytic enzymes including pyruvate dehydrogenase kinase 1 (PDK1). Pharmacological inhibition of PDK1 completely reversed HIF1α-driven bone formation in vivo. Thus, HIF1α stimulates osteoblast formation through direct activation of glycolysis, and alterations in cellular metabolism may be a broadly applicable mechanism for regulating cell differentiation.

  9. DptR2, a DeoR-type auto-regulator, is required for daptomycin production in Streptomyces roseosporus.

    PubMed

    Wang, Feng; Ren, Ni-Ni; Luo, Shuai; Chen, Xiao-Xia; Mao, Xu-Ming; Li, Yong-Quan

    2014-07-10

    Daptomycin, a novel cyclic lipopeptide antibiotic against Gram-positive bacteria, is produced by Streptomyces roseosporus. Though its biosynthetic mechanism, structural shuffling and fermentation optimization have been extensively studied, little is understood about its production regulation at the transcriptional levels. Here we reported that dptR2, encoding a DeoR-type regulator located close to the daptomycin biosynthesis gene cluster in S. roseosporus SW0702, is required for daptomycin production, but not for the expression of daptomycin gene cluster, suggesting that DptR2 was not a pathway-specific regulator. Furthermore, EMSA and qRT-PCR analysis suggested that DptR2 was positively auto-regulated by binding to its own promoter. Meanwhile, the binding sites on the dptR2 promoter were determined by a DNase I footprinting assay, and the essentiality of the inverted complementary sequences in the protected region for DptR2 binding was assessed. Our results for the first time reported the regulation of daptomycin production at the transcriptional level in S. roseosporus.

  10. Pet-1 is required across different stages of life to regulate serotonergic function

    PubMed Central

    Liu, Chen; Maejima, Takashi; Wyler, Steven C.; Casadesus, Gemma; Herlitze, Stefan; Deneris, Evan S.

    2010-01-01

    Transcriptional cascades are required for specification of 5-HT neurons and 5-HT modulated behaviors. Expression of several cascade factors extends across lifespan suggesting their control of behavior may not be temporally restricted to programming normal numbers of 5-HT neurons. We applied new mouse conditional targeting approaches to investigate ongoing requirements for Pet-1, a cascade factor required for the initiation of 5-HT synthesis but whose expression persists into adulthood. We found that Pet-1 was required after 5-HT neuron generation, for multiple steps in 5-HT neuron maturation including axonal innervation to the somatosensory cortex, firing properties, and 5-HT1A and 5-HT1B autoreceptor expression. Targeting Pet-1 in adult 5-HT neurons showed that it was still needed to preserve normal anxiety-related behaviors through direct autoregulated control of serotonergic gene expression. These findings show that Pet-1 is required across lifespan and therefore behavioral pathogenesis can result from both developmental and adult-onset alterations in serotonergic transcription. PMID:20818386

  11. Identification of TyeA residues required to interact with YopN and to regulate Yop secretion.

    PubMed

    Joseph, Sabrina S; Plano, Gregory V

    2007-01-01

    The secretion of Yops via the Yersinia type III secretion system (T3SS) is controlled, in part, by a cytoplasmic YopN/TyeA complex. This complex is required to prevent Yop secretion in the presence of extracellular calcium and prior to contact between the bacterium and a eukaryotic cell. In this study we utilized site-directed mutagenesis to analyze the role of specific TyeA regions and residues in the regulation of Yop secretion. We identified two spatially distinct, surface-exposed regions of the TyeA molecule that were required to regulate Yop secretion. One region, identified by residues M51, F55 and P56, was required for TyeA to interact with YopN. A second region, identified by residues R19, W20 and D25 was not involved in the interaction of TyeA with YopN, but may be required for the YopN/TyeA complex to interact with the T3S apparatus in a manner that blocks Yop secretion.

  12. Requirements for Ion and Solute Transport, and pH Regulation During Enamel Maturation

    PubMed Central

    LACRUZ, RODRIGO S.; SMITH, CHARLES E.; MOFFATT, PIERRE; CHANG, EUGENE H.; BROMAGE, TIMOTHY G.; BRINGAS, PABLO; NANCI, ANTONIO; BANIWAL, SANJEEV K.; ZABNER, JOSEPH; WELSH, MICHAEL J.; KURTZ, IRA; PAINE, MICHAEL L.

    2012-01-01

    Transcellular bicarbonate transport is suspected to be an important pathway used by ameloblasts to regulate extracellular pH and support crystal growth during enamel maturation. Proteins that play a role in amelogenesis include members of the ABC transporters (SLC gene family and CFTR). A number of carbonic anhydrases (CAs) have also been identified. The defined functions of these genes are likely interlinked during enamel mineralization. The purpose of this study is to quantify relative mRNA levels of individual SLC, Cftr, and CAs in enamel cells obtained from secretory and maturation stages on rat incisors. We also present novel data on the enamel phenotypes for two animal models, amutant porcine(CFTR-ΔF508) and the NBCe1-null mouse.Our data show that two SLCs(AE2 and NBCe1),Cftr,and Car2, Car3,Car6,and Car12 are all significantly up-regulated at the onset of the maturation stage of amelogenesis when compared to the secretory stage. The remaining SLCs and CA gene transcripts showed negligible expression or no significant change in expression from secretory to maturation stages. The enamel of Cftr-ΔF508 adult pigs was hypomineralized and showed abnormal crystal growth. NBCe1-null mice enamel was structurally defective and had a marked decrease in mineral content relative to wild-type. These data demonstrate the importance of many non-matrix proteins to amelogenesis and that the expression levels of multiple genes regulating extracellular pH are modulated during enamel maturation in response to an increased need for pH buffering during hydroxyapatite crystal growth. PMID:21732355

  13. Requirements for cell rounding and surface protein down-regulation by Ebola virus glycoprotein.

    PubMed

    Francica, Joseph R; Matukonis, Meghan K; Bates, Paul

    2009-01-20

    Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of beta1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects.

  14. Cell cycle regulation of a mouse histone H4 gene requires the H4 promoter.

    PubMed Central

    Seiler-Tuyns, A; Paterson, B M

    1987-01-01

    The mouse histone H4 gene, when stably transformed into L cells on the PSV2gpt shuttle vector, is cell cycle regulated in parallel with the endogenous H4 genes. This was determined in exponentially growing pools of transformants fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cell cycle without the use of treatments that arrest growth. Linker additions in the 5' noncoding region of the H4 RNA or in the coding region of the gene did not affect the cell cycle-regulated expression of the modified H4 gene even though the overall level of expression was altered. However, replacing the H4 promoter with the human alpha-2 globin promoter, so that the histone transcript produced by the chimeric gene remains essentially unchanged, resulted in the constitutive expression of H4 mRNA during all phases of the cell cycle with no net increase in H4 mRNA levels during the G1-to-S transition. From these results we conclude that all the information necessary for the cell cycle-regulated expression of the H4 gene is contained in the 5.2-kilobase subclone used in these studies with 228 nucleotides of 5'-flanking DNA and that the increase in H4 mRNA during the G1-to-S transition in the cell cycle is mediated by the H4 promoter and not by the increased stability of the H4 RNA. Images PMID:3561406

  15. Phosphorylation sites required for regulation of cardiac calcium channels in the fight-or-flight response.

    PubMed

    Fu, Ying; Westenbroek, Ruth E; Scheuer, Todd; Catterall, William A

    2013-11-26

    L-type Ca(2+) currents conducted by CaV1.2 channels initiate excitation-contraction coupling in the heart. Their activity is increased by β-adrenergic/cAMP signaling via phosphorylation by PKA in the fight-or-flight response, but the sites of regulation are unknown. We describe the functional role of phosphorylation of Ser1700 and Thr1704-sites of phosphorylation by PKA and casein kinase II at the interface between the proximal and distal C-terminal regulatory domains. Mutation of both residues to Ala in STAA mice reduced basal L-type Ca(2+) currents, due to a small decrease in expression and a substantial decrease in functional activity. The increase in L-type Ca(2+) current caused by isoproterenol was markedly reduced at physiological levels of stimulation (3-10 nM). Maximal increases in calcium current at nearly saturating concentrations of isoproterenol (100 nM) were also significantly reduced, but the mutation effects were smaller, suggesting that alternative regulatory mechanisms are engaged at maximal levels of stimulation. The β-adrenergic increase in cell contraction was also diminished. STAA ventricular myocytes exhibited arrhythmic contractions in response to isoproterenol, and up to 20% of STAA cells failed to sustain contractions when stimulated at 1 Hz. STAA mice have reduced exercise capacity, and cardiac hypertrophy is evident at 3 mo. We conclude that phosphorylation of Ser1700 and Thr1704 is essential for regulation of basal activity of CaV1.2 channels and for up-regulation by β-adrenergic signaling at physiological levels of stimulation. Disruption of phosphorylation at those sites leads to impaired cardiac function in vivo, as indicated by reduced exercise capacity and cardiac hypertrophy.

  16. Regulation of Innate Immune Responses is Required for S. mansoni Development

    DTIC Science & Technology

    2013-01-07

    71 Figure 8. Chronic stimulation of TLR2 with bacterial Pam3CSK4 facilitates S. mansoni development in RAG-/- mice and results in regulation of pro...prepatent infection…… ……………………………………………..…..138 Figure 18. Chronic stimulation of TLR4 with bacterial LPS facilitates S. mansoni Development in γc...shift in regards to schistosome vaccines, from the idea of achieving sterile immunity as is generally the goal of bacterial and viral vaccine

  17. Questions and Answers on the Requirements of Operating Permits Program Regulations

    EPA Pesticide Factsheets

    This document may be of assistance in applying the Title V air operating permit regulations. This document is part of the Title V Policy and Guidance Database available at www2.epa.gov/title-v-operating-permits/title-v-operating-permit-policy-and-guidance-document-index. 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.

  18. Leptin is required for hypothalamic regulation of miRNAs targeting POMC 3'UTR.

    PubMed

    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.

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

    PubMed Central

    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

  20. Quorum Sensing Regulators Are Required for Metabolic Fitness in Vibrio parahaemolyticus.

    PubMed

    Kalburge, Sai Siddarth; Carpenter, Megan R; Rozovsky, Sharon; Boyd, E Fidelma

    2017-03-01

    Quorum sensing (QS) is a process by which bacteria alter gene expression in response to cell density changes. In Vibrio species, at low cell density, the sigma 54-dependent response regulator LuxO is active and regulates the two QS master regulators AphA, which is induced, and OpaR, which is repressed. At high cell density the opposite occurs: LuxO is inactive, and therefore OpaR is induced while AphA is repressed. In Vibrio parahaemolyticus, a significant enteric pathogen of humans, the roles of these regulators in pathogenesis are less known. We examined deletion mutants of luxO, opaR, and aphA for in vivo fitness using an adult mouse model. We found that the luxO and aphA mutants were defective in colonization compared to levels in the wild type. The opaR mutant did not show any defect in vivo Colonization was restored to wild-type levels in a luxO opaR double mutant and was also increased in an opaR aphA double mutant. These data suggest that AphA is important and that overexpression of opaR is detrimental to in vivo fitness. Transcriptome sequencing (RNA-Seq) analysis of the wild type and luxO mutant grown in mouse intestinal mucus showed that 60% of the genes that were downregulated in the luxO mutant were involved in amino acid and sugar transport and metabolism. These data suggest that the luxO mutant has a metabolic disadvantage, which was confirmed by growth pattern analysis using phenotype microarrays. Bioinformatics analysis revealed OpaR binding sites in the regulatory region of 55 carbon transporter and metabolism genes. Biochemical analysis of five representatives of these regulatory regions demonstrated direct binding of OpaR in all five tested. These data demonstrate the role of OpaR in carbon utilization and metabolic fitness, an overlooked role in the QS regulon.

  1. FOXO1 is Required for Binding of PR on IRF4, Novel Transcriptional Regulator of Endometrial Stromal Decidualization

    PubMed Central

    Vasquez, Yasmin M.; Mazur, Erik C.; Li, Xilong; Kommagani, Ramakrishna; Jiang, Lichun; Chen, Rui; Lanz, Rainer B.; Kovanci, Ertug; Gibbons, William E.

    2015-01-01

    The forkhead box O1A (FOXO1) is an early-induced target of the protein kinase A pathway during the decidualization of human endometrial stromal cells (HESCs). In this study we identified the cistrome and transcriptome of FOXO1 and its role as a transcriptional regulator of the progesterone receptor (PR). Direct targets of FOXO1 were identified by integrating RNA sequencing with chromatin immunoprecipitation followed by deep sequencing. Gene ontology analysis demonstrated that FOXO1 regulates a subset of genes in decidualization such as those involved in cancer, p53 signaling, focal adhesions, and Wnt signaling. An overlap of the FOXO1 and PR chromatin immunoprecipitation followed by deep sequencing intervals revealed the co-occupancy of FOXO1 in more than 75% of PR binding intervals. Among these intervals were highly enriched motifs for the interferon regulatory factor member 4 (IRF4). IRF4 was determined to be a genomic target of both FOXO1 and PR and also to be differentially regulated in HESCs treated with small interfering RNA targeting FOXO1 or PR prior to decidualization stimulus. Ablation of FOXO1 was found to abolish binding of PR to the shared binding interval downstream of the IRF4 gene. Finally, small interfering RNA-mediated ablation of IRF4 was shown to compromise morphological transformation of decidualized HESCs and to attenuate the expression of the decidual markers IGFBP1, PRL, and WNT4. These results provide the first evidence that FOXO1 is functionally required for the binding of PR to genomic targets. Most notably, FOXO1 and PR are required for the regulation of IRF4, a novel transcriptional regulator of decidualization in HESCs. PMID:25584414

  2. 75 FR 12518 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 237...

    Federal Register 2010, 2011, 2012, 2013, 2014

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  3. 77 FR 16196 - Delaying the Compliance Date for Certain Requirements of the Regulations Implementing Titles II...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-20

    ... for existing swimming pools and spas. Concurrently with the publication of this Notice of Proposed... existing swimming pools, wading pools, and spas to May 21, 2012 in order to allow additional time to... proposed suspension of the applicability of the requirements in the 2010 Standards to swimming pools and...

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    ... collection requirements addressed in this notice are available on the World Wide Web at: http://www.acq.osd..., buildings, equipment). DoD needs this information to develop appropriate profit objectives when negotiating... carries the facilities ] capital employed amount to DD Form 1547 to develop a profit objective. When the...

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    2010-08-27

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    Federal Register 2010, 2011, 2012, 2013, 2014

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    Federal Register 2010, 2011, 2012, 2013, 2014

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    Federal Register 2010, 2011, 2012, 2013, 2014

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    ..., DC 20417. ATTN: Hada Flowers/IC 9000-0061, Transportation Requirements. Instructions: Please submit... FAR Part 47 contains policies and procedures for applying transportation and traffic management considerations in the acquisition of supplies. The FAR part also contains policies and procedures when acquiring...

  13. 78 FR 20624 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-05

    ... of material containing RFID tag data. DoD receiving personnel use the advance shipment notice to associate the unique identification encoded on the RFID tag with the corresponding shipment. Use of the RFID... ensure that the data on each passive RFID tag are unique and conforms to the requirements that they...

  14. 78 FR 69267 - Defense Federal Acquisition Regulation Supplement: Requirements Relating to Supply Chain Risk...

    Federal Register 2010, 2011, 2012, 2013, 2014

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    ... 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...

  15. Beyond Transcription Factors: The Role of Chromatin Modifying Enzymes in Regulating Transcription Required for Memory

    ERIC Educational Resources Information Center

    Barrett, Ruth M.; Wood, Marcelo A.

    2008-01-01

    One of the alluring aspects of examining chromatin modifications in the role of modulating transcription required for long-term memory processes is that these modifications may provide transient and potentially stable epigenetic marks in the service of activating and/or maintaining transcriptional processes. These, in turn, may ultimately…

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    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

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    2013-11-22

    ..., Department of Defense (DoD). ACTION: Notice and request for comments regarding a proposed extension of an... Paperwork Reduction Act of 1995 (44 U.S.C. chapter 35), DoD announces the proposed extension of a public information collection requirement and seeks public comment on the provisions thereof. DoD invites comments...

  18. 49 CFR 192.13 - What general requirements apply to pipelines regulated under this part?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

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    Code of Federal Regulations, 2011 CFR

    2011-10-01

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    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 3 2012-10-01 2012-10-01 false What general requirements apply to pipelines... (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) PIPELINE SAFETY TRANSPORTATION OF NATURAL AND OTHER GAS BY PIPELINE: MINIMUM FEDERAL SAFETY...

  1. 2 CFR 25.200 - Requirements for program announcements, regulations, and application instructions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 2 Grants and Agreements 1 2012-01-01 2012-01-01 false Requirements for program announcements...) Is issued on or after the effective date of this part; or (2) Has application or plan due dates after... an application or plan; (2) Maintain an active CCR registration with current information at all times...

  2. 2 CFR 25.200 - Requirements for program announcements, regulations, and application instructions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 2 Grants and Agreements 1 2011-01-01 2011-01-01 false Requirements for program announcements...) Is issued on or after the effective date of this part; or (2) Has application or plan due dates after... an application or plan; (2) Maintain an active CCR registration with current information at all times...

  3. 2 CFR 25.200 - Requirements for program announcements, regulations, and application instructions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 2 Grants and Agreements 1 2013-01-01 2013-01-01 false Requirements for program announcements...) Is issued on or after the effective date of this part; or (2) Has application or plan due dates after... an application or plan; (2) Maintain an active CCR registration with current information at all times...

  4. 2 CFR 25.200 - Requirements for program announcements, regulations, and application instructions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 2 Grants and Agreements 1 2014-01-01 2014-01-01 false Requirements for program announcements... on or after the effective date of this part; or (2) Has application or plan due dates after October 1... application or plan; (2) Maintain an active CCR registration with current information at all times during...

  5. 76 FR 53886 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Special...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-30

    ... and quantity data on any Government orders for the replenishment part issued within the most recent 12... review in connection with the contract. Paragraph (d) of 217.7505, Acquisition of Replenishment Parts... replenishment parts, a provision requiring that the offeror supply with its proposal, price and quantity data...

  6. Beyond Transcription Factors: The Role of Chromatin Modifying Enzymes in Regulating Transcription Required for Memory

    ERIC Educational Resources Information Center

    Barrett, Ruth M.; Wood, Marcelo A.

    2008-01-01

    One of the alluring aspects of examining chromatin modifications in the role of modulating transcription required for long-term memory processes is that these modifications may provide transient and potentially stable epigenetic marks in the service of activating and/or maintaining transcriptional processes. These, in turn, may ultimately…

  7. 75 FR 34277 - Federal Acquisition Regulation; FAR Case 2008-007, Additional Requirements for Market Research

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-16

    ... the simplified acquisition threshold. In addition, a prime contractor with a contract in excess of $5... officers and affected prime contractors the relevant regulatory guidance needed when addressing the...). * * * * * (d) See 44.402(a)(2) and 52.244-6 (Alternate I) for the requirement for a prime contractor to perform...

  8. Possible monitoring requirements for the disinfectants and disinfection by-products (D/DBP) regulations

    SciTech Connect

    Not Available

    1993-01-01

    The monitoring requirements presented in the report were developed by EPA before a negotiated Disinfectants and Disinfection By-Products (D/DBP) rule was considered. The framework described herein may be substantially changed as a result of the negotiated rulemaking process. The document is useful to consider in developing various monitoring options during the negotiated rulemaking process.

  9. The Centrosomal Protein C-Nap1 Is Required for Cell Cycle–Regulated Centrosome Cohesion

    PubMed Central

    Mayor, Thibault; Stierhof, York-Dieter; Tanaka, Kayoko; Fry, Andrew M.; Nigg, Erich A.

    2000-01-01

    Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle–regulated manner. To test this model, we have performed a detailed structure–function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle–regulated structure that mediates centriole–centriole cohesion. PMID:11076968

  10. The SUMO pathway is developmentally regulated and required for programmed DNA elimination in Paramecium tetraurelia.

    PubMed

    Matsuda, Atsushi; Forney, James D

    2006-05-01

    Extensive genome-wide remodeling occurs during the formation of the somatic macronuclei from the germ line micronuclei in ciliated protozoa. This process is limited to sexual reproduction and includes DNA amplification, chromosome fragmentation, and the elimination of internal segments of DNA. Our efforts to define the pathways regulating these events revealed a gene encoding a homologue of ubiquitin activating enzyme 2 (UBA2) that is upregulated at the onset of macronuclear development in Paramecium tetraurelia. Uba2 enzymes are known to activate the protein called small ubiquitin-related modifier (SUMO) that is covalently attached to target proteins. Consistent with this relationship, Northern analysis showed increased abundance of SUMO transcripts during sexual reproduction in Paramecium. RNA interference (RNAi) against UBA2 or SUMO during vegetative growth had little effect on cell survival or fission rates. In contrast, RNAi of mating cells resulted in failure to form a functional macronucleus. Despite normal amplification of the genome, excision of internal eliminated sequences was completely blocked. Additional experiments showed that the homologous UBA2 and SUMO genes in Tetrahymena thermophila are also upregulated during conjugation. These results provide evidence for the developmental regulation of the SUMO pathway in ciliates and suggest a key role for the pathway in controlling genome remodeling.

  11. The SUMO Pathway Is Developmentally Regulated and Required for Programmed DNA Elimination in Paramecium tetraurelia† ‡

    PubMed Central

    Matsuda, Atsushi; Forney, James D.

    2006-01-01

    Extensive genome-wide remodeling occurs during the formation of the somatic macronuclei from the germ line micronuclei in ciliated protozoa. This process is limited to sexual reproduction and includes DNA amplification, chromosome fragmentation, and the elimination of internal segments of DNA. Our efforts to define the pathways regulating these events revealed a gene encoding a homologue of ubiquitin activating enzyme 2 (UBA2) that is upregulated at the onset of macronuclear development in Paramecium tetraurelia. Uba2 enzymes are known to activate the protein called small ubiquitin-related modifier (SUMO) that is covalently attached to target proteins. Consistent with this relationship, Northern analysis showed increased abundance of SUMO transcripts during sexual reproduction in Paramecium. RNA interference (RNAi) against UBA2 or SUMO during vegetative growth had little effect on cell survival or fission rates. In contrast, RNAi of mating cells resulted in failure to form a functional macronucleus. Despite normal amplification of the genome, excision of internal eliminated sequences was completely blocked. Additional experiments showed that the homologous UBA2 and SUMO genes in Tetrahymena thermophila are also upregulated during conjugation. These results provide evidence for the developmental regulation of the SUMO pathway in ciliates and suggest a key role for the pathway in controlling genome remodeling. PMID:16682458

  12. The splicing regulator PTBP2 controls a program of embryonic splicing required for neuronal maturation

    PubMed Central

    Li, Qin; Zheng, Sika; Han, Areum; Lin, Chia-Ho; Stoilov, Peter; Fu, Xiang-Dong; Black, Douglas L

    2014-01-01

    We show that the splicing regulator PTBP2 controls a genetic program essential for neuronal maturation. Depletion of PTBP2 in developing mouse cortex leads to degeneration of these tissues over the first three postnatal weeks, a time when the normal cortex expands and develops mature circuits. Cultured Ptbp2−/− neurons exhibit the same initial viability as wild type, with proper neurite outgrowth and marker expression. However, these mutant cells subsequently fail to mature and die after a week in culture. Transcriptome-wide analyses identify many exons that share a pattern of mis-regulation in the mutant brains, where isoforms normally found in adults are precociously expressed in the developing embryo. These transcripts encode proteins affecting neurite growth, pre- and post-synaptic assembly, and synaptic transmission. Our results define a new genetic regulatory program, where PTBP2 acts to temporarily repress expression of adult protein isoforms until the final maturation of the neuron. DOI: http://dx.doi.org/10.7554/eLife.01201.001 PMID:24448406

  13. The Ustilago maydis Nit2 Homolog Regulates Nitrogen Utilization and Is Required for Efficient Induction of Filamentous Growth

    PubMed Central

    Horst, Robin J.; Zeh, Christine; Saur, Alexandra; Sonnewald, Sophia; Sonnewald, Uwe

    2012-01-01

    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. PMID:22247264

  14. Regulation of asymmetric cell division and polarity by Scribble is not required for humoral immunity.

    PubMed

    Hawkins, Edwin D; Oliaro, Jane; Kallies, Axel; Belz, Gabrielle T; Filby, Andrew; Hogan, Thea; Haynes, Nicole; Ramsbottom, Kelly M; Van Ham, Vanessa; Kinwell, Tanja; Seddon, Benedict; Davies, Derek; Tarlinton, David; Lew, Andrew M; Humbert, Patrick O; Russell, Sarah M

    2013-01-01

    The production of protective antibody requires effective signalling of naive B cells following encounter with antigen, and the divergence of responding B lymphocytes into distinct lineages. Polarity proteins have recently been proposed as important mediators of both the initial B cell response, and potentially of asymmetric cell division. Here we show that, although polarity proteins of the Scribble complex, Scribble, Dlg1 and Lgl1, are expressed and polarized during early B cell activation, their deficiency has no effect on the in vivo outcome of immunization or challenge with influenza infection. Furthermore, we find a striking correlation in the differentiation outcome of daughters of single founder B cells in vitro. Taken together, our results indicate that B cell differentiation does not require polarity proteins of the Scribble complex, and the findings do not support a role for asymmetric cell division in B cell activation and differentiation.

  15. 75 FR 3617 - Outer Continental Shelf Air Regulations Update To Include New Jersey State Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-22

    ..., Reporting and recordkeeping requirements, Sulfur oxides. Dated: December 30, 2009. Judith A. Enck, Regional.... Variances N.J.A.C. 7:27-6.7. Exceptions Chapter 27 Subchapter 7--Sulfur (Effective 3/1/67) N.J.A.C. 7:27-7.1. Definitions N.J.A.C. 7:27-7.2. Control and prohibition of air pollution from sulfur compounds Chapter 27...

  16. The RNA Export Factor, Nxt1, Is Required for Tissue Specific Transcriptional Regulation

    PubMed Central

    Jiang, Jianqiao; White-Cooper, Helen

    2013-01-01

    The highly conserved, Nxf/Nxt (TAP/p15) RNA nuclear export pathway is important for export of most mRNAs from the nucleus, by interacting with mRNAs and promoting their passage through nuclear pores. Nxt1 is essential for viability; using a partial loss of function allele, we reveal a role for this gene in tissue specific transcription. We show that many Drosophila melanogaster testis-specific mRNAs require Nxt1 for their accumulation. The transcripts that require Nxt1 also depend on a testis-specific transcription complex, tMAC. We show that loss of Nxt1 leads to reduced transcription of tMAC targets. A reporter transcript from a tMAC-dependent promoter is under-expressed in Nxt1 mutants, however the same transcript accumulates in mutants if driven by a tMAC-independent promoter. Thus, in Drosophila primary spermatocytes, the transcription factor used to activate expression of a transcript, rather than the RNA sequence itself or the core transcription machinery, determines whether this expression requires Nxt1. We additionally find that transcripts from intron-less genes are more sensitive to loss of Nxt1 function than those from intron-containing genes and propose a mechanism in which transcript processing feeds back to increase activity of a tissue specific transcription complex. PMID:23754955

  17. RhoE is required for contact inhibition and negatively regulates tumor initiation and progression.

    PubMed

    Hernández-Sánchez, Marta; Poch, Enric; Guasch, Rosa M; Ortega, Joaquín; López-Almela, Inmaculada; Palmero, Ignacio; Pérez-Roger, Ignacio

    2015-07-10

    RhoE is a small GTPase involved in the regulation of actin cytoskeleton dynamics, cell cycle and apoptosis. The role of RhoE in cancer is currently controversial, with reports of both oncogenic and tumor-suppressive functions for RhoE. Using RhoE-deficient mice, we show here that the absence of RhoE blunts contact-inhibition of growth by inhibiting p27Kip1 nuclear translocation and cooperates in oncogenic transformation of mouse primary fibroblasts. Heterozygous RhoE+/gt mice are more susceptible to chemically induced skin tumors and RhoE knock-down results in increased metastatic potential of cancer cells. These results indicate that RhoE plays a role in suppressing tumor initiation and progression.

  18. FDA regulation of dietary supplements and requirements regarding adverse event reporting.

    PubMed

    Frankos, V H; Street, D A; O'Neill, R K

    2010-02-01

    In 1994, the Dietary Supplement Health and Education Act (DSHEA) amended the Federal Food, Drug, and Cosmetic Act (FDC Act) to set up a distinct regulatory framework for what we now call dietary supplements. The DSHEA was passed with the intent of striking a balance between providing consumers access to safe dietary supplements to help maintain or improve their health and giving the US Food and Drug Administration (FDA) authority to regulate and take action against manufacturers of supplements or supplement ingredients that present safety problems, are presented with false or misleading claims, or are adulterated or misbranded. This article will present FDA's recent experience in collecting and evaluating dietary supplement adverse event data for the purpose of assuring the public that the dietary supplements they purchase are safe.

  19. RhoE is required for contact inhibition and negatively regulates tumor initiation and progression

    PubMed Central

    Hernández-Sánchez, Marta; Poch, Enric; Guasch, Rosa M.; Ortega, Joaquín; López-Almela, Inmaculada; Palmero, Ignacio; Pérez-Roger, Ignacio

    2015-01-01

    RhoE is a small GTPase involved in the regulation of actin cytoskeleton dynamics, cell cycle and apoptosis. The role of RhoE in cancer is currently controversial, with reports of both oncogenic and tumor-suppressive functions for RhoE. Using RhoE-deficient mice, we show here that the absence of RhoE blunts contact-inhibition of growth by inhibiting p27Kip1 nuclear translocation and cooperates in oncogenic transformation of mouse primary fibroblasts. Heterozygous RhoE+/gt mice are more susceptible to chemically induced skin tumors and RhoE knock-down results in increased metastatic potential of cancer cells. These results indicate that RhoE plays a role in suppressing tumor initiation and progression. PMID:26036260

  20. Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi.

    PubMed

    Zuber, Johannes; McJunkin, Katherine; Fellmann, Christof; Dow, Lukas E; Taylor, Meredith J; Hannon, Gregory J; Lowe, Scott W

    2011-01-01

    Short hairpin RNAs (shRNAs) are versatile tools for analyzing loss-of-function phenotypes in vitro and in vivo. However, their use for studying genes involved in proliferation and survival, which are potential therapeutic targets in cancer and other diseases, is confounded by the strong selective advantage of cells in which shRNA expression is inefficient. We therefore developed a toolkit that combines Tet-regulated miR30-shRNA technology, robust transactivator expression and two fluorescent reporters to track and isolate cells with potent target knockdown. We demonstrated that this system improves the study of essential genes and was sufficiently robust to eradicate aggressive cancer in mice by suppressing a single gene. Further, we applied this system for in vivo negative-selection screening with pooled shRNAs and propose a streamlined, inexpensive workflow that will facilitate the use of RNA interference (RNAi) for the identification and evaluation of essential therapeutic targets.

  1. SUMOylation Is Developmentally Regulated and Required for Cell Pairing during Conjugation in Tetrahymena thermophila

    PubMed Central

    Nasir, Amjad M.; Yang, Qianyi

    2014-01-01

    The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins regulates numerous nuclear events in eukaryotes, including transcription, mitosis and meiosis, and DNA repair. Despite extensive interest in nuclear pathways within the field of ciliate molecular biology, there have been no investigations of the SUMO pathway in Tetrahymena. The developmental program of sexual reproduction of this organism includes cell pairing, micronuclear meiosis, and the formation of a new somatic macronucleus. We identified the Tetrahymena thermophila SMT3 (SUMO) and UBA2 (SUMO-activating enzyme) genes and demonstrated that the corresponding green fluorescent protein (GFP) tagged gene products are found predominantly in the somatic macronucleus during vegetative growth. Use of an anti-Smt3p antibody to perform immunoblot assays with whole-cell lysates during conjugation revealed a large increase in SUMOylation that peaked during formation of the new macronucleus. Immunofluorescence using the same antibody showed that the increase was localized primarily within the new macronucleus. To initiate functional analysis of the SUMO pathway, we created germ line knockout cell lines for both the SMT3 and UBA2 genes and found both are essential for cell viability. Conditional Smt3p and Uba2p cell lines were constructed by incorporation of the cadmium-inducible metallothionein promoter. Withdrawal of cadmium resulted in reduced cell growth and increased sensitivity to DNA-damaging agents. Interestingly, Smt3p and Uba2p conditional cell lines were unable to pair during sexual reproduction in the absence of cadmium, consistent with a function early in conjugation. Our studies are consistent with multiple roles for SUMOylation in Tetrahymena, including a dynamic regulation associated with the sexual life cycle. PMID:25527524

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

    PubMed

    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.

  3. The Endoplasmic Reticulum Resident Protein AGR3. Required for Regulation of Ciliary Beat Frequency in the Airway.

    PubMed

    Bonser, Luke R; Schroeder, Bradley W; Ostrin, Lisa A; Baumlin, Nathalie; Olson, Jean L; Salathe, Matthias; Erle, David J

    2015-10-01

    Protein disulfide isomerase (PDI) family members regulate protein folding and calcium homeostasis in the endoplasmic reticulum (ER). The PDI family member anterior gradient (AGR) 3 is expressed in the airway, but the localization, regulation, and function of AGR3 are poorly understood. Here we report that AGR3, unlike its closest homolog AGR2, is restricted to ciliated cells in the airway epithelium and is not induced by ER stress. Mice lacking AGR3 are viable and develop ciliated cells with normal-appearing cilia. However, ciliary beat frequency was lower in airways from AGR3-deficient mice compared with control mice (20% lower in the absence of stimulation and 35% lower after ATP stimulation). AGR3 deficiency had no detectable effects on ciliary beat frequency (CBF) when airways were perfused with a calcium-free solution, suggesting that AGR3 is required for calcium-mediated regulation of ciliary function. Decreased CBF was associated with impaired mucociliary clearance in AGR3-deficient airways. We conclude that AGR3 is a specialized member of the PDI family that plays an unexpected role in the regulation of CBF and mucociliary clearance in the airway.

  4. Noncoding 3' sequences of the transferrin receptor gene are required for mRNA regulation by iron.

    PubMed Central

    Owen, D; Kühn, L C

    1987-01-01

    The cell-surface receptor for transferrin mediates cellular uptake of iron from serum. Transferrin receptor protein and mRNA levels are increased in cells treated with iron chelating agents, and are decreased by treatment with iron salts or hemin. Here we report that expression of human transferrin receptor cDNA constructions in stably transfected mouse fibroblasts is regulated both by the iron chelator, desferrioxamine, and by hemin. We found that sequences within the 3' noncoding region are required for the iron-dependent feed-back regulation of receptor expression, whereas the presence of the transferrin receptor promoter region is not necessary. Regulation by iron is observed when transcription is initiated at either the SV-40 early promoter or the transferrin receptor promoter, but deletion of a 2.3 kb fragment within the 2.6 kb 3' noncoding region of the cDNA abolishes regulation and increases the constitutive level of receptor expression. Furthermore, the 3' deletion does not affect the decrease in receptors which is observed in response to growth arrest, indicating that transferrin receptor expression is controlled by at least two distinct mechanisms. Images Fig. 3. Fig. 6. Fig. 8. PMID:3608980

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

    PubMed

    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.

  6. A dynamic set point for thermal adaptation requires phospholipase C-mediated regulation of TRPM8 in vivo

    PubMed Central

    Brenner, Daniel S.; Golden, Judith P.; Vogt, Sherri K.; Dhaka, Ajay; Story, Gina M.; Gereau, Robert W.

    2014-01-01

    The ability to sense and respond to thermal stimuli at varied environmental temperatures is essential for survival in seasonal areas. In this study, we show that mice respond similarly to ramping changes in temperature from a wide range of baseline temperatures. Further investigation suggests that this ability to adapt to different ambient environments is based on rapid adjustments made to a dynamic temperature setpoint. The adjustment of this setpoint requires TRPM8 but not TRPA1 and is regulated by phospholipase C (PLC) activity. Overall, our findings suggest that temperature response thresholds in mice are dynamic, and that this ability to adapt to environmental temperature seems to mirror the in vitro findings that PLC-mediated hydrolysis of phosphoinositol 4,5-bisphosphate modulates TRPM8 activity and thereby regulates the response thresholds to cold stimuli. PMID:25109670

  7. Tectonic, a novel regulator of the Hedgehog pathway required for both activation and inhibition

    PubMed Central

    Reiter, Jeremy F.; Skarnes, William C.

    2006-01-01

    We report the identification of a novel protein that participates in Hedgehog-mediated patterning of the neural tube. This protein, named Tectonic, is the founding member of a previously undescribed family of evolutionarily conserved secreted and transmembrane proteins. During neural tube development, mouse Tectonic is required for formation of the most ventral cell types and for full Hedgehog (Hh) pathway activation. Epistasis analyses reveal that Tectonic modulates Hh signal transduction downstream of Smoothened (Smo) and Rab23. Interestingly, characterization of Tectonic Shh and Tectonic Smo double mutants indicates that Tectonic plays an additional role in repressing Hh pathway activity. PMID:16357211

  8. Tectonic, a novel regulator of the Hedgehog pathway required for both activation and inhibition.

    PubMed

    Reiter, Jeremy F; Skarnes, William C

    2006-01-01

    We report the identification of a novel protein that participates in Hedgehog-mediated patterning of the neural tube. This protein, named Tectonic, is the founding member of a previously undescribed family of evolutionarily conserved secreted and transmembrane proteins. During neural tube development, mouse Tectonic is required for formation of the most ventral cell types and for full Hedgehog (Hh) pathway activation. Epistasis analyses reveal that Tectonic modulates Hh signal transduction downstream of Smoothened (Smo) and Rab23. Interestingly, characterization of Tectonic Shh and Tectonic Smo double mutants indicates that Tectonic plays an additional role in repressing Hh pathway activity.

  9. Financial Management Regulation. Volume 1. General Financial Management Information, Systems, and Requirements

    DTIC Science & Technology

    1993-05-01

    INFORMATION, SYSTEMS, AND REQUIREMENTS MAY 1993 D117C QUALITy IISPECTED 3 NTIS CRA&I L)IW TAB SU : ,flro•!n c.:d 1) c’ bu To f HA,(dIL,)!l’+Y Codes Avail jrVlh...Payment Policy and Procedures 2. Budget Formulation and Presentation 11. Reimbursable Operations, Policy and Procedures 3 . Budget Execution - Availability...1 0102 AUTi IORITIES AND FUNCTIONS 1 0103 CHIEF FINANCIAL OFFICERS COUNCIL 3 0104 DEPUTY CHIEF FINANCIAL OFFICER OF THE DEPARTMENT OF DEFENSE 3 0105

  10. Requirement for MLL3 in p53 regulation of hepatic expression of small heterodimer partner and bile acid homeostasis.

    PubMed

    Kim, Dae-Hwan; Kim, Juhee; Lee, Jae W

    2011-12-01

    The histone H3-lysine-4 methyltransferase mixed-lineage leukemia 3 (MLL3) belongs to a large complex that functions as a coactivator of multiple transcription factors, including the bile acid (BA)-activated nuclear receptor, farnesoid X receptor (FXR), a critical player in BA homeostasis. BA-activated FXR induces hepatic expression of small heterodimer partner (SHP), which in turn suppresses expression of BA synthesis genes, Cyp7a1 and Cyp8b1. Thus, MLL3(Δ/Δ) mice that express a catalytically inactive mutant form of MLL3 display increased BA levels. Recently, we have discovered a distinct regulatory pathway for BA homeostasis, in which p53 independently up-regulates SHP expression in the liver. Here, we show that the MLL3 complex is also essential for p53 transactivation of SHP. Although activated p53 signaling in MLL3(+/+) mice results in decreased BA levels through hepatic up-regulation of SHP, these changes are abolished in MLL3(Δ/Δ) mice. For both HepG2 cells and mouse liver, we also demonstrate that p53 directs the recruitment of different components of the MLL3 complex to the p53-response elements of SHP and that p53-dependent H3-lysine-4-trimethylation of SHP requires MLL3. From these results, we conclude that both FXR- and p53-dependent regulatory pathways for SHP expression in BA homeostasis require the MLL3 complex; thus, the MLL3 complex is likely a master regulator of BA homeostasis. Using a common coregulator complex for multiple transcription factors, which independently control expression of the same gene, might be a prevalent theme in gene regulation and may also play critical roles in assigning a specific biological function to a coregulator complex.

  11. Impacts & Compliance Implementation Plans & Required Deviations for Toxic Substance Control Act (TSCA) Regulation of Double Shell Tanks (DST)

    SciTech Connect

    MULKEY, C.H.

    2000-08-22

    In May 2000, the U.S. Department of Energy, Office of River Protection (DOE-ORP) and the U.S. Environmental Protection Agency (EPA) held meetings regarding the management of polychlorinated biphenyls (PCBs) in the Hanford tank waste. It was decided that the radioactive waste currently stored in the double-shell tanks (DSTs) contain waste which will become subject to the Toxic Substance Control Act (TSCA) (40 CFR 761). As a result, DOE-ORP directed the River Protection Project tank farm contractor (TFC) to prepare plans for managing the PCB inventory in the DSTs. Two components of the PCB management plans are this assessment of the operational impacts of TSCA regulation and the identifications of deviations from TSCA that are required to accommodate tank farm unique limitations. This plan provides ORP and CH2M HILL Hanford Group, Inc. (CHG) with an outline of TSCA PCB requirements and their applicability to tank farm activities, and recommends a compliance/implementation approach. Where strict compliance is not possible, the need for deviations from TSCA PCB requirements is identified. The purpose of assembling this information is to enhance the understanding of PCB management requirements, identify operational impacts and select impact mitigation strategies. This information should be useful in developing formal agreements with EPA where required.

  12. APSR1, a novel gene required for meristem maintenance, is negatively regulated by low phosphate availability.

    PubMed

    González-Mendoza, Víctor; Zurita-Silva, Andrés; Sánchez-Calderón, Lenin; Sánchez-Sandoval, María Eugenia; Oropeza-Aburto, Araceli; Gutiérrez-Alanís, Dolores; Alatorre-Cobos, Fulgencio; Herrera-Estrella, Luis

    2013-05-01

    Proper root growth is crucial for anchorage, exploration, and exploitation of the soil substrate. Root growth is highly sensitive to a variety of environmental cues, among them water and nutrient availability have a great impact on root development. Phosphorus (P) availability is one of the most limiting nutrients that affect plant growth and development under natural and agricultural environments. Root growth in the direction of the long axis proceeds from the root tip and requires the coordinated activities of cell proliferation, cell elongation and cell differentiation. Here we report a novel gene, APSR1 (Altered Phosphate Starvation Response1), involved in root meristem maintenance. The loss of function mutant apsr1-1 showed a reduction in primary root length and root apical meristem size, short differentiated epidermal cells and long root hairs. Expression of APSR1 gene decreases in response to phosphate starvation and apsr1-1 did not show the typical progressive decrease of undifferentiated cells at root tip when grown under P limiting conditions. Interestingly, APSR1 expression pattern overlaps with root zones of auxin accumulation. Furthermore, apsr1-1 showed a clear decrease in the level of the auxin transporter PIN7. These data suggest that APSR1 is required for the coordination of cell processes necessary for correct root growth in response to phosphate starvation conceivably by direct or indirect modulation of PIN7. We also propose, based on its nuclear localization and structure, that APSR1 may potentially be a member of a novel group of transcription factors.

  13. DNA Replication Is Required for Circadian Clock Function by Regulating Rhythmic Nucleosome Composition.

    PubMed

    Liu, Xiao; Dang, Yunkun; Matsu-Ura, Toru; He, Yubo; He, Qun; Hong, Christian I; Liu, Yi

    2017-07-20

    Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription. DNA replication is essential for the rhythmic changes of nucleosome composition at the frq promoter. The FACT complex, known to be involved in histone disassembly/reassembly, is required for clock function and is recruited to the frq promoter in a replication-dependent manner to promote replacement of histone H2A.Z by H2A. Finally, deletion of H2A.Z uncoupled the dependence of the circadian clock on DNA replication. Together, these results establish circadian clock and cell cycle as interdependent coupled oscillators and identify DNA replication as a critical process in the circadian mechanism. Published by Elsevier Inc.

  14. Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum

    PubMed Central

    Casey, Amanda K.; Chen, Shuliang; Novick, Peter; Ferro-Novick, Susan; Wente, Susan R.

    2015-01-01

    The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system and yet have distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles, including Rtn1, Yop1, Pom33, and Per33. Of interest, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1's role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions. PMID:26041935

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

    PubMed

    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.

  16. Vangl2-Regulated Polarisation of Second Heart Field-Derived Cells Is Required for Outflow Tract Lengthening during Cardiac Development

    PubMed Central

    Rhee, Hong Jun; Eley, Lorraine; Phillips, Helen M.; Rigby, Hannah F.; Dean, Charlotte; Chaudhry, Bill; Henderson, Deborah J.

    2014-01-01

    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. PMID:25521757

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

    PubMed

    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 (p<0.05; >10-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.

  18. The HIP1 binding site is required for growth regulation of the dihydrofolate reductase gene promoter.

    PubMed Central

    Means, A L; Slansky, J E; McMahon, S L; Knuth, M W; Farnham, P J

    1992-01-01

    The transcription rate of the dihydrofolate reductase (DHFR) gene increases at the G1/S boundary of the proliferative cell cycle. Through analysis of transiently and stably transfected NIH 3T3 cells, we have now demonstrated that DHFR promoter sequences extending from -270 to +20 are sufficient to confer similar regulation on a reporter gene. Mutation of a protein binding site that spans sequences from -16 to +11 in the DHFR promoter resulted in loss of the transcriptional increase at the G1/S boundary. Purification of an activity from HeLa nuclear extract that binds to this region enriched for a 180-kDa polypeptide (HIP1). Using this HIP1 preparation, we have identified specific positions within the binding site that are critical for efficient protein-DNA interactions. An analysis of association and dissociation rates suggests that bound HIP1 protein can exchange rapidly with free protein. This rapid exchange may facilitate the burst of transcriptional activity from the DHFR promoter at the G1/S boundary. Images PMID:1545788

  19. HIRA Is Required for Heart Development and Directly Regulates Tnni2 and Tnnt3

    PubMed Central

    Dilg, Daniel; Saleh, Rasha Noureldin M.; Phelps, Sarah Elizabeth Lee; Rose, Yoann; Dupays, Laurent; Murphy, Cian; Mohun, Timothy; Anderson, Robert H.; Scambler, Peter J.; Chapgier, Ariane L. A.

    2016-01-01

    Chromatin remodelling is essential for cardiac development. Interestingly, the role of histone chaperones has not been investigated in this regard. HIRA is a member of the HUCA (HIRA/UBN1/CABIN1/ASF1a) complex that deposits the variant histone H3.3 on chromatin independently of replication. Lack of HIRA has general effects on chromatin and gene expression dynamics in embryonic stem cells and mouse oocytes. Here we describe the conditional ablation of Hira in the cardiogenic mesoderm of mice. We observed surface oedema, ventricular and atrial septal defects and embryonic lethality. We identified dysregulation of a subset of cardiac genes, notably upregulation of troponins Tnni2 and Tnnt3, involved in cardiac contractility and decreased expression of Epha3, a gene necessary for the fusion of the muscular ventricular septum and the atrioventricular cushions. We found that HIRA binds GAGA rich DNA loci in the embryonic heart, and in particular a previously described enhancer of Tnni2/Tnnt3 (TTe) bound by the transcription factor NKX2.5. HIRA-dependent H3.3 enrichment was observed at the TTe in embryonic stem cells (ESC) differentiated toward cardiomyocytes in vitro. Thus, we show here that HIRA has locus-specific effects on gene expression and that histone chaperone activity is vital for normal heart development, impinging on pathways regulated by an established cardiac transcription factor. PMID:27518902

  20. Key events during the transition from rapid growth to quiescence in budding yeast require posttranscriptional regulators

    PubMed Central

    Li, Lihong; Miles, Shawna; Melville, Zephan; Prasad, Amalthiya; Bradley, Graham; Breeden, Linda L.

    2013-01-01

    Yeast that naturally exhaust the glucose from their environment differentiate into three distinct cell types distinguishable by flow cytometry. Among these is a quiescent (Q) population, which is so named because of its uniform but readily reversed G1 arrest, its fortified cell walls, heat tolerance, and longevity. Daughter cells predominate in Q-cell populations and are the longest lived. The events that differentiate Q cells from nonquiescent (nonQ) cells are initiated within hours of the diauxic shift, when cells have scavenged all the glucose from the media. These include highly asymmetric cell divisions, which give rise to very small daughter cells. These daughters modify their cell walls by Sed1- and Ecm33-dependent and dithiothreitol-sensitive mechanisms that enhance Q-cell thermotolerance. Ssd1 speeds Q-cell wall assembly and enables mother cells to enter this state. Ssd1 and the related mRNA-binding protein Mpt5 play critical overlapping roles in Q-cell formation and longevity. These proteins deliver mRNAs to P-bodies, and at least one P-body component, Lsm1, also plays a unique role in Q-cell longevity. Cells lacking Lsm1 and Ssd1 or Mpt5 lose viability under these conditions and fail to enter the quiescent state. We conclude that posttranscriptional regulation of mRNAs plays a crucial role in the transition in and out of quiescence. PMID:24088570

  1. Cas9-dependent endogenous gene regulation is required for bacterial virulence.

    PubMed

    Sampson, Timothy R; Weiss, David S

    2013-12-01

    CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems are known to mediate bacterial defence against foreign nucleic acids. We recently demonstrated a non-canonical role for a CRISPR-Cas system in controlling endogenous gene expression, which had not previously been appreciated. In the present article, we describe the studies that led to this discovery, beginning with an unbiased genome-wide screen to identify virulence genes in the intracellular pathogen Francisella novicida. A gene annotated as encoding a hypothetical protein, but which we now know encodes the Cas protein Cas9, was identified as one of the most critical to the ability of F. novicida to replicate and survive during murine infection. Subsequent studies revealed a role for this protein in evasion of the host innate immune response. Specifically, Cas9 represses the expression of a BLP (bacterial lipoprotein) that could otherwise be recognized by TLR2 (Toll-like receptor 2), a host protein involved in initiating an antibacterial pro-inflammatory response. By repressing BLP levels, Cas9 mediates evasion of TLR2, promoting bacterial virulence. Finally, we described the molecular mechanism by which Cas9 functions in complex with two small RNAs to target the mRNA encoding the BLP for degradation. This work greatly broadened the paradigm for CRISPR-Cas function, highlighting a role in gene regulation that could be conserved in numerous bacteria, and elucidating its integral contribution to bacterial pathogenesis.

  2. Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation

    PubMed Central

    Hodgson, Andrea; Wier, Eric M.; Wen, Matthew G.; Kamenyeva, Olena; Xia, Xue; Koo, Lily Y.

    2016-01-01

    The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production. PMID:27635653

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

    PubMed

    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.

  4. Angiopoietin receptor Tie2 is required for vein specification and maintenance via regulating COUP-TFII

    PubMed Central

    Chu, Man; Li, Taotao; Shen, Bin; Cao, Xudong; Zhong, Haoyu; Zhang, Luqing; Zhou, Fei; Ma, Wenjuan; Jiang, Haijuan; Xie, Pancheng; Liu, Zhengzheng; Dong, Ningzheng; Xu, Ying; Zhao, Yun; Xu, Guoqiang; Lu, Peirong; Luo, Jincai; Wu, Qingyu; Alitalo, Kari; Koh, Gou Young; Adams, Ralf H; He, Yulong

    2016-01-01

    Mechanisms underlying the vein development remain largely unknown. Tie2 signaling mediates endothelial cell (EC) survival and vascular maturation and its activating mutations are linked to venous malformations. Here we show that vein formation are disrupted in mouse skin and mesentery when Tie2 signals are diminished by targeted deletion of Tek either ubiquitously or specifically in embryonic ECs. Postnatal Tie2 attenuation resulted in the degeneration of newly formed veins followed by the formation of haemangioma-like vascular tufts in retina and venous tortuosity. Mechanistically, Tie2 insufficiency compromised venous EC identity, as indicated by a significant decrease of COUP-TFII protein level, a key regulator in venogenesis. Consistently, angiopoietin-1 stimulation increased COUP-TFII in cultured ECs, while Tie2 knockdown or blockade of Tie2 downstream PI3K/Akt pathway reduced COUP-TFII which could be reverted by the proteasome inhibition. Together, our results imply that Tie2 is essential for venous specification and maintenance via Akt mediated stabilization of COUP-TFII. DOI: http://dx.doi.org/10.7554/eLife.21032.001 PMID:28005008

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

    PubMed Central

    Thompson, Eloise; Breil, Florence; Lorthiois, Audrey; Dupuy, Florian; Cummings, Ross; Duffier, Yoann; Corbett, Yolanda; Mercereau-Puijalon, Odile; Vernick, Kenneth; Taramelli, Donatella; Baker, David A.; Langsley, Gordon; Lavazec, Catherine

    2015-01-01

    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. PMID:25951195

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

    PubMed Central

    Xu, Kewei; Harrison, Rene E.

    2015-01-01

    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. PMID:26082487

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

    PubMed

    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. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. Non-nuclear Pool of Splicing Factor SFPQ Regulates Axonal Transcripts Required for Normal Motor Development.

    PubMed

    Thomas-Jinu, Swapna; Gordon, Patricia M; Fielding, Triona; Taylor, Richard; Smith, Bradley N; Snowden, Victoria; Blanc, Eric; Vance, Caroline; Topp, Simon; Wong, Chun-Hao; Bielen, Holger; Williams, Katherine L; McCann, Emily P; Nicholson, Garth A; Pan-Vazquez, Alejandro; Fox, Archa H; Bond, Charles S; Talbot, William S; Blair, Ian P; Shaw, Christopher E; Houart, Corinne

    2017-04-04

    Recent progress revealed the complexity of RNA processing and its association to human disorders. Here, we unveil a new facet of this complexity. Complete loss of function of the ubiquitous splicing factor SFPQ affects zebrafish motoneuron differentiation cell autonomously. In addition to its nuclear localization, the protein unexpectedly localizes to motor axons. The cytosolic version of SFPQ abolishes motor axonal defects, rescuing key transcripts, and restores motility in the paralyzed sfpq null mutants, indicating a non-nuclear processing role in motor axons. Novel variants affecting the conserved coiled-coil domain, so far exclusively found in fALS exomes, specifically affect the ability of SFPQ to localize in axons. They broadly rescue morphology and motility in the zebrafish mutant, but alter motor axon morphology, demonstrating functional requirement for axonal SFPQ. Altogether, we uncover the axonal function of the splicing factor SFPQ in motor development and highlight the importance of the coiled-coil domain in this process.

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

    PubMed

    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.

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

    PubMed

    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.

  11. A requirement for protein phosphorylation in regulating the meiotic and mitotic cell cycles in echinoderms.

    PubMed

    Néant, I; Charbonneau, M; Guerrier, P

    1989-04-01

    Populations of hormone-stimulated starfish oocytes and fertilized sea urchin eggs undergo synchronous meiotic and mitotic divisions. We have studied the requirement for protein phosphorylation during these events by testing the effects of 6-dimethylaminopurine (6-DMAP) upon the incorporation of [32P]orthophosphate. It was found that 6-DMAP blocked meiosis reinitiation and early cleavage and simultaneously inhibited protein phosphorylation, without changing the rate of [35S]methionine incorporation or pattern of protein synthesis. The protein, cyclin (54 kDa in starfish and 57 kDa in sea urchin), continues to be synthesized in the presence of 6-DMAP. This protein is destroyed at first and second cell cycles when 6-DMAP is added 30 min following fertilization but not when this drug is present before fertilization. Thus, cyclin breakdown does not depend on the completion of the nuclear events of M-phase, and its time of breakdown is set at an early step between fertilization and first cleavage. Using tubulin immunostaining, we found that 6-DMAP did not affect the cortical microtubules and resting female centrioles of prophase-arrested starfish oocytes, whereas it induced a precocious disappearance of spindle fibers when applied to hormone-stimulated oocytes. While an early addition of 6-DMAP precluded nuclear breakdown and spindle formation in both systems, a late treatment always allowed chromosome separation and centriole separation. Under these conditions pericentriolar tubulin persisted and could organize new spindles after the inhibitor was removed. It is suggested that (1) the assembly of cortical and centriolar-associated microtubules is not controlled by the same factors as spindle-associated tubulin; (2) specific proteins which are required for the cell to enter the following M-phase can become operative only via a process depending upon protein phosphorylation; (3) microtubule-associated kinases may play an important role in MPF function and spindle dynamics.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26002901

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

    PubMed Central

    Mittra, Bidyottam; Laranjeira-Silva, Maria Fernanda; Perrone Bezerra de Menezes, Juliana; Jensen, Jennifer; Michailowsky, Vladimir; Andrews, Norma W.

    2016-01-01

    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. PMID:26741360

  14. Nuclear localization signal sequence is required for VACM-1/CUL5-dependent regulation of cellular growth.

    PubMed

    Willis, Angelica N; Dean, Shirley E Bradley; Habbouche, Joe A; Kempers, Brian T; Ludwig, Megan L; Sayfie, Aaron D; Lewis, Steven P; Harrier, Stephanie; DeBruine, Zachary J; Garrett, Richard; Burnatowska-Hledin, Maria A

    2017-04-01

    VACM-1/CUL5 is a member of the cullin family of proteins involved in the E3 ligase-dependent degradation of diverse proteins that regulate cellular proliferation. The ability of VACM-1/CUL5 to inhibit cellular growth is affected by its posttranslational modifications and its localization to the nucleus. Since the mechanism of VACM-1/CUL5 translocation to the nucleus is not clear, the goal of this project was to determine the role that the putative nuclear localization signal (NLS) we identified in the VACM-1/CUL5 ((640)PKLKRQ(646)) plays in the cellular localization of VACM-1/CUL5 and its effect on cellular growth. We used site-directed mutagenesis to change Lys642 and Lys644 to Gly and the mutated cDNA constructs were transfected into COS-1 cells. Mutation of the NLS in VACM-1/CUL5 significantly reduced its localization to the nucleus and compromised its effect on cellular growth. We have shown previously that the antiproliferative effect of VACM-1/CUL5 could be reversed by mutation of PKA-specific phosphorylation sequence ((S730A)VACM-1/CUL5), which was associated with its increased nuclear localization and modification by NEDD8. Thus, we examined whether these properties can be controlled by the NLS. The mutation of NLS in (S730A)VACM-1/CUL5 cDNA compromised its proliferative effect and reduced its localization to the nucleus. The immunocytochemistry results showed that, in cells transfected with the mutant cDNAs, the nuclear NEDD8 signal was decreased. Western blot analysis of total cell lysates, however, showed that VACM-1/CUL5 neddylation was not affected. Together, these results suggest that the presence of the NLS, both in VACM-1/CUL5 and in (S730A)VACM-1/CUL5 sequences, is critical for their control of cell proliferation.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  18. SGK1 is not required for regulation of colonic ENaC activity.

    PubMed

    Rexhepaj, Rexhep; Artunc, Ferruh; Grahammer, Florian; Nasir, Omaima; Sandu, Ciprian; Friedrich, Björn; Kuhl, Dietmar; Lang, Florian

    2006-10-01

    , transepithelial potential and amiloride-sensitive short circuit current are enhanced in the colonic epithelium of SGK1-deficient mice. Thus, lack of SGK1 does not disrupt colonic ENaC activity and its regulation by salt depletion.

  19. Viral safety of human plasma-derived medicinal products: impact of regulation requirements.

    PubMed

    Velthove, Karin J; Over, Jan; Abbink, Kristiena; Janssen, Mart P

    2013-07-01

    The viral safety of plasma-derived medicinal products is of paramount importance. This article aims to provide insight into the relative impact of different safety measures on achieving viral safety of finished products, derived from human plasma. Virus removal and/or inactivation during the production process is the most important safety measure, and model-based risk estimates show that with current safety measures, the risk of transmission of known blood-borne pathogens to plasma product recipients is extremely low. However, because the residual risk of virus transmissions is also influenced by the incidence rate of infection in the donor population, it makes sense to control these incidence rates, as well. The current measures are aiming in the right direction, but integration of guidelines is required to adequately address their common goal: controlling the risk of infectious disease transmission by plasma-derived medicinal products. By integration of guidelines, the combination of various types of safety measures to prevent virus transmission-donor selection, donation screening, quarantining, and virus removal and/or inactivation during production-may be consistently interpreted and adequately assessed.

  20. Desnutrin/ATGL is Regulated by AMPK and is Required for a Brown Adipose Phenotype

    PubMed Central

    Ahmadian, Maryam; Abbott, Marcia J.; Tang, Tianyi; Hudak, Carolyn S.S.; Kim, Yangha; Bruss, Matthew; Hellerstein, Marc K.; Lee, Hui-Young; Samuel, Varman T.; Shulman, Gerald I.; Wang, Yuhui; Duncan, Robin E.; Kang, Chulho; Sul, Hei Sook

    2011-01-01

    SUMMARY While fatty acids (FAs) released by white adipose tissue (WAT) provide energy for other organs, lipolysis is also critical in brown adipose tissue (BAT), generating FAs for oxidation and UCP-1 activation for thermogenesis. Here, we show that adipose-specific ablation of desnutrin/ATGL in mice converts BAT to a WAT-like tissue. These mice exhibit severely impaired thermogenesis with increased expression of WAT-enriched genes but decreased BAT genes including UCP-1 with lower PPARα binding to its promoter, revealing the requirement of desnutrin-catalyzed lipolysis for maintaining BAT phenotype. We also show that desnutrin is phosphorylated by AMPK at S406, increasing TAG hydrolase activity, and provide evidence for increased lipolysis by AMPK phosphorylation of desnutrin in adipocytes and in vivo. Despite adiposity and impaired BAT function, desnutrin-ASKO mice have improved hepatic insulin sensitivity with lower DAG levels. Overall, desnutrin is phosphorylated/activated by AMPK to increase lipolysis and brings FA oxidation and UCP-1 induction for thermogenesis. PMID:21641555

  1. Mitochondrial inheritance is required for MEN-regulated cytokinesis in budding yeast.

    PubMed

    García-Rodríguez, Luis J; Crider, David G; Gay, Anna Card; Salanueva, Iñigo J; Boldogh, Istvan R; Pon, Liza A

    2009-11-03

    Mitochondrial inheritance, the transfer of mitochondria from mother to daughter cell during cell division, is essential for daughter cell viability. The mitochore, a mitochondrial protein complex containing Mdm10p, Mdm12p, and Mmm1p, is required for mitochondrial motility leading to inheritance in budding yeast. We observe a defect in cytokinesis in mitochore mutants and another mutant (mmr1Delta gem1Delta) with impaired mitochondrial inheritance. This defect is not observed in yeast that have no mitochondrial DNA or defects in mitochondrial protein import or assembly of beta-barrel proteins in the mitochondrial outer membrane. Deletion of MDM10 inhibits contractile-ring closure, but does not inhibit contractile-ring assembly, localization of a chromosomal passenger protein to the spindle during early anaphase, spindle alignment, nucleolar segregation, or nuclear migration during anaphase. Release of the mitotic exit network (MEN) component, Cdc14p, from the nucleolus during anaphase is delayed in mdm10Delta cells. Finally, hyperactivation of the MEN by deletion of BUB2 restores defects in cytokinesis in mdm10Delta and mmr1Delta gem1Delta cells and reduces the fidelity of mitochondrial segregation between mother and daughter cells in wild-type and mdm10Delta cells. Our studies identify a novel MEN-linked regulatory system that inhibits cytokinesis in response to defects in mitochondrial inheritance in budding yeast.

  2. Desnutrin/ATGL is regulated by AMPK and is required for a brown adipose phenotype.

    PubMed

    Ahmadian, Maryam; Abbott, Marcia J; Tang, Tianyi; Hudak, Carolyn S S; Kim, Yangha; Bruss, Matthew; Hellerstein, Marc K; Lee, Hui-Young; Samuel, Varman T; Shulman, Gerald I; Wang, Yuhui; Duncan, Robin E; Kang, Chulho; Sul, Hei Sook

    2011-06-08

    While fatty acids (FAs) released by white adipose tissue (WAT) provide energy for other organs, lipolysis is also critical in brown adipose tissue (BAT), generating FAs for oxidation and UCP-1 activation for thermogenesis. Here we show that adipose-specific ablation of desnutrin/ATGL in mice converts BAT to a WAT-like tissue. These mice exhibit severely impaired thermogenesis with increased expression of WAT-enriched genes but decreased BAT genes, including UCP-1 with lower PPARα binding to its promoter, revealing the requirement of desnutrin-catalyzed lipolysis for maintaining a BAT phenotype. We also show that desnutrin is phosphorylated by AMPK at S406, increasing TAG hydrolase activity, and provide evidence for increased lipolysis by AMPK phosphorylation of desnutrin in adipocytes and in vivo. Despite adiposity and impaired BAT function, desnutrin-ASKO mice have improved hepatic insulin sensitivity with lower DAG levels. Overall, desnutrin is phosphorylated/activated by AMPK to increase lipolysis and brings FA oxidation and UCP-1 induction for thermogenesis. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Osmotic Regulation Is Required for Cancer Cell Survival under Solid Stress.

    PubMed

    McGrail, Daniel J; McAndrews, Kathleen M; Brandenburg, Chandler P; Ravikumar, Nithin; Kieu, Quang Minh N; Dawson, Michelle R

    2015-10-06

    For a solid tumor to grow, it must be able to support the compressive stress that is generated as it presses against the surrounding tissue. Although the literature suggests a role for the cytoskeleton in counteracting these stresses, there has been no systematic evaluation of which filaments are responsible or to what degree. Here, using a three-dimensional spheroid model, we show that cytoskeletal filaments do not actively support compressive loads in breast, ovarian, and prostate cancer. However, modulation of tonicity can induce alterations in spheroid size. We find that under compression, tumor cells actively efflux sodium to decrease their intracellular tonicity, and that this is reversible by blockade of sodium channel NHE1. Moreover, although polymerized actin does not actively support the compressive load, it is required for sodium efflux. Compression-induced cell death is increased by both sodium blockade and actin depolymerization, whereas increased actin polymerization offers protective effects and increases sodium efflux. Taken together, these results demonstrate that cancer cells modulate their tonicity to survive under compressive solid stress. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. Osmotic Regulation Is Required for Cancer Cell Survival under Solid Stress

    PubMed Central

    McGrail, Daniel J.; McAndrews, Kathleen M.; Brandenburg, Chandler P.; Ravikumar, Nithin; Kieu, Quang Minh N.; Dawson, Michelle R.

    2015-01-01

    For a solid tumor to grow, it must be able to support the compressive stress that is generated as it presses against the surrounding tissue. Although the literature suggests a role for the cytoskeleton in counteracting these stresses, there has been no systematic evaluation of which filaments are responsible or to what degree. Here, using a three-dimensional spheroid model, we show that cytoskeletal filaments do not actively support compressive loads in breast, ovarian, and prostate cancer. However, modulation of tonicity can induce alterations in spheroid size. We find that under compression, tumor cells actively efflux sodium to decrease their intracellular tonicity, and that this is reversible by blockade of sodium channel NHE1. Moreover, although polymerized actin does not actively support the compressive load, it is required for sodium efflux. Compression-induced cell death is increased by both sodium blockade and actin depolymerization, whereas increased actin polymerization offers protective effects and increases sodium efflux. Taken together, these results demonstrate that cancer cells modulate their tonicity to survive under compressive solid stress. PMID:26445434

  5. MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration

    PubMed Central

    Aranda, Juan F.; Reglero-Real, Natalia; Kremer, Leonor; Marcos-Ramiro, Beatriz; Ruiz-Sáenz, Ana; Calvo, María; Enrich, Carlos; Correas, Isabel; Millán, Jaime; Alonso, Miguel A.

    2011-01-01

    Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes. PMID:21325632

  6. Neuronal PINCH is Regulated by TNF-α and is Required for Neurite Extension

    PubMed Central

    Jatiani, Asavari; Pannizzo, Paola; Gualco, Elisa; Del-Valle, Luis

    2011-01-01

    During HIV infection of the CNS, neurons are damaged by viral proteins, such as Tat and gp120, or by inflammatory factors, such as TNF-α, that are released from infected and/or activated glial cells. Host responses to this damage may include the induction of survival or repair mechanisms. In this context, previous studies report robust expression of a protein called particularly interesting new cysteine histidine-rich protein (PINCH), in the neurons of HIV patients’ brains, compared with nearly undetectable levels in HIV-negative individuals (Rearden et al., J Neurosci Res 86:2535–2542, 2008), suggesting PINCH’s involvement in neuronal signaling during HIV infection of the brain. To address potential triggers for PINCH induction in HIV patients’ brains, an in vitro system mimicking some aspects of HIV infection of the CNS was utilized. We investigated neuronal PINCH expression, subcellular distribution, and biological consequences of PINCH sequestration upon challenge with Tat, gp120, and TNF-α. Our results indicate that in neurons, TNF-α stimulation increases PINCH expression and changes its subcellular localization. Furthermore, PINCH mobility is required to maintain neurite extension upon challenge with TNF-α. PINCH may function as a neuron-specific host-mediated response to challenge by HIV-related factors in the CNS. PMID:20689998

  7. Extracellular signal-regulated kinase (ERK) activation is required for itch sensation in the spinal cord.

    PubMed

    Zhang, Ling; Jiang, Guan-Yu; Song, Ning-Jing; Huang, Ying; Chen, Jia-Yin; Wang, Qing-Xiu; Ding, Yu-Qiang

    2014-04-03

    Itch, chronic itch in particular, can have a significant negative impact on an individual's quality of life. However, the molecular mechanisms underlying itch processing in the central nervous system remain largely unknown. We report here that activation of ERK signaling in the spinal cord is required for itch sensation. ERK activation, as revealed by anti-phosphorylated ERK1/2 immunostaining, is observed in the spinal dorsal horn of mice treated with intradermal injections of histamine and compound 48/80 but not chloroquine or SLIGRL-NH2, indicating that ERK activation only occurs in histamine-dependent acute itch. In addition, ERK activation is also observed in 2, 4-dinitrofluorobenzene (DNFB)-induced itch. Consistently, intrathecal administration of the ERK phosphorylation inhibitor U0126 dramatically reduces the scratching behaviors induced by histamine and DNFB, but not by chloroquine. Furthermore, administration of the histamine receptor H1 antagonist chlorpheniramine decreases the scratching behaviors and ERK activation induced by histamine, but has no effect on DNFB-induced itch responses. Finally, the patch-clamp recording shows that in histamine-, chloroquine- and DNFB-treated mice the spontaneous excitatory postsynaptic current (sEPSC) of dorsal horn neurons is increased, and the decrease of action potential threshold is largely prevented by bathing of U0126 in histamine- and DNFB-treated mice but not those treated with chloroquine. Our results demonstrate a critical role for ERK activation in itch sensation at the spinal level.

  8. Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1.

    PubMed Central

    Ferrigno, P; Posas, F; Koepp, D; Saito, H; Silver, P A

    1998-01-01

    MAP kinase signaling modules serve to transduce extracellular signals to the nucleus of eukaryotic cells, but little is known about how signals cross the nuclear envelope. Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 MAP kinase cascade, which is composed of three tiers of protein kinases, namely the SSK2, SSK22 and STE11 MAPKKKs, the PBS2 MAPKK, and the HOG1 MAPK. Using green fluorescent protein (GFP) fusions of these kinases, we found that HOG1, PBS2 and STE11 localize to the cytoplasm of unstressed cells. Following osmotic stress, HOG1, but neither PBS2 nor STE11, translocates into the nucleus. HOG1 translocation occurs very rapidly, is transient, and correlates with the phosphorylation and activation of the MAP kinase by its MAPKK. HOG1 phosphorylation is necessary and sufficient for nuclear translocation, because a catalytically inactive kinase when phosphorylated is translocated to the nucleus as efficiently as the wild-type. Nuclear import of the MAPK under stress conditions requires the activity of the small GTP binding protein Ran-GSP1, but not the NLS-binding importin alpha/beta heterodimer. Rather, HOG1 import requires the activity of a gene, NMD5, that encodes a novel importin beta homolog. Similarly, export of dephosphorylated HOG1 from the nucleus requires the activity of the NES receptor XPO1/CRM1. Our findings define the requirements for the regulated nuclear transport of a stress-activated MAP kinase. PMID:9755161

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

    PubMed

    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.

  10. Requirement of Split ends for Epigenetic Regulation of Notch Signal-Dependent Genes during Infection-Induced Hemocyte Differentiation▿ †

    PubMed Central

    Jin, Li Hua; Choi, Jung Kyoon; Kim, Byungil; Cho, Hwan Sung; Kim, Jihyun; Kim-Ha, Jeongsil; Kim, Young-Joon

    2009-01-01

    Drosophila producing a mutant form of the putative transcription coregulator, Split ends (Spen), originally identified in the analysis of neuronal development, display diverse immune defects. In order to understand the role of Spen in the innate immune response, we analyzed the transcriptional defects associated with spen mutant hemocytes and their relationship to the Notch signaling pathways. Spen is regulated by the Notch pathway in the lymph glands and is required for Notch-dependent activation of a large number of genes involved in energy metabolism and differentiation. Analysis of the epigenetic marks associated with Spen-dependent genes indicates that Spen performs its function as a coactivator by regulating chromatin modification. Intriguingly, expression of the Spen-dependent genes was transiently downregulated in a Notch-dependent manner by the Dif activated upon recognition of pathogen-associated molecules, demonstrating the existence of cross talk between hematopoietic regulation and the innate immune response. Our observations reveal a novel connection between the Notch and Toll/IMD signaling pathways and demonstrate a coactivating role for Spen in activating Notch-dependent genes in differentiating cells. PMID:19139277

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

    PubMed

    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.

  12. The Extracellular-Regulated Kinase Effector Lk6 is Required for Glutamate Receptor Localization at the Drosophila Neuromuscular Junction

    PubMed Central

    Hussein, Nizar A.; Delaney, Taylor L.; Tounsel, Brittany L.; Liebl, Faith L.W.

    2016-01-01

    The proper localization and synthesis of postsynaptic glutamate receptors are essential for synaptic plasticity. Synaptic translation initiation is thought to occur via the target of rapamycin (TOR) and mitogen-activated protein kinase signal-integrating kinase (Mnk) signaling pathways, which is downstream of extracellular-regulated kinase (ERK). We used the model glutamatergic synapse, the Drosophila neuromuscular junction, to better understand the roles of the Mnk and TOR signaling pathways in synapse development. These synapses contain non-NMDA receptors that are most similar to AMPA receptors. Our data show that Lk6, the Drosophila homolog of Mnk1 and Mnk2, is required in either presynaptic neurons or postsynaptic muscle for the proper localization of the GluRIIA glutamate receptor subunit. Lk6 may signal through eukaryotic initiation factor (eIF) 4E to regulate the synaptic levels of GluRIIA as either interfering with eIF4E binding to eIF4G or expression of a nonphosphorylatable isoform of eIF4E resulted in a significant reduction in GluRIIA at the synapse. We also find that Lk6 and TOR may independently regulate synaptic levels of GluRIIA. PMID:27199570

  13. Urban renewal in the nucleus: is protein turnover by proteasomes absolutely required for nuclear receptor-regulated transcription?

    PubMed

    Nawaz, Zafar; O'Malley, Bert W

    2004-03-01

    The importance of the ubiquitin proteasome pathway in higher eukaryotes has been well established in cell cycle regulation, signal transduction, and cell differentiation, but has only recently been linked to nuclear hormone receptor-regulated gene transcription. Characterization of a number of ubiquitin proteasome pathway enzymes as coactivators and observations that several nuclear receptors are ubiquitinated and degraded in the course of their nuclear activities provide evidence that ubiquitin proteasome-mediated protein degradation plays an integral role in eukaryotic transcription. In addition to receptors, studies have revealed that coactivators are ubiquitinated and degraded via the proteasome. The notion that the ubiquitin proteasome pathway is involved in gene transcription is further strengthened by the fact that ubiquitin proteasome pathway enzymes are recruited to the promoters of target genes and that proteasome-dependent degradation of nuclear receptors is required for efficient transcriptional activity. These findings suggest that protein degradation is coupled with nuclear receptor coactivation activity. It is possible that the ubiquitin proteasome pathway modulates transcription by promoting remodeling and turnover of the nuclear receptor-transcription complex. In this review, we discus the possible role of the ubiquitin proteasome pathway in nuclear hormone receptor-regulated gene transcription.

  14. Inositol-requiring enzyme 1α is a key regulator of angiogenesis and invasion in malignant glioma

    PubMed Central

    Auf, Gregor; Jabouille, Arnaud; Guérit, Sylvaine; Pineau, Raphaël; Delugin, Maylis; Bouchecareilh, Marion; Magnin, Noël; Favereaux, Alexandre; Maitre, Marlène; Gaiser, Timo; von Deimling, Andreas; Czabanka, Marcus; Vajkoczy, Peter; Chevet, Eric; Bikfalvi, Andreas; Moenner, Michel

    2010-01-01

    Inositol-requiring enzyme 1 (IRE1) is a proximal endoplasmic reticulum (ER) stress sensor and a central mediator of the unfolded protein response. In a human glioma model, inhibition of IRE1α correlated with down-regulation of prevalent proangiogenic factors such as VEGF-A, IL-1β, IL-6, and IL-8. Significant up-regulation of antiangiogenic gene transcripts was also apparent. These transcripts encode SPARC, decorin, thrombospondin-1, and other matrix proteins functionally linked to mesenchymal differentiation and glioma invasiveness. In vivo, using both the chick chorio-allantoic membrane assay and a mouse orthotopic brain model, we observed in tumors underexpressing IRE1: (i) reduction of angiogenesis and blood perfusion, (ii) a decreased growth rate, and (iii) extensive invasiveness and blood vessel cooption. This phenotypic change was consistently associated with increased overall survival in glioma-implanted recipient mice. Ectopic expression of IL-6 in IRE1-deficient tumors restored angiogenesis and neutralized vessel cooption but did not reverse the mesenchymal/infiltrative cell phenotype. The ischemia-responsive IRE1 protein is thus identified as a key regulator of tumor neovascularization and invasiveness. PMID:20702765

  15. Ciliary transcription factors and miRNAs precisely regulate Cp110 levels required for ciliary adhesions and ciliogenesis

    PubMed Central

    Walentek, Peter; Quigley, Ian K; Sun, Dingyuan I; Sajjan, Umeet K; Kintner, Christopher; Harland, Richard M

    2016-01-01

    Upon cell cycle exit, centriole-to-basal body transition facilitates cilia formation. The centriolar protein Cp110 is a regulator of this process and cilia inhibitor, but its positive roles in ciliogenesis remain poorly understood. Using Xenopus we show that Cp110 inhibits cilia formation at high levels, while optimal levels promote ciliogenesis. Cp110 localizes to cilia-forming basal bodies and rootlets, and is required for ciliary adhesion complexes that facilitate Actin interactions. The opposing roles of Cp110 in ciliation are generated in part by coiled-coil domains that mediate preferential binding to centrioles over rootlets. Because of its dual role in ciliogenesis, Cp110 levels must be precisely controlled. In multiciliated cells, this is achieved by both transcriptional and post-transcriptional regulation through ciliary transcription factors and microRNAs, which activate and repress cp110 to produce optimal Cp110 levels during ciliogenesis. Our data provide novel insights into how Cp110 and its regulation contribute to development and cell function. DOI: http://dx.doi.org/10.7554/eLife.17557.001 PMID:27623009

  16. Requirement of Split ends for epigenetic regulation of Notch signal-dependent genes during infection-induced hemocyte differentiation.

    PubMed

    Jin, Li Hua; Choi, Jung Kyoon; Kim, Byungil; Cho, Hwan Sung; Kim, Jihyun; Kim-Ha, Jeongsil; Kim, Young-Joon

    2009-03-01

    Drosophila producing a mutant form of the putative transcription coregulator, Split ends (Spen), originally identified in the analysis of neuronal development, display diverse immune defects. In order to understand the role of Spen in the innate immune response, we analyzed the transcriptional defects associated with spen mutant hemocytes and their relationship to the Notch signaling pathways. Spen is regulated by the Notch pathway in the lymph glands and is required for Notch-dependent activation of a large number of genes involved in energy metabolism and differentiation. Analysis of the epigenetic marks associated with Spen-dependent genes indicates that Spen performs its function as a coactivator by regulating chromatin modification. Intriguingly, expression of the Spen-dependent genes was transiently downregulated in a Notch-dependent manner by the Dif activated upon recognition of pathogen-associated molecules, demonstrating the existence of cross talk between hematopoietic regulation and the innate immune response. Our observations reveal a novel connection between the Notch and Toll/IMD signaling pathways and demonstrate a coactivating role for Spen in activating Notch-dependent genes in differentiating cells.

  17. DDX5 regulates DNA replication and is required for cell proliferation in a subset of breast cancer cells.

    PubMed

    Mazurek, Anthony; Luo, Weijun; Krasnitz, Alexander; Hicks, James; Powers, R Scott; Stillman, Bruce

    2012-09-01

    Understanding factors required for DNA replication will enrich our knowledge of this important process and potentially identify vulnerabilities that can be exploited in cancer therapy. We applied an assay that measures the stability of maintenance of an episomal plasmid in human tissue culture cells to screen for new DNA replication factors. We identify an important role for DDX5 in G(1)-S-phase progression where it directly regulates DNA replication factor expression by promoting the recruitment of RNA polymerase II to E2F-regulated gene promoters. We find that the DDX5 locus is frequently amplified in breast cancer and that breast cancer-derived cells with amplification of DDX5 are much more sensitive to its depletion than breast cancer cells and a breast epithelial cell line that lacks DDX5 amplification. Our results show a novel role for DDX5 in cancer cell proliferation and suggest DDX5 as a therapeutic target in breast cancer treatment. DDX5 is required for cell proliferation by controlling the transcription of genes expressing DNA replication proteins in cancer cells in which the DDX5 locus is amplified, and this has uncovered a dependence on DDX5 for cell proliferation. Given the high frequency of DDX5 amplification in breast cancer, our results highlight DDX5 as a promising candidate for targeted therapy of breast tumors with DDX5 amplification, and indeed we show that DDX5 inhibition sensitizes a subset of breast cancer cells to trastuzumab.

  18. Bidirectional regulation of synaptic transmission by BRAG1/IQSEC2 and its requirement in long-term depression

    PubMed Central

    Brown, Joshua C.; Petersen, Amber; Zhong, Ling; Himelright, Miranda L.; Murphy, Jessica A.; Walikonis, Randall S.; Gerges, Nashaat Z.

    2016-01-01

    Dysfunction of the proteins regulating synaptic function can cause synaptic plasticity imbalance that underlies neurological disorders such as intellectual disability. A study found that four distinct mutations within BRAG1, an Arf-GEF synaptic protein, each led to X-chromosome-linked intellectual disability (XLID). Although the physiological functions of BRAG1 are poorly understood, each of these mutations reduces BRAG1's Arf-GEF activity. Here we show that BRAG1 is required for the activity-dependent removal of AMPA receptors in rat hippocampal pyramidal neurons. Moreover, we show that BRAG1 bidirectionally regulates synaptic transmission. On one hand, BRAG1 is required for the maintenance of synaptic transmission. On the other hand, BRAG1 expression enhances synaptic transmission, independently of BRAG1 Arf-GEF activity or neuronal activity, but dependently on its C-terminus interactions. This study demonstrates a dual role of BRAG1 in synaptic function and highlights the functional relevance of reduced BRAG1 Arf-GEF activity as seen in the XLID-associated human mutations. PMID:27009485

  19. Regulation of CD34 transcription by Sp1 requires sites upstream and downstream of the transcription start site.

    PubMed

    Taranenko, N; Krause, D S

    2000-08-01

    CD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells, but not on fully differentiated cells in the peripheral blood. To better understand the molecular regulation of early hematopoiesis, we are elucidating the mechanisms of CD34 transcriptional regulation. By deletion analysis we identify a 39-bp element in the proximal region of murine CD34 promoter that is critical for promoter activity. Electromobility shift assays indicate that nuclear proteins of hematopoietic cells bind to this domain; however, the presence of this binding activity does not correlate directly with CD34 expression.Using methylation interference, the DNA binding site for this activity was localized to four guanine residues within the GGGGTCGG sequence from -48 to -54 bp. When the four contact guanines were mutated, both protein binding and promoter activity were abolished. Although this sequence does not contain a standard consensus for Sp1, this transcription factor binds specifically to the 39-bp region and stimulates promoter activity in both hematopoietic cells and in Sp1 null Drosophila S2 cells. In addition, Ku binds to this domain in a sequence-specific manner. Activation of the CD34 promoter by Sp1 requires the presence of a binding domain at -48 bp as well as the 5' untranslated region, which also binds Sp1.A functional interaction between regulatory regions upstream and downstream of the transcription start site is required for CD34 gene expression.

  20. Cortactin Is Required for N-cadherin Regulation of Kv1.5 Channel Function*

    PubMed Central

    Cheng, Lan; Yung, Aaron; Covarrubias, Manuel; Radice, Glenn L.

    2011-01-01

    The intercalated disc serves as an organizing center for various cell surface components at the termini of the cardiomyocyte, thus ensuring proper mechanoelectrical coupling throughout the myocardium. The cell adhesion molecule, N-cadherin, is an essential component of the intercalated disc. Cardiac-specific deletion of N-cadherin leads to abnormal electrical conduction and sudden arrhythmic death in mice. The mechanisms linking the loss of N-cadherin in the heart and spontaneous malignant ventricular arrhythmias are poorly understood. To investigate whether ion channel remodeling contributes to arrhythmogenesis in N-cadherin conditional knock-out (N-cad CKO) mice, cardiac myocyte excitability and voltage-gated potassium channel (Kv), as well as inwardly rectifying K+ channel remodeling, were investigated in N-cad CKO cardiomyocytes by whole cell patch clamp recordings. Action potential duration was prolonged in N-cad CKO ventricle myocytes compared with wild type. Relative to wild type, IK,slow density was significantly reduced consistent with decreased expression of Kv1.5 and Kv accessory protein, Kcne2, in the N-cad CKO myocytes. The decreased Kv1.5/Kcne2 expression correlated with disruption of the actin cytoskeleton and reduced cortactin at the sarcolemma. Biochemical experiments revealed that cortactin co-immunoprecipitates with Kv1.5. Finally, cortactin was required for N-cadherin-mediated enhancement of Kv1.5 channel activity in a heterologous expression system. Our results demonstrate a novel mechanistic link among the cell adhesion molecule, N-cadherin, the actin-binding scaffold protein, cortactin, and Kv channel remodeling in the heart. These data suggest that in addition to gap junction remodeling, aberrant Kv1.5 channel function contributes to the arrhythmogenic phenotype in N-cad CKO mice. PMID:21507952

  1. Cortactin is required for N-cadherin regulation of Kv1.5 channel function.

    PubMed

    Cheng, Lan; Yung, Aaron; Covarrubias, Manuel; Radice, Glenn L

    2011-06-10

    The intercalated disc serves as an organizing center for various cell surface components at the termini of the cardiomyocyte, thus ensuring proper mechanoelectrical coupling throughout the myocardium. The cell adhesion molecule, N-cadherin, is an essential component of the intercalated disc. Cardiac-specific deletion of N-cadherin leads to abnormal electrical conduction and sudden arrhythmic death in mice. The mechanisms linking the loss of N-cadherin in the heart and spontaneous malignant ventricular arrhythmias are poorly understood. To investigate whether ion channel remodeling contributes to arrhythmogenesis in N-cadherin conditional knock-out (N-cad CKO) mice, cardiac myocyte excitability and voltage-gated potassium channel (Kv), as well as inwardly rectifying K(+) channel remodeling, were investigated in N-cad CKO cardiomyocytes by whole cell patch clamp recordings. Action potential duration was prolonged in N-cad CKO ventricle myocytes compared with wild type. Relative to wild type, I(K,slow) density was significantly reduced consistent with decreased expression of Kv1.5 and Kv accessory protein, Kcne2, in the N-cad CKO myocytes. The decreased Kv1.5/Kcne2 expression correlated with disruption of the actin cytoskeleton and reduced cortactin at the sarcolemma. Biochemical experiments revealed that cortactin co-immunoprecipitates with Kv1.5. Finally, cortactin was required for N-cadherin-mediated enhancement of Kv1.5 channel activity in a heterologous expression system. Our results demonstrate a novel mechanistic link among the cell adhesion molecule, N-cadherin, the actin-binding scaffold protein, cortactin, and Kv channel remodeling in the heart. These data suggest that in addition to gap junction remodeling, aberrant Kv1.5 channel function contributes to the arrhythmogenic phenotype in N-cad CKO mice.

  2. Vav family proteins are required for optimal regulation of PLCγ2 by integrin αIIbβ3

    PubMed Central

    Pearce, Andrew C.; Mccarty, Owen J. T.; Calaminus, Simon D. J.; Vigorito, Elena; Turner, Martin; Watson, Steve P.

    2006-01-01

    Vav proteins belong to the family of guanine-nucleotide-exchange factors for the Rho/Rac family of small G-proteins. In addition, they serve as important adapter proteins for the activation of PLCγ (phospholipase Cγ) isoforms by ITAM (immunoreceptor tyrosine-based activation motif) receptors, including the platelet collagen receptor GPVI (glycoprotein VI). Vav proteins are also regulated downstream of integrins, including the major platelet integrin αIIbβ3, which has recently been shown to regulate PLCγ2. In the present study, we have investigated the role of Vav family proteins in filopodia and lamellipodia formation on fibrinogen using platelets deficient in Vav1 and Vav3. Wild-type mouse platelets undergo a limited degree of spreading on fibrinogen, characterized by the formation of numerous filopodia and limited lamellipodia structures. Platelets deficient in Vav1 and Vav3 exhibit reduced filopodia and lamellipodia formation during spreading on fibrinogen. This is accompanied by reduced αIIbβ3-mediated PLCγ2 tyrosine phosphorylation and reduced Ca2+ mobilization. In contrast, the G-protein agonist thrombin stimulates full spreading of control and Vav1/3-deficient platelets. Consistent with this, stimulation of F-actin (filamentous actin) formation and Rac activation by thrombin is not altered in Vav-deficient cells. These results demonstrate that Vav1 and Vav3 are required for optimal spreading and regulation of PLCγ2 by integrin αIIbβ3, but that their requirement is by-passed upon G-protein receptor activation. PMID:17054426

  3. High power valve regulated lead-acid batteries for new vehicle requirements

    NASA Astrophysics Data System (ADS)

    Trinidad, Francisco; Sáez, Francisco; Valenciano, Jesús

    The performance of high power VRLA ORBITAL™ batteries is presented. These batteries have been designed with isolated cylindrical cells, providing high reliability to the recombination process, while maintaining, at the same time, a very high compression (>80 kPa) over the life of the battery. Hence, the resulting VRLA modules combine a high rate capability with a very good cycle performance. Two different electrochemically active material compositions have been developed: high porosity and low porosity for starting and deep cycle applications, respectively (depending on the power demand and depth of discharge). Although, the initial performance of the starting version is higher, after a few cycles the active material of the deep cycle version is fully developed, and this achieves the same high rate capability. Both types are capable of supplying the necessary reliability for cranking at the lowest temperature (-40°C). Specific power of over 500 W/kg is achievable at a much lower cost than for nickel-metal hydride systems. Apart from the initial performance, an impressive behaviour of the cycling version has been found in deep cycle applications, due to the highly compressed and high density active material. When submitted to continuous discharge-charge cycles at 75% (IEC 896-2 specification) and 100% (BCI deep cycle) DoD, it has been found that the batteries are still healthy after more than 1000 and 700 cycles, respectively. However, it has been proven that the application of an IUi algorithm (up to 110% of overcharging) with a small constant current charging period at the end of the charge is absolutely necessary to achieve the above results. Without the final boosting period, the cycle life of the battery could be substantially shortened. The high specific power and reliability observed in the tests carried out, would allow ORBITAL™ batteries to comply with the more demanding requirements that are being introduced in conventional and future hybrid electric

  4. Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

    PubMed Central

    Wold, Mitchell S.; Gong, Shiaoching; Phillips, Greg R.; Dou, Zhixun; Zhao, Yanxiang; Heintz, Nathaniel; Zong, Wei-Xing; Yue, Zhenyu

    2014-01-01

    Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis. PMID:25275521

  5. PB1 Domain-Dependent Signaling Complex Is Required for Extracellular Signal-Regulated Kinase 5 Activation

    PubMed Central

    Nakamura, Kazuhiro; Uhlik, Mark T.; Johnson, Nancy L.; Hahn, Klaus M.; Johnson, Gary L.

    2006-01-01

    MEKK2, MEK5, and extracellular signal-regulated kinase 5 (ERK5) are members of a three-kinase cascade for the activation of ERK5. MEK5 is the only MAP2K to express a PB1 domain, and we have shown that it heterodimerizes with the PB1 domain of MEKK2. Here we demonstrate the MEK5 PB1 domain is a scaffold that also binds ERK5, functionally forming a MEKK2-MEK5-ERK5 complex. Reconstitution assays and CFP/YFP imaging (fluorescence resonance energy transfer [FRET]) measuring YFP-MEKK2/CFP-MEK5 and CFP-MEK5/YFP-ERK5 interactions define distinct MEK5 PB1 domain binding sites for MEKK2 and ERK5, with a C-terminal extension of the PB1 domain contributing to ERK5 binding. Stimulus-dependent CFP/YFP FRET in combination with mutational analysis was used to define MEK5 PB1 domain residues critical for the interaction of MEKK2/MEK5 and MEK5/ERK5 required for activation of the ERK5 pathway in living cells. Fusion of the MEK5 PB1 domain to the N terminus of MEK1 confers ERK5 regulation by a MAP2K normally regulating only ERK1/2. The MEK5 PB1 domain confers stringent MAP3K regulation of ERK5 relative to more promiscuous MAP3K control of ERK1/2, JNK, and p38. PMID:16507987

  6. Beclin 1 is required for neuron viability and regulates endosome pathways via the UVRAG-VPS34 complex.

    PubMed

    McKnight, Nicole C; Zhong, Yun; Wold, Mitchell S; Gong, Shiaoching; Phillips, Greg R; Dou, Zhixun; Zhao, Yanxiang; Heintz, Nathaniel; Zong, Wei-Xing; Yue, Zhenyu

    2014-10-01

    Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis.

  7. Regulation of mitosis in response to damaged or incompletely replicated DNA require different levels of Grapes (Drosophila Chk1)

    PubMed Central

    Purdy, Amanda; Uyetake, Lyle; Cordeiro, Melissa Garner; Su, Tin Tin

    2011-01-01

    Summary Checkpoints monitor the state of DNA and can delay or arrest the cell cycle at multiple points including G1-S transition, progress through S phase and G2-M transition. Regulation of progress through mitosis, specifically at the metaphase-anaphase transition, occurs after exposure to ionizing radiation (IR) in Drosophila and budding yeast, but has not been conclusively demonstrated in mammals. Here we report that regulation of metaphase-anaphase transition in Drosophila depends on the magnitude of radiation dose and time in the cell cycle at which radiation is applied, which may explain the apparent differences among experimental systems and offer an explanation as to why this regulation has not been seen in mammalian cells. We further document that mutants in Drosophila Chk1 (Grapes) that are capable of delaying the progress through mitosis in response to IR are incapable of delaying progress through mitosis when DNA synthesis is blocked by mutations in an essential replication factor encoded by double park (Drosophila Cdt1). We conclude that DNA damage and replication checkpoints operating in the same cell cycle at the same developmental stage in Drosophila can exhibit differential requirements for the Chk1 homolog. The converse situation exists in fission yeast where loss of Chk1 is more detrimental to the DNA damage checkpoint than to the DNA replication checkpoint. It remains to be seen which of these two different uses of Chk1 homologs are conserved in mammals. Finally, our results demonstrate that Drosophila provides a unique opportunity to study the regulation of the entry into, and progress through, mitosis by DNA structure checkpoints in metazoa. PMID:16079276

  8. Tight regulation of SpSoxB factors is required for patterning and morphogenesis in sea urchin embryos.

    PubMed

    Kenny, Alan P; Oleksyn, David W; Newman, Laurel A; Angerer, Robert C; Angerer, Lynne M

    2003-09-15

    Previous studies in sea urchin embryos have demonstrated that nuclearization of beta-catenin is essential for initial steps in the specification of endoderm and mesenchyme, which are derived from vegetal blastomeres. This process begins at the 4th and extends through the 9th cleavage stage, an interval in which the SpSoxB1 transcription regulator is downregulated by beta-catenin-dependent gene products that include the transcription repressor SpKrl. These observations raise the possibility that SpSoxB1 removal is required to allow vegetal development to proceed. Here we show that elevated and ectopic expression of this factor suppresses differentiation of all vegetal cell types, a phenotype that is very similar to that caused by the suppression of beta-catenin nuclear function by cadherin overexpression. Suppression of vegetal fates involves interference at the protein-protein level because a mutation of SpSoxB1 that prevents its binding to DNA does not significantly reduce this activity. Reduction in SpSoxB1 level results in elevated TCF/Lef-beta-catenin-dependent expression of a luciferase reporter gene in vivo, indicating that in the normal embryo this protein suppresses the primary vegetal signaling mechanism that is required for specification of mesenchyme and endoderm. Surprisingly, normal expression of SpSoxB1 is required for gastrulation and endoderm differentiation, as shown by both morpholino-mediated translational interference and expression of a dominant negative protein. Similar gain-of-function and loss-of-function assays of a closely related factor, SpSoxB2, demonstrate that it, too, is required for gastrulation and that its overexpression can suppress vegetal development. However, significant phenotypic differences are apparent in the two perturbations, indicating that SpSoxB1 and SpSoxB2 have at least some distinct developmental functions. The results of all these studies support a model in which the concentration of SpSoxB factors must be tightly

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

    SciTech Connect

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; 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. Moreover, 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. Our 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.

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

    DOE PAGES

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; ...

    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 PilZmore » domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, 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. Our 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.« less

  11. Dimeric c-di-GMP Is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa*

    PubMed Central

    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

    2015-01-01

    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. PMID:25817996

  12. The ESCRT-0 Component HRS is Required for HIV-1 Vpu-Mediated BST-2/Tetherin Down-Regulation

    PubMed Central

    Janvier, Katy; Pelchen–Matthews, Annegret; Renaud, Jean-Baptiste; Caillet, Marina; Marsh, Mark; Berlioz-Torrent, Clarisse

    2011-01-01

    The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery, a highly conserved set of four hetero-oligomeric protein complexes, is required for multivesicular body formation, sorting ubiquitinylated membrane proteins for lysosomal degradation, cytokinesis and the final stages of assembly of a number of enveloped viruses, including the human immunodeficiency viruses. Here, we show an additional role for the ESCRT machinery in HIV-1 release. BST-2/tetherin is a restriction factor that impedes HIV release by tethering mature virus particles to the plasma membrane. We found that HRS, a key component of the ESCRT-0 complex, promotes efficient release of HIV-1 and that siRNA-mediated HRS depletion induces a BST-2/tetherin phenotype. This activity is related to the ability of the HIV-1 Vpu protein to down-regulate BST-2/tetherin. We found that BST-2/tetherin undergoes constitutive ESCRT-dependent sorting for lysosomal degradation and that this degradation is enhanced by Vpu expression. We demonstrate that Vpu-mediated BST-2/tetherin down-modulation and degradation require HRS (ESCRT-0) function and that knock down of HRS increases cellular levels of BST-2/tetherin and restricts virus release. Furthermore, HRS co-precipitates with Vpu and BST-2. Our results provide further insight into the mechanism by which Vpu counteracts BST-2/tetherin and promotes HIV-1 dissemination, and they highlight an additional role for the ESCRT machinery in virus release. PMID:21304933

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

    EPA Pesticide Factsheets

    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.

  14. 25 CFR 518.7 - If a tribe holds a certificate of self-regulation, is it required to report information to the...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ....7 Indians NATIONAL INDIAN GAMING COMMISSION, DEPARTMENT OF THE INTERIOR GENERAL PROVISIONS SELF REGULATION OF CLASS II GAMING § 518.7 If a tribe holds a certificate of self-regulation, is it required to... signed by an authorized tribal official, which explains how tribal net gaming revenues were used in...

  15. Casein kinase II is required for the spindle assembly checkpoint by regulating Mad2p in fission yeast

    SciTech Connect

    Shimada, Midori; Yamamoto, Ayumu; Murakami-Tonami, Yuko; Nakanishi, Makoto; Yoshida, Takashi; Aiba, Hirofumi; Murakami, Hiroshi

    2009-10-23

    The spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis. Here we show that fission yeast casein kinase II (CK2) is required for this checkpoint function. In the CK2 mutants mitosis occurs in the presence of a spindle defect, and the spindle checkpoint protein Mad2p fails to localize to unattached kinetochores. The CK2 mutants are sensitive to the microtubule depolymerising drug thiabendazole, which is counteracted by ectopic expression of mad2{sup +}. The level of Mad2p is low in the CK2 mutants. These results suggest that CK2 has a role in the spindle checkpoint by regulating Mad2p.

  16. shot regulates the microtubule reorganization required for localization of axis-determining mRNAs during oogenesis.

    PubMed

    Lee, Jiyeon; Lee, Sujung; Chen, Cheng; Shim, Hyeran; Kim-Ha, Jeongsil

    2016-02-01

    The Drosophila mid-oogenesis stages are notable as the time when most maternal mRNAs become localized at discrete regions of the oocyte. Microtubule rearrangement occurs during this period and is critical for the localization of axis-determining maternal mRNAs. We have identified shot as a key player in establishing the cytoskeletal arrangement required for the spatial localization of axis-determining maternal mRNAs. We also found that the spatial distribution of the Shot protein is regulated by its mRNA localization. Our results suggest that the RNA localization mechanism is used not only for restricted accumulation of patterning molecules but also for the microtubule organization that leads to the initial development of oocyte polarity.

  17. A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis

    PubMed Central

    Zhang, Yongping; Shang, Weina; Nagarkar Jaiswal, Sonal; di Ronza, Alberto; Jaiswal, Manish; Yamamoto, Shinya; Sandoval, Hector; Duraine, Lita; Sardiello, Marco; Sillitoe, Roy V.; Venkatachalam, Kartik; Fan, Hengyu; Bellen, Hugo J.; Tong, Chao

    2015-01-01

    Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis. PMID:25811491

  18. Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells.

    PubMed

    Gidda, Satinder K; Park, Sunjung; Pyc, Michal; Yurchenko, Olga; Cai, Yingqi; Wu, Peng; Andrews, David W; Chapman, Kent D; Dyer, John M; Mullen, Robert T

    2016-04-01

    Eukaryotic cells compartmentalize neutral lipids into organelles called lipid droplets (LDs), and while much is known about the role of LDs in storing triacylglycerols in seeds, their biogenesis and function in nonseed tissues are poorly understood. Recently, we identified a class of plant-specific, lipid droplet-associated proteins (LDAPs) that are abundant components of LDs in nonseed cell types. Here, we characterized the three LDAPs in Arabidopsis (Arabidopsis thaliana) to gain insight to their targeting, assembly, and influence on LD function and dynamics. While all three LDAPs targeted specifically to the LD surface, truncation analysis of LDAP3 revealed that essentially the entire protein was required for LD localization. The association of LDAP3 with LDs was detergent sensitive, but the protein bound with similar affinity to synthetic liposomes of various phospholipid compositions, suggesting that other factors contributed to targeting specificity. Investigation of LD dynamics in leaves revealed that LD abundance was modulated during the diurnal cycle, and characterization of LDAP misexpression mutants indicated that all three LDAPs were important for this process. LD abundance was increased significantly during abiotic stress, and characterization of mutant lines revealed that LDAP1 and LDAP3 were required for the proper induction of LDs during heat and cold temperature stress, respectively. Furthermore, LDAP1 was required for proper neutral lipid compartmentalization and triacylglycerol degradation during postgerminative growth. Taken together, these studies reveal that LDAPs are required for the maintenance and regulation of LDs in plant cells and perform nonredundant functions in various physiological contexts, including stress response and postgerminative growth. © 2016 American Society of Plant Biologists. All Rights Reserved.

  19. Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells1

    PubMed Central

    Park, Sunjung; Wu, Peng

    2016-01-01

    Eukaryotic cells compartmentalize neutral lipids into organelles called lipid droplets (LDs), and while much is known about the role of LDs in storing triacylglycerols in seeds, their biogenesis and function in nonseed tissues are poorly understood. Recently, we identified a class of plant-specific, lipid droplet-associated proteins (LDAPs) that are abundant components of LDs in nonseed cell types. Here, we characterized the three LDAPs in Arabidopsis (Arabidopsis thaliana) to gain insight to their targeting, assembly, and influence on LD function and dynamics. While all three LDAPs targeted specifically to the LD surface, truncation analysis of LDAP3 revealed that essentially the entire protein was required for LD localization. The association of LDAP3 with LDs was detergent sensitive, but the protein bound with similar affinity to synthetic liposomes of various phospholipid compositions, suggesting that other factors contributed to targeting specificity. Investigation of LD dynamics in leaves revealed that LD abundance was modulated during the diurnal cycle, and characterization of LDAP misexpression mutants indicated that all three LDAPs were important for this process. LD abundance was increased significantly during abiotic stress, and characterization of mutant lines revealed that LDAP1 and LDAP3 were required for the proper induction of LDs during heat and cold temperature stress, respectively. Furthermore, LDAP1 was required for proper neutral lipid compartmentalization and triacylglycerol degradation during postgerminative growth. Taken together, these studies reveal that LDAPs are required for the maintenance and regulation of LDs in plant cells and perform nonredundant functions in various physiological contexts, including stress response and postgerminative growth. PMID:26896396

  20. The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development

    PubMed Central

    Benadiba, Carine; Magnani, Dario; Niquille, Mathieu; Morlé, Laurette; Valloton, Delphine; Nawabi, Homaira; Ait-Lounis, Aouatef; Otsmane, Belkacem; Reith, Walter; Theil, Thomas; Hornung, Jean-Pierre

    2012-01-01

    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. PMID:22479201

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

    PubMed Central

    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

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

    SciTech Connect

    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.

  3. Pdlim7 Regulates Arf6-Dependent Actin Dynamics and Is Required for Platelet-Mediated Thrombosis in Mice

    PubMed Central

    Miller, Kaylie P.; Krcmery, Jennifer; Simon, Hans-Georg

    2016-01-01

    Upon vessel injury, platelets become activated and rapidly reorganize their actin cytoskeleton to adhere to the site of endothelial damage, triggering the formation of a fibrin-rich plug to prevent further blood loss. Inactivation of Pdlim7 provides the new perspective that regulation of actin cytoskeletal changes in platelets is dependent on the encoded PDZ-LIM protein. Loss-of-function of Pdlim7 triggers hypercoagulopathy and causes significant perinatal lethality in mice. Our in vivo and in vitro studies reveal that Pdlim7 is dynamically distributed along actin fibers, and lack of Pdlim7 leads to a marked inability to rearrange the actin cytoskeleton. Specifically, the absence of Pdlim7 prevents platelets from bundling actin fibers into a concentric ring that defines the round spread shape of activated platelets. Similarly, in mouse embryonic fibroblasts, loss of Pdlim7 abolishes the formation of stress fibers needed to adopt the typical elongated fibroblast shape. In addition to revealing a fundamental cell biological role in actin cytoskeletal organization, we also demonstrate a function of Pdlim7 in regulating the cycling between the GTP/GDP-bound states of Arf6. The small GTPase Arf6 is an essential factor required for actin dynamics, cytoskeletal rearrangements, and platelet activation. Consistent with our findings of significantly elevated initial F-actin ratios and subsequent morphological aberrations, loss of Pdlim7 causes a shift in balance towards an increased Arf6-GTP level in resting platelets. These findings identify a new Pdlim7-Arf6 axis controlling actin dynamics and implicate Pdlim7 as a primary endogenous regulator of platelet-dependent hemostasis. PMID:27792740

  4. DICER1 regulated let-7 expression levels in p53-induced cancer repression requires cyclin D1

    PubMed Central

    Sun, Xin; Tang, Shou-Ching; Xu, Chongwen; Wang, Chenguang; Qin, Sida; Du, Ning; Liu, Jian; Zhang, Yiwen; Li, Xiang; Luo, Gang; Zhou, Jie; Xu, Fei; Ren, Hong

    2015-01-01

    Let-7 miRNAs act as tumour suppressors by directly binding to the 3′UTRs of downstream gene products. The regulatory role of let-7 in downstream gene expression has gained much interest in the cancer research community, as it controls multiple biological functions and determines cell fates. For example, one target of the let-7 family is cyclin D1, which promotes G0/S cell cycle progression and oncogenesis, was correlated with endoribonuclease DICER1, another target of let-7. Down-regulated let-7 has been identified in many types of tumours, suggesting a feedback loop may exist between let-7 and cyclin D1. A potential player in the proposed feedback relationship is Dicer, a central regulator of miRNA expression through sequence-specific silencing. We first identified that DICER1 is the key downstream gene for cyclin D1-induced let-7 expression. In addition, we found that let-7 miRNAs expression decreased because of the p53-induced cell death response, with deregulated cyclin D1. Our results also showed that cyclin D1 is required for Nutlin-3 and TAX-induced let-7 expression in cancer repression and the cell death response. For the first time, we provide evidence that let-7 and cyclin D1 form a feedback loop in regulating therapy response of cancer cells and cancer stem cells, and importantly, that alteration of let-7 expression, mainly caused by cyclin D1, is a sensitive indicator for better chemotherapies response. PMID:25702703

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

    PubMed

    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.

  6. Dynamic regulation of the PR-Set7 histone methyltransferase is required for normal cell cycle progression

    PubMed Central

    Wu, Shumin; Wang, Weiping; Kong, Xiangduo; Congdon, Lauren M.; Yokomori, Kyoko; Kirschner, Marc W.; Rice, Judd C.

    2010-01-01

    Although the PR-Set7/Set8/KMT5a histone H4 Lys 20 monomethyltransferase (H4K20me1) plays an essential role in mammalian cell cycle progression, especially during G2/M, it remained unknown how PR-Set7 itself was regulated. In this study, we discovered the mechanisms that govern the dynamic regulation of PR-Set7 during mitosis, and that perturbation of these pathways results in defective mitotic progression. First, we found that PR-Set7 is phosphorylated at Ser 29 (S29) specifically by the cyclin-dependent kinase 1 (cdk1)/cyclinB complex, primarily from prophase through early anaphase, subsequent to global accumulation of H4K20me1. While S29 phosphorylation did not affect PR-Set7 methyltransferase activity, this event resulted in the removal of PR-Set7 from mitotic chromosomes. S29 phosphorylation also functions to stabilize PR-Set7 by directly inhibiting its interaction with the anaphase-promoting complex (APC), an E3 ubiquitin ligase. The dephosphorylation of S29 during late mitosis by the Cdc14 phosphatases was required for APCcdh1-mediated ubiquitination of PR-Set7 and subsequent proteolysis. This event is important for proper mitotic progression, as constitutive phosphorylation of PR-Set7 resulted in a substantial delay between metaphase and anaphase. Collectively, we elucidated the molecular mechanisms that control PR-Set7 protein levels during mitosis, and demonstrated that its orchestrated regulation is important for normal mitotic progression. PMID:20966048

  7. The kinesin-2 family member KIF3C regulates microtubule dynamics and is required for axon growth and regeneration.

    PubMed

    Gumy, Laura F; Chew, Daniel J; Tortosa, Elena; Katrukha, Eugene A; Kapitein, Lukas C; Tolkovsky, Aviva M; Hoogenraad, Casper C; Fawcett, James W

    2013-07-10

    Axon regeneration after injury requires the extensive reconstruction, reorganization, and stabilization of the microtubule cytoskeleton in the growth cones. Here, we identify KIF3C as a key regulator of axonal growth and regeneration by controlling microtubule dynamics and organization in the growth cone. KIF3C is developmentally regulated. Rat embryonic sensory axons and growth cones contain undetectable levels of KIF3C protein that is locally translated immediately after injury. In adult neurons, KIF3C is axonally transported from the cell body and is enriched at the growth cone where it preferentially binds to tyrosinated microtubules. Functionally, the interaction of KIF3C with EB3 is necessary for its localization at the microtubule plus-ends in the growth cone. Depletion of KIF3C in adult neurons leads to an increase in stable, overgrown and looped microtubules because of a strong decrease in the microtubule frequency of catastrophes, suggesting that KIF3C functions as a microtubule-destabilizing factor. Adult axons lacking KIF3C, by RNA interference or KIF3C gene knock-out, display an impaired axonal outgrowth in vitro and a delayed regeneration after injury both in vitro and in vivo. Murine KIF3C knock-out embryonic axons grow normally but do not regenerate after injury because they are unable to locally translate KIF3C. These data show that KIF3C is an injury-specific kinesin that contributes to axon growth and regeneration by regulating and organizing the microtubule cytoskeleton in the growth cone.

  8. Iron-induced turnover of the Arabidopsis IRON-REGULATED TRANSPORTER1 metal transporter requires lysine residues.

    PubMed

    Kerkeb, Loubna; Mukherjee, Indrani; Chatterjee, Iera; Lahner, Brett; Salt, David E; Connolly, Erin L

    2008-04-01

    Iron is an essential micronutrient but is toxic if accumulated at high levels. Thus, iron uptake and distribution in plants are controlled by precise regulatory mechanisms. IRON-REGULATED TRANSPORTER1 (IRT1) is the major high affinity iron transporter responsible for iron uptake from the soil in Arabidopsis (Arabidopsis thaliana). Previously, we showed that IRT1 is subject to posttranscriptional regulation; when expressed from the constitutive cauliflower mosaic virus 35S promoter, IRT1 protein accumulates only in iron-deficient roots. IRT1 contains an intracellular loop that may be critical for posttranslational regulation by metals. Of particular interest are a histidine (His) motif (HGHGHGH) that might bind metals and two lysine residues that could serve as attachment sites for ubiquitin. We constructed a set of mutant IRT1 alleles: IRT1H154Q, IRT1H156Q, IRT1H158Q, IRT1H160Q, IRT14HQ (quadruple His mutant), IRT1K146R, IRT1K171R, and a double mutant (IRT1K146R,K171R). Mutation of the His or lysine residues did not eliminate the ability of IRT1 to transport iron or zinc. Expression of each of the IRT1 variants and an IRT1intact construct in plants from the 35S promoter revealed that either K146 or K171 is required for iron-induced protein turnover, and 35S-IRT1K146R,K171R plants contain higher levels of iron as compared to 35S-IRT1 and wild type. Furthermore, accumulation of metals in 35S-IRT1K146R,K171R plants was not associated with an increase in ferric chelate reductase activity; this result indicates that, at least under conditions when iron is abundant, reduction of ferric iron may not be the rate-limiting step in iron uptake by strategy I plants such as Arabidopsis.

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

    PubMed

    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.

  10. Interleukin33 Is Required for Disposal of Unnecessary Cells during Ovarian Atresia through Regulation of Autophagy and Macrophage Migration

    PubMed Central

    Wu, Jean; Carlock, Colin; Zhou, Cindy; Nakae, Susumu; Hicks, John; Adams, Henry P.; Lou, Yahuan

    2015-01-01

    Physiological processes such as ovarian follicle atresia generate large amounts of unnecessary cells or tissue detritus, which needs to be disposed of rapidly. Interleukin33 (IL33) is a member of the IL1 cytokine gene family. Consecutive expression of IL33 in a wide range of tissues has hinted at its role beyond immune defense. We have previously reported a close correlation between IL33 expression patterns and ovarian atresia. Here, we demonstrated that IL33 is required for disposal of degenerative tissue during ovarian atresia using Il33−/− mice. Deletion of the Il33 gene impaired normal disposal of atretic follicles, resulting in massive accumulations of tissue wastes abundant with aging-related catabolic wastes such as lipofuscin. Accumulation of tissue wastes in Il33−/− mice, in turn, accelerated ovarian aging and functional decline. Thus, their reproductive lifespan was shortened to 2/3 of that for Il33+/− littermates. IL33 orchestrated disposal mechanism through regulation of autophagy in degenerating tissues and macrophage migration into the tissues. Our study provided direct evidence supporting an expanded role of IL33 in tissue integrity and aging through regulating disposal of unnecessary tissues or cells. PMID:25617473

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

    PubMed

    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.

  12. Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response

    PubMed Central

    Edmonds, Matthew J.; Carter, Rachel J.; Nickson, Catherine M.; Williams, Sarah C.; Parsons, Jason L.

    2017-01-01

    Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo. We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response. PMID:27924031

  13. Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly

    PubMed Central

    Racine, Pierre-Jean; Chamontin, Célia; de Rocquigny, Hugues; Bernacchi, Serena; Paillart, Jean-Christophe; Mougel, Marylène

    2016-01-01

    HIV-1 is a retrovirus replicating within cells by reverse transcribing its genomic RNA (gRNA) into DNA. Within cells, virus assembly requires the structural Gag proteins with few accessory proteins, notably the viral infectivity factor (Vif) and two copies of gRNA as well as cellular factors to converge to the plasma membrane. In this process, the nucleocapsid (NC) domain of Gag binds to the packaging signal of gRNA which consists of a series of stem-loops (SL1-SL3) ensuring gRNA selection and packaging into virions. Interestingly, mutating NC activates a late-occurring reverse transcription (RT) step in producer cells, leading to the release of DNA-containing HIV-1 particles. In order to decipher the molecular mechanism regulating this late RT, we explored the role of several key partners of NC, such as Vif, gRNA and the cellular cytidine deaminase APOBEC3G that restricts HIV-1 infection by targeting the RT. By studying combinations of deletions of these putative players, we revealed that NC, SL1-SL3 and in lesser extent Vif, but not APOBEC3G, interplay regulates the late RT. PMID:27273064

  14. Regulation of Anticancer Styrylpyrone Biosynthesis in the Medicinal Mushroom Inonotus obliquus Requires Thioredoxin Mediated Transnitrosylation of S-nitrosoglutathione Reductase.

    PubMed

    Zhao, Yanxia; He, Meihong; Ding, Jianing; Xi, Qi; Loake, Gary J; Zheng, Weifa

    2016-11-21

    The medicinal macrofungus Inonotus obliquus widely utilized as folk medicine in Russia and Baltic countries is a source of phenylpropanoid-derived styrylpyrone polyphenols that can inhibit tumor proliferation. Insights into the regulatory machinery that controls I. obliquus styrylpyrone polyphenol biosynthesis will enable strategies to increase the production of these molecules. Here we show that Thioredoxin (Trx) mediated transnitrosylation of S-nitrosoglutathione reductase (GSNOR) underpins the regulation of styrylpyrone production, driven by nitric oxide (NO) synthesis triggered by P. morii coculture. NO accumulation results in the S-nitrosylation of PAL and 4CL required for the synthesis of precursor phenylpropanoids and styrylpyrone synthase (SPS), integral to the production of styrylpyrone, inhibiting their activities. These enzymes are targeted for denitrosylation by Trx proteins, which restore their activity. Further, this Trx S-nitrosothiol (SNO) reductase activity was potentiated following S-nitrosylation of Trx proteins at a non-catalytic cysteine (Cys) residue. Intriguingly, this process was counterbalanced by Trx denitrosylation, mediated by Trx-dependent transnitrosylation of GSNOR. Thus, unprecedented interplay between Trx and GSNOR oxidoreductases regulates the biosynthesis of styrylpyrone polyphenols in I. obliquus.

  15. Gene-specific requirement of a nuclear protein, IkappaB-zeta, for promoter association of inflammatory transcription regulators.

    PubMed

    Yamazaki, Soh; Matsuo, Susumu; Muta, Tatsushi; Yamamoto, Masahiro; Akira, Shizuo; Takeshige, Koichiro

    2008-11-21

    Expression of many inflammatory genes is induced through activation of the transcription factor NF-kappaB. In contrast to the advanced understanding of cytoplasmic control of NF-kappaB activation, its regulation in the nucleus has not been fully understood despite its importance in selective gene expression. We previously identified an inducible nuclear protein, IkappaB-zeta, and demonstrated that this molecule is indispensable for the expression of a group of NF-kappaB-regulated genes. In this study, we established a unique gene induction system, in which IkappaB-zeta is expressed independently of inflammatory stimuli, to specifically investigate the molecular basis underlying IkappaB-zeta-mediated gene activation. We show that in the presence of IkappaB-zeta other primary response genes are dispensable for the expression of the target secondary response genes. ChIP analyses revealed that IkappaB-zeta is required for stimulus-induced recruitment of NF-kappaB onto the target promoter in a gene-specific manner. Surprisingly, IkappaB-zeta is also necessary for the gene-selective promoter recruitment of another inflammatory transcription factor, C/EBPbeta, and the chromatin remodeling factor Brg1. We propose a new gene regulatory mechanism underlying the selective expression of inflammatory genes.

  16. Regulation of Anticancer Styrylpyrone Biosynthesis in the Medicinal Mushroom Inonotus obliquus Requires Thioredoxin Mediated Transnitrosylation of S-nitrosoglutathione Reductase

    PubMed Central

    Zhao, Yanxia; He, Meihong; Ding, Jianing; Xi, Qi; Loake, Gary J.; Zheng, Weifa

    2016-01-01

    The medicinal macrofungus Inonotus obliquus widely utilized as folk medicine in Russia and Baltic countries is a source of phenylpropanoid-derived styrylpyrone polyphenols that can inhibit tumor proliferation. Insights into the regulatory machinery that controls I. obliquus styrylpyrone polyphenol biosynthesis will enable strategies to increase the production of these molecules. Here we show that Thioredoxin (Trx) mediated transnitrosylation of S-nitrosoglutathione reductase (GSNOR) underpins the regulation of styrylpyrone production, driven by nitric oxide (NO) synthesis triggered by P. morii coculture. NO accumulation results in the S-nitrosylation of PAL and 4CL required for the synthesis of precursor phenylpropanoids and styrylpyrone synthase (SPS), integral to the production of styrylpyrone, inhibiting their activities. These enzymes are targeted for denitrosylation by Trx proteins, which restore their activity. Further, this Trx S-nitrosothiol (SNO) reductase activity was potentiated following S-nitrosylation of Trx proteins at a non-catalytic cysteine (Cys) residue. Intriguingly, this process was counterbalanced by Trx denitrosylation, mediated by Trx-dependent transnitrosylation of GSNOR. Thus, unprecedented interplay between Trx and GSNOR oxidoreductases regulates the biosynthesis of styrylpyrone polyphenols in I. obliquus. PMID:27869186

  17. NADPH oxidase- generated ROS are required for SDF-1α-stimulated angiogenesis Short title: NOX is an angiogenic regulator

    PubMed Central

    Pi, Xinchun; Xie, Liang; Portbury, Andrea L.; Kumar, Sarayu; Lockyer, Pamela; Li, Xi; Patterson, Cam

    2014-01-01

    Objective Reactive oxygen species (ROS) act as signaling molecules during angiogenesis, however, the mechanisms used for such signaling events remain unclear. Stromal cell-derived factor-1α (SDF-1α) is one of the most potent angiogenic chemokines. Here we examined the role of ROS in the regulation of SDF-1α-dependent angiogenesis. Approach and results Bovine aortic endothelial cells (BAECs) were treated with SDF-1α and intracellular ROS generation was monitored. SDF-1α treatment induced BAEC migration and ROS generation, with the majority of ROS generated by BAECs at the leading edge of the migratory cells. Antioxidants and NADPH oxidase (NOX) inhibitors blocked SDF-1α-induced endothelial migration. Furthermore, knockdown of either NOX5 or p22phox (a requisite subunit for NOX1/2/4 activation) significantly impaired endothelial motility and tube formation, suggesting that multiple NOXs regulate SDF-1α-dependent angiogenesis. Our previous study demonstrated that JNK3 activity is essential for SDF-1α-dependent angiogenesis. Here, we identified that NOX5 is the dominant NOX required for SDF-1α-induced JNK3 activation and that NOX5 and MKP7 (the JNK3 phosphatase) associate with one another but decrease this interaction upon SDF-1α treatment. Furthermore, MKP7 activity was inhibited by SDF-1α and this inhibition was relieved by NOX5 knockdown, indicating that NOX5 promotes JNK3 activation by blocking MKP7 activity. Conclusions We conclude that NOX is required for SDF-1α signaling and that intracellular redox balance is critical for SDF-1α-induced endothelial migration and angiogenesis. PMID:24990230

  18. Identification of TSG101 Functional Domains and p21 Loci Required for TSG101-Mediated p21 Gene Regulation

    PubMed Central

    Lin, Yu-Shiuan; Chen, Yin-Ju; Cohen, Stanley N.; Cheng, Tzu-Hao

    2013-01-01

    TSG101 (tumor susceptibility gene 101) is a multi-domain protein known to act in the cell nucleus, cytoplasm, and periplasmic membrane. Remarkably, TSG101, whose location within cells varies with the stage of the cell cycle, affects biological events as diverse as cell growth and proliferation, gene expression, cytokinesis, and endosomal trafficking. The functions of TSG101 additionally are recruited for viral and microvesicle budding and for intracellular survival of invading bacteria. Here we report that the TSG101 protein also interacts with and down-regulates the promoter of the p21CIP1/WAF1tumor suppressor gene, and identify a p21 locus and TSG101 domains that mediate this interaction. TSG101 deficiency in Saos-2 human osteosarcoma cells was accompanied by an increased abundance of p21 mRNA and protein and the retardation of cell proliferation. A cis-acting element in the p21 promoter that interacts with TSG101 and is required for promoter repression was located using chromatin immunoprecipitation (ChIP) analysis and p21-driven luciferase reporter gene expression, respectively. Additional analysis of TSG101 deletion mutants lacking specific domains established the role of the central TSG101 domains in binding to the p21 promoter and demonstrated the additional essentiality of the TSG101 C-terminal steadiness box (SB) in the repression of p21 promoter activity. Neither binding of TSG101 to the p21 promoter nor repression of this promoter required the TSG101 N-terminal UEV domain, which mediates the ubiquitin-recognition functions of TSG101 and its actions as a member of ESCRT endocytic trafficking complexes, indicating that regulation of the p21 promoter by TSG101 is independent of its role in such trafficking. PMID:24244542

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

    SciTech Connect

    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.

  20. Regulation of vitellogenin gene expression in transgenic Caenorhabditis elegans: short sequences required for activation of the vit-2 promoter.

    PubMed Central

    MacMorris, M; Broverman, S; Greenspoon, S; Lea, K; Madej, C; Blumenthal, T; Spieth, J

    1992-01-01

    The Caenorhabditis elegans vitellogenin genes are subject to sex-, stage-, and tissue-specific regulation: they are expressed solely in the adult hermaphrodite intestine. Comparative sequence analysis of the DNA immediately upstream of these genes revealed the presence of two repeated heptameric elements, vit promoter element 1 (VPE1) and VPE2. VPE1 has the consensus sequence TGTCAAT, while VPE2, CTGATAA, shares the recognition sequence of the GATA family of transcription factors. We report here a functional analysis of the VPEs within the 5'-flanking region of the vit-2 gene using stable transgenic lines. The 247 upstream bp containing the VPEs was sufficient for high-level, regulated expression. Furthermore, none of the four deletion mutations or eight point mutations tested resulted in expression of the reporter gene in larvae, males, or inappropriate hermaphrodite tissues. Mutation of the VPE1 closest to the TATA box inactivated the promoter, in spite of the fact that four additional close matches to the VPE1 consensus sequence are present within the 5'-flanking 200 bp. Each of these upstream VPE1-like sequences could be mutated without loss of high-level transgene expression, suggesting that if these VPE1 sequences play a role in regulating vit-2, their effects are more subtle. A site-directed mutation in the overlapping VPE1 and VPE2 at -98 was sufficient to inactivate the promoter, indicating that one or both of these VPEs must be present for activation of vit-2 transcription. Similarly, a small perturbation of the VPE2 at -150 resulted in reduction of fp155 expression, while a more extensive mutation in this element eliminated expression. On the other hand, deletion of this VPE2 and all upstream DNA still permitted correctly regulated expression, although at a very low level, suggesting that this VPE2 performs an important role in activation of vit-2 expression but may not be absolutely required. The results, taken together, demonstrate that both VPE1 and

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

    PubMed

    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.

  2. Regulation of Cellular Diacylglycerol through Lipid Phosphate Phosphatases Is Required for Pathogenesis of the Rice Blast Fungus, Magnaporthe oryzae

    PubMed Central

    Mir, Albely Afifa; Choi, Jaeyoung; Choi, Jaehyuk; Lee, Yong-Hwan

    2014-01-01

    Considering implication of diacylglycerol in both metabolism and signaling pathways, maintaining proper levels of diacylglycerol (DAG) is critical to cellular homeostasis and development. Except the PIP2-PLC mediated pathway, metabolic pathways leading to generation of DAG converge on dephosphorylation of phosphatidic acid catalyzed by lipid phosphate phosphatases. Here we report the role of such enzymes in a model plant pathogenic fungus, Magnaporthe oryzae. We identified five genes encoding putative lipid phosphate phosphatases (MoLPP1 to MoLPP5). Targeted disruption of four genes (except MoLPP4) showed that MoLPP3 and MoLPP5 are required for normal progression of infection-specific development and proliferation within host plants, whereas MoLPP1 and MoLPP2 are indispensable for fungal pathogenicity. Reintroduction of MoLPP3 and MoLPP5 into individual deletion mutants restored all the defects. Furthermore, exogenous addition of saturated DAG not only restored defect in appressorium formation but also complemented reduced virulence in both mutants. Taken together, our data indicate differential roles of lipid phosphate phosphatase genes and requirement of proper regulation of cellular DAGs for fungal development and pathogenesis. PMID:24959955

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

    PubMed

    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.

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

    PubMed Central

    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

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

    PubMed

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    PubMed

    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.

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

    PubMed Central

    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

  8. Regulation of Fuel and Fuel Additives: Refiner and Importer Quality Assurance Requirements for Downstream Oxygenate Blending and Requirements for Disposition of Pipeline Interfaces Additional Resources

    EPA Pesticide Factsheets

    EPA has published a Direct Final Rule that addresses requirements for parties that handle pipeline interface as well as addresses downstream quality assurance requirements for refiners (EPA publication # EPA-420-F-06-039).

  9. ELT-5 and ELT-6 are required continuously to regulate epidermal seam cell differentiation and cell fusion in C. elegans.

    PubMed

    Koh, K; Rothman, J H

    2001-08-01

    The C. elegans epidermis is a simple epithelium comprised of three major cell types, the seam, syncytial and P cells. While specification of all major epidermal cells is known to require the ELT-1 GATA transcription factor, little is known about how the individual epidermal cell types are specified. We report that elt-5 and -6, adjacent genes encoding GATA factors, are essential for the development of the lateral epidermal cells, the seam cells. Inhibition of elt-5 and -6 function by RNA-mediated interference results in penetrant late embryonic and early larval lethality. Seam cells in affected animals do not differentiate properly: the alae, seam-specific cuticular structures, are generally absent and expression of several seam-specific markers is blocked. In addition, elt-3, which encodes another GATA factor normally expressed in non-seam epidermis, is often ectopically expressed in the seam cells of affected animals, demonstrating that ELT-5 and -6 repress elt-3 expression in wild-type seam cells. Seam cells in affected animals often undergo inappropriate fusion with the epidermal syncytia. Interference of elt-5 and -6 function during larval development can cause fusion of all seam cells with the surrounding syncytia and pronounced defects in molting. elt-5 and -6 are both expressed in seam cells and many other cells, and are apparently functionally interchangeable. Their expression is controlled by separable tissue-specific regulatory elements and the apportionment of monocistronic versus dicistronic transcription of both genes appears to be subject to cell-type-specific regulation. Collectively, these findings indicate that elt-5 and -6 function continuously throughout C. elegans development to regulate seam cell differentiation and cell fusion.

  10. VdNUC-2, the Key Regulator of Phosphate Responsive Signaling Pathway, Is Required for Verticillium dahliae Infection

    PubMed Central

    Deng, Sheng; Wang, Cai-yue; Zhang, Xin; Wang, Qing; Lin, Ling

    2015-01-01

    In fungal cells, a phosphate (Pi) responsive signaling and metabolism (PHO) pathway regulates Pi-homeostasis. NUC-2/PHO81 and its homologs are one of the most important components in the regulation pathway. In soil-borne phytopathogenic fungus Verticillium dahliae, we identified a Neurospora crassa nuc-2 homolog gene VdNUC-2. VdNUC-2 is composed of 1,018 amino acids, and is highly conserved in tested filamentous fungi. Under conditions of Pi-starvation, compared with the wild-type strain and ectopic complementation strains, the VdNUC-2 knocked out mutants exhibited reduced radial growth, decreased production of conidia and microsclerotia, and were more sensitive to hydrogen peroxide stress. The virulence of VdNUC-2 defective mutants was significantly compromised, and that was unable to be restored by exogenous application of extra Pi. Additionally, the deletion mutants of VdNUC-1, a key transcription factor gene positively controlled by VdNUC-2 in the PHO pathway, showed the similar cultural phenotypes as VdNUC-2 mutants when both of them grew in Pi-limited conditions. However, the virulence of VdNUC-1 mutants was comparable to the wild-type strain. These evidences indicated that the virulence reduction in VdNUC-2 mutants is not due to the interruptions in the PHO pathway or the disturbance of Pi-homeostasis in V. dahliae cytoplasm. VdNUC-2 is not only a crucial gene in the PHO pathway in V. dahliae, but also is required for the full virulence during host-infection. PMID:26670613

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

    PubMed

    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.

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

    PubMed

    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.

  13. Neuroplasticity of the Hypothalamic–Pituitary–Adrenal Axis Early in Life Requires Recurrent Recruitment of Stress-Regulating Brain Regions

    PubMed Central

    Fenoglio, Kristina A.; Chen, Yuncai; Baram, Tallie Z.

    2008-01-01

    An eloquent example of experience-induced neuroplasticity involves the enduring effects of daily “handling” of rat pups on the expression of genes regulating hormonal and behavioral responses to stress. Handling-evoked augmentation of maternal care of pups induces long-lasting reduction of hypothalamic corticotropin releasing hormone (CRH) expression and upregulates hippocampal glucocorticoid receptor levels. These changes promote a lifelong attenuation of hormonal stress responses. We have found previously that handling-evoked downregulation of CRH expression occurs already by postnatal day 9, implicating it as an early step in this experience-induced neuroplasticity. Here, we investigated the neuronal pathways and cellular mechanisms involved. CRH mRNA expression in hypothalamic paraventricular nucleus (PVN) diminished after daily handling but not after handling once only, indicating that “recurrent” handling was required for this effect. Return of handled pups to their cage provoked a burst of nurturing behavior in dams that, in turn, induced transient, coordinate Fos expression in selected regions of the pups’ brains. These included central nucleus of the amygdala (ACe) and bed nucleus of the stria terminals (BnST), regions that are afferent to PVN and influence CRH expression there. Whereas handling once sufficed to evoke Fos expression within ACe and BnST, expression in thalamic paraventricular nucleus, a region involved in storing and processing stress-related experience, required recurrent handling. Fos induction in all three regions elicited reduced transcription factor phosphorylation, followed by attenuated activation of CRH gene transcription within the PVN. These studies provide a neurobiological foundation for the profound neuroplasticity of stress-related genes evoked by early-life experience. PMID:16510721

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

    PubMed Central

    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

  15. Perlecan is required for the chondrogenic differentiation of synovial mesenchymal cells through regulation of Sox9 gene expression.

    PubMed

    Sadatsuki, Ryo; Kaneko, Haruka; Kinoshita, Mayuko; Futami, Ippei; Nonaka, Risa; Culley, Kirsty L; Otero, Miguel; Hada, Shinnosuke; Goldring, Mary B; Yamada, Yoshihiko; Kaneko, Kazuo; Arikawa-Hirasawa, Eri; Ishijima, Muneaki

    2017-04-01

    We previously reported that perlecan, a heparan-sulfate proteoglycan (Hspg2), expressed in the synovium at the cartilage-synovial junction, is required for osteophyte formation in knee osteoarthritis. To examine the mechanism underlying this process, we examined the role of perlecan in the proliferation and differentiation of synovial mesenchymal cells (SMCs), using a recently established mouse synovial cell culture method. Primary SMCs isolated from Hspg2(-/-) -Tg (Hspg2(-/-) ;Col2a1-Hspg2(Tg/-) ) mice, in which the perlecan-knockout was rescued from perinatal lethality, lack perlecan. The chondrogenic-, osteogenic-, and adipogenic-potentials were examined in the Hspg2(-/-) -Tg SMCs compared to the control SMCs prepared from wild-type Hspg2(+/+) -Tg (Hspg2(+/+) ;Col2a1-Hspg2(Tg/-) ) littermates. In a culture condition permitting proliferation, both control and Hspg2(-/-) -Tg SMCs showed similar rates of proliferation and expression of cell surface markers. However, in micromass cultures, the cartilage matrix production and Sox9 and Col2a1 mRNA levels were significantly reduced in Hspg2(-/-) -Tg SMCs, compared with control SMCs. The reduced level of Sox9 mRNA was restored by the supplementation with exogenous perlecan protein. There was no difference in osteogenic differentiation between the control and Hspg2(-/-) -Tg SMCs, as measured by the levels of Runx2 and Col1a1 mRNA. The adipogenic induction and PPARγ mRNA levels were significantly reduced in Hspg2(-/-) -Tg SMCs compared to control SMCs. The reduction of PPARγ mRNA levels in Hspg2(-/-) -Tg SMCs was restored by supplementation of perlecan. Perlecan is required for the chondrogenic and adipogenic differentiation from SMCs via its regulation of the Sox9 and PPARγ gene expression, but not for osteogenic differentiation via Runx2. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:837-846, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  16. The heterozygous abp1/ABP1 insertional mutant has defects in functions requiring polar auxin transport and in regulation of early auxin-regulated genes.

    PubMed

    Effendi, Yunus; Rietz, Steffen; Fischer, Urs; Scherer, Günther F E

    2011-01-01

    AUXIN-BINDING PROTEIN 1 (ABP1) is not easily accessible for molecular studies because the homozygous T-DNA insertion mutant is embryo-lethal. We found that the heterozygous abp1/ABP1 insertion mutant has defects in auxin physiology-related responses: higher root slanting angles, longer hypocotyls, agravitropic roots and hypocotyls, aphototropic hypocotyls, and decreased apical dominance. Heterozygous plants flowered earlier than wild-type plants under short-day conditions. The length of the main root, the lateral root density and the hypocotyl length were little altered in the mutant in response to auxin. Compared to wild-type plants, transcription of early auxin-regulated genes (IAA2, IAA11, IAA13, IAA14, IAA19, IAA20, SAUR9, SAUR15, SAUR23, GH3.5 and ABP1) was less strongly up-regulated in the mutant by 0.1, 1 and 10 μm IAA. Surprisingly, ABP1 was itself an early auxin-up-regulated gene. IAA uptake into the mutant seedlings during auxin treatments was indistinguishable from wild-type. Basipetal auxin transport in young roots was slower in the mutant, indicating a PIN2/EIR1 defect, while acropetal transport was indistinguishable from wild-type. In the eir1 background, three of the early auxin-regulated genes tested (IAA2, IAA13 and ABP1) were more strongly induced by 1 μm IAA in comparison to wild-type, but eight of them were less up-regulated in comparison to wild-type. Similar but not identical disturbances in regulation of early auxin-regulated genes indicate tight functional linkage of ABP1 and auxin transport regulation. We hypothesize that ABP1 is involved in the regulation of polar auxin transport, and thus affects local auxin concentration and early auxin gene regulation. In turn, ABP1 itself is under the transcriptional control of auxin.

  17. Zinc fingers 1, 2, 5 and 6 of transcriptional regulator, PRDM4, are required for its nuclear localisation

    SciTech Connect

    Tunbak, Hale; Georgiou, Christiana; Guan, Cui; Richardson, William David; Chittka, Alexandra

    2016-05-27

    PRDM4 is a member of the PRDM family of transcriptional regulators which control various aspects of cellular differentiation and proliferation. PRDM proteins exert their biological functions both in the cytosol and the nucleus of cells. All PRDM proteins are characterised by the presence of two distinct structural motifs, the PR/SET domain and the zinc finger (ZF) motifs. We previously observed that deletion of all six zinc fingers found in PRDM4 leads to its accumulation in the cytosol, whereas overexpressed full length PRDM4 is found predominantly in the nucleus. Here, we investigated the requirements for single zinc fingers in the nuclear localisation of PRDM4. We demonstrate that ZF's 1, 2, 5 and 6 contribute to the accumulation of PRDM4 in the nucleus. Their effect is additive as deleting either ZF1-2 or ZF 5–6 redistributes PRDM4 protein from being almost exclusively nuclear to cytosolic and nuclear. We investigated the potential mechanism of nuclear shuttling of PRDM4 via the importin α/β-mediated pathway and find that PRDM4 nuclear targeting is independent of α/β-mediated nuclear import. -- Highlights: •Zinc fingers 1, 2, 5, and 6 are necessary for efficient nuclear localisation of PRDM4. •Zinc fingers 3 and 4 are dispensable for nuclear localisation of PRDM4. •Zinc knuckle is dispensable for nuclear localisation of PRDM4. •PRDM4 nuclear transport is independent of importin α/β-mediated pathway of nuclear import.

  18. HIF- and Non-HIF-Regulated Hypoxic Responses Require the Estrogen-Related Receptor in Drosophila melanogaster

    PubMed Central

    Li, Yan; Padmanabha, Divya; Gentile, Luciana B.; Dumur, Catherine I.; Beckstead, Robert B.; Baker, Keith D.

    2013-01-01

    Low-oxygen tolerance is supported by an adaptive response that includes a coordinate shift in metabolism and the activation of a transcriptional program that is driven by the hypoxia-inducible factor (HIF) pathway. The precise contribution of HIF-1a in the adaptive response, however, has not been determined. Here, we investigate how HIF influences hypoxic adaptation throughout Drosophila melanogaster development. We find that hypoxic-induced transcriptional changes are comprised of HIF-dependent and HIF-independent pathways that are distinct and separable. We show that normoxic set-points of carbohydrate metabolites are significantly altered in sima mutants and that these animals are unable to mobilize glycogen in hypoxia. Furthermore, we find that the estrogen-related receptor (dERR), which is a global regulator of aerobic glycolysis in larvae, is required for a competent hypoxic response. dERR binds to dHIFa and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFa in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including upregulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs. PMID:23382692

  19. Down-regulation of NR3A-containing NMDARs is required for synapse maturation and memory consolidation

    PubMed Central

    Roberts, Adam C.; Díez-García, Javier; Rodriguiz, Ramona M.; López, Iciar Paula; Luján, Rafael; Martínez-Turrillas, Rebeca; Picó, Esther; Henson, Maile A.; Bernardo, Danilo R.; Jarrett, Thomas M.; Clendeninn, Dallis J.; López-Mascaraque, Laura; Feng, Guoping; Lo, Donald C.; Wesseling, John F.; Wetsel, William C.; Philpot, Benjamin D.; Pérez-Otaño, Isabel

    2012-01-01

    SUMMARY NR3A is the only NMDA receptor (NMDAR) subunit that down-regulates sharply prior to the onset of sensitive periods for plasticity, yet the functional importance of this transient expression remains largely unknown. To investigate the possibility that removal/replacement of juvenile NR3A-containing NMDARs is involved in experience-driven synapse maturation, we used a reversible transgenic system that allowed persistent NR3A expression in the postnatal forebrain. We found that removal of NR3A is required to develop strong NMDAR currents, full expression of long-term synaptic plasticity, a mature synaptic organization characterized by more synapses and larger postsynaptic densities, and the ability to form long-term memories. Deficits associated with prolonged NR3A were reversible, as late-onset suppression of transgene expression rescued both the synaptic and memory impairments. Our results suggest that NR3A behaves as a molecular brake to prevent the premature strengthening and stabilization of excitatory synapses, and that NR3A removal might thereby initiate critical stages of synapse maturation during early postnatal neural development. PMID:19679074

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

    PubMed Central

    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

  1. SUMO modification is required for in vivo Hox gene regulation by the Caenorhabditis elegans Polycomb group protein SOP-2.

    PubMed

    Zhang, Hong; Smolen, Gromoslaw A; Palmer, Rachel; Christoforou, Andrea; van den Heuvel, Sander; Haber, Daniel A

    2004-05-01

    Post-translational modification of proteins by the ubiquitin-like molecule SUMO (sumoylation) regulates their subcellular localization and affects their functional properties in vitro, but the physiological function of sumoylation in multicellular organisms is largely unknown. Here, we show that the C. elegans Polycomb group (PcG) protein SOP-2 interacts with the SUMO-conjugating enzyme UBC-9 through its evolutionarily conserved SAM domain. Sumoylation of SOP-2 is required for its localization to nuclear bodies in vivo and for its physiological repression of Hox genes. Global disruption of sumoylation phenocopies a sop-2 mutation by causing ectopic Hox gene expression and homeotic transformations. Chimeric constructs in which the SOP-2 SAM domain is replaced with that derived from fruit fly or mammalian PcG proteins, but not those in which the SOP-2 SAM domain is replaced with the SAM domains of non-PcG proteins, confer appropriate in vivo nuclear localization and Hox gene repression. These observations indicate that sumoylation of PcG proteins, modulated by their evolutionarily conserved SAM domain, is essential to their physiological repression of Hox genes.

  2. Distinct p21 requirements for regulating normal and self-reactive T cells through IFN-γ production.

    PubMed

    Daszkiewicz, Lidia; Vázquez-Mateo, Cristina; Rackov, Gorjana; Ballesteros-Tato, André; Weber, Kathrin; Madrigal-Avilés, Adrián; Di Pilato, Mauro; Fotedar, Arun; Fotedar, Rati; Flores, Juana M; Esteban, Mariano; Martínez-A, Carlos; Balomenos, Dimitrios

    2015-01-09

    Self/non-self discrimination characterizes immunity and allows responses against pathogens but not self-antigens. Understanding the principles that govern this process is essential for designing autoimmunity treatments. p21 is thought to attenuate autoreactivity by limiting T cell expansion. Here, we provide direct evidence for a p21 role in controlling autoimmune T cell autoreactivity without affecting normal T cell responses. We studied C57BL/6, C57BL/6/lpr and MRL/lpr mice overexpressing p21 in T cells, and showed reduced autoreactivity and lymphadenopathy in C57BL/6/lpr, and reduced mortality in MRL/lpr mice. p21 inhibited effector/memory CD4(+) CD8(+) and CD4(-)CD8(-) lpr T cell accumulation without altering defective lpr apoptosis. This was mediated by a previously non-described p21 function in limiting T cell overactivation and overproduction of IFN-γ, a key lupus cytokine. p21 did not affect normal T cell responses, revealing differential p21 requirements for autoreactive and normal T cell activity regulation. The underlying concept of these findings suggests potential treatments for lupus and autoimmune lymphoproliferative syndrome, without compromising normal immunity.

  3. LET-711, the Caenorhabditis elegans NOT1 ortholog, is required for spindle positioning and regulation of microtubule length in embryos.

    PubMed

    DeBella, Leah R; Hayashi, Adam; Rose, Lesilee S

    2006-11-01

    Spindle positioning is essential for the segregation of cell fate determinants during asymmetric division, as well as for proper cellular arrangements during development. In Caenorhabditis elegans embryos, spindle positioning depends on interactions between the astral microtubules and the cell cortex. Here we show that let-711 is required for spindle positioning in the early embryo. Strong loss of let-711 function leads to sterility, whereas partial loss of function results in embryos with defects in the centration and rotation movements that position the first mitotic spindle. let-711 mutant embryos have longer microtubules that are more cold-stable than in wild type, a phenotype opposite to the short microtubule phenotype caused by mutations in the C. elegans XMAP215 homolog ZYG-9. Simultaneous reduction of both ZYG-9 and LET-711 can rescue the centration and rotation defects of both single mutants. let-711 mutant embryos also have larger than wild-type centrosomes at which higher levels of ZYG-9 accumulate compared with wild type. Molecular identification of LET-711 shows it to be an ortholog of NOT1, the core component of the CCR4/NOT complex, which plays roles in the negative regulation of gene expression at transcriptional and post-transcriptional levels in yeast, flies, and mammals. We therefore propose that LET-711 inhibits the expression of ZYG-9 and potentially other centrosome-associated proteins, in order to maintain normal centrosome size and microtubule dynamics during early embryonic divisions.

  4. Trophic factor-induced activity 'signature' regulates the functional expression of postsynaptic excitatory acetylcholine receptors required for synaptogenesis.

    PubMed

    Luk, Collin C; Lee, Arthur J; Wijdenes, Pierre; Zaidi, Wali; Leung, Andrew; Wong, Noelle Y; Andrews, Joseph; Syed, Naweed I

    2015-04-01

    Highly coordinated and coincidental patterns of activity-dependent mechanisms ("fire together wire together") are thought to serve as inductive signals during synaptogenesis, enabling neuronal pairing between specific sub-sets of excitatory partners. However, neither the nature of activity triggers, nor the "activity signature" of long-term neuronal firing in developing/regenerating neurons have yet been fully defined. Using a highly tractable model system comprising of identified cholinergic neurons from Lymnaea, we have discovered that intrinsic trophic factors present in the Lymnaea brain-conditioned medium (CM) act as a natural trigger for activity patterns in post- but not the presynaptic neuron. Using microelectrode array recordings, we demonstrate that trophic factors trigger stereotypical activity patterns that include changes in frequency, activity and variance. These parameters were reliable indicators of whether a neuron expressed functional excitatory or inhibitory nAChRs and synapse formation. Surprisingly, we found that the post- but not the presynaptic cell exhibits these changes in activity patterns, and that the functional expression of excitatory nAChRs required neuronal somata, de novo protein synthesis and voltage gated calcium channels. In summary, our data provides novel insights into trophic factor mediated actions on neuronal activity and its specific regulation of nAChR expression.

  5. Sustained Induction of Collagen Synthesis by TGF-β Requires Regulated Intramembrane Proteolysis of CREB3L1

    PubMed Central

    Chen, Qiuyue; Lee, Ching-En; Denard, Bray; Ye, Jin

    2014-01-01

    CREB3L1 (cAMP response element binding protein 3-like 1), a transcription factor synthesized as a membrane-bound precursor and activated through Regulated Intramembrane Proteolysis (RIP), is essential for collagen production by osteoblasts during bone development. Here, we show that TGF-β (transforming growth factor-β), a cytokine known to stimulate production of collagen during wound healing and fibrotic diseases, induces proteolytic activation of CREB3L1 in human A549 cells. This activation results from inhibition of expression of TM4SF20 (transmembrane 4 L6 family member 20), which normally inhibits RIP of CREB3L1. Cleavage of CREB3L1 releases its NH2-terminal domain from membranes, allowing it to enter the nucleus where it binds to Smad4 to activate transcription of genes encoding proteins required for assembly of collagen-containing extracellular matrix. Our findings raise the possibility that inhibition of RIP of CREB3L1 could prevent excess deposition of collagen in certain fibrotic diseases. PMID:25310401

  6. The extracellular domain of Smoothened regulates ciliary localization and is required for high-level Hh signaling.

    PubMed

    Aanstad, Pia; Santos, Nicole; Corbit, Kevin C; Scherz, Paul J; Trinh, Le A; Salvenmoser, Willi; Huisken, Jan; Reiter, Jeremy F; Stainier, Didier Y R

    2009-06-23

    Members of the Hedgehog (Hh) family of secreted proteins function as morphogens to pattern developing tissues and control cell proliferation. The seven-transmembrane domain (7TM) protein Smoothened (Smo) is essential for the activation of all levels of Hh signaling. However, the mechanisms by which Smo differentially activates low- or high-level Hh signaling are not known. Here we show that a newly identified mutation in the extracellular domain (ECD) of zebrafish Smo attenuates Smo signaling. The Smo agonist purmorphamine induces the stabilization, ciliary translocation, and high-level signaling of wild-type Smo. In contrast, purmorphamine induces the stabilization but not the ciliary translocation or high-level signaling of the Smo ECD mutant protein. Surprisingly, a truncated form of Smo that lacks the cysteine-rich domain of the ECD localizes to the cilium but is unable to activate high-level Hh signaling. We also present evidence that cilia may be required for Hh signaling in early zebrafish embryos. These data indicate that the ECD, previously thought to be dispensable for vertebrate Smo function, both regulates Smo ciliary localization and is essential for high-level Hh signaling.

  7. MinD-dependent conformational changes in MinE required for the Min oscillator to spatially regulate cytokinesis

    PubMed Central

    Park, Kyung-Tae; Wu, Wei; Battaile, Kevin P.; Lovell, Scott; Holyoak, Todd; Lutkenhaus, Joe

    2011-01-01

    Summary MinD recruits MinE to the membrane leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring in E. coli. How these proteins interact, however, is not clear since the MinD binding regions of MinE are sequestered within a 6-stranded β-sheet and masked by N-terminal helices. Here, minE mutations are isolated that restore interaction to some MinD and MinE mutants. These mutations alter the MinE structure releasing the MinD binding regions and N-terminal helices that bind MinD and the membrane, respectively. Crystallization of MinD-MinE complexes reveals a 4-stranded β-sheet MinE dimer with the released β strands (MinD binding regions) converted to α-helices bound to MinD dimers. These results suggest a 6 stranded, β-sheet dimer of MinE ‘senses’ MinD and switches to a 4-stranded β-sheet dimer that binds MinD and contributes to membrane binding. Also, the results indicate how MinE persists at the MinD-membrane surface. PMID:21816275

  8. OsJAR1 is required for JA-regulated floret opening and anther dehiscence in rice.

    PubMed

    Xiao, Yuguo; Chen, Yi; Charnikhova, Tatsiana; Mulder, Patrick P J; Heijmans, Jeroen; Hoogenboom, Angela; Agalou, Adamantia; Michel, Corinne; Morel, Jean-Benoit; Dreni, Ludovico; Kater, Martin M; Bouwmeester, Harro; Wang, Mei; Zhu, Zhen; Ouwerkerk, Pieter B F

    2014-09-01

    Jasmonates are important phytohormones regulating reproductive development. We used two recessive rice Tos17 alleles of OsJAR1, osjar1-2 and osjar1-3, to study the biological function of jasmonates in rice anthesis. The florets of both osjar1 alleles stayed open during anthesis because the lodicules, which control flower opening in rice, were not withering on time. Furthermore, dehiscence of the anthers filled with viable pollen, was impaired, resulting in lower fertility. In situ hybridization and promoter GUS transgenic analysis confirmed OsJAR1 expression in these floral tissues. Flower opening induced by exogenous applied methyl jasmonate was impaired in osjar1 plants and was restored in a complementation experiment with transgenics expressing a wild type copy of OsJAR1 controlled by a rice actin promoter. Biochemical analysis showed that OsJAR1 encoded an enzyme conjugating jasmonic acid (JA) to at least Ile, Leu, Met, Phe, Trp and Val and both osjar1 alleles had substantial reduction in content of JA-Ile, JA-Leu and JA-Val in florets. We conclude that OsJAR1 is a JA-amino acid synthetase that is required for optimal flower opening and closing and anther dehiscence in rice.

  9. Dynein-2 affects the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena thermophila.

    PubMed

    Rajagopalan, Vidyalakshmi; Subramanian, Aswati; Wilkes, David E; Pennock, David G; Asai, David J

    2009-01-01

    Eukaryotic cilia and flagella are assembled and maintained by the bidirectional intraflagellar transport (IFT). Studies in alga, nematode, and mouse have shown that the heavy chain (Dyh2) and the light intermediate chain (D2LIC) of the cytoplasmic dynein-2 complex are essential for retrograde intraflagellar transport. In these organisms, disruption of either dynein-2 component results in short cilia/flagella with bulbous tips in which excess IFT particles have accumulated. In Tetrahymena, the expression of the DYH2 and D2LIC genes increases during reciliation, consistent with their roles in IFT. However, the targeted elimination of either DYH2 or D2LIC gene resulted in only a mild phenotype. Both knockout cell lines assembled motile cilia, but the cilia were of more variable lengths and less numerous than wild-type controls. Electron microscopy revealed normally shaped cilia with no swelling and no obvious accumulations of material in the distal ciliary tip. These results demonstrate that dynein-2 contributes to the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena.

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

    PubMed

    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.

  11. LET-711, the Caenorhabditis elegans NOT1 Ortholog, Is Required for Spindle Positioning and Regulation of Microtubule Length in Embryos

    PubMed Central

    DeBella, Leah R.; Hayashi, Adam

    2006-01-01

    Spindle positioning is essential for the segregation of cell fate determinants during asymmetric division, as well as for proper cellular arrangements during development. In Caenorhabditis elegans embryos, spindle positioning depends on interactions between the astral microtubules and the cell cortex. Here we show that let-711 is required for spindle positioning in the early embryo. Strong loss of let-711 function leads to sterility, whereas partial loss of function results in embryos with defects in the centration and rotation movements that position the first mitotic spindle. let-711 mutant embryos have longer microtubules that are more cold-stable than in wild type, a phenotype opposite to the short microtubule phenotype caused by mutations in the C. elegans XMAP215 homolog ZYG-9. Simultaneous reduction of both ZYG-9 and LET-711 can rescue the centration and rotation defects of both single mutants. let-711 mutant embryos also have larger than wild-type centrosomes at which higher levels of ZYG-9 accumulate compared with wild type. Molecular identification of LET-711 shows it to be an ortholog of NOT1, the core component of the CCR4/NOT complex, which plays roles in the negative regulation of gene expression at transcriptional and post-transcriptional levels in yeast, flies, and mammals. We therefore propose that LET-711 inhibits the expression of ZYG-9 and potentially other centrosome-associated proteins, in order to maintain normal centrosome size and microtubule dynamics during early embryonic divisions. PMID:16971515

  12. Regulation of expression of rice thaumatin-like protein: inducibility by elicitor requires promoter W-box elements.

    PubMed

    Hiroyuki, Kanzaki; Terauchi, Ryohei

    2008-09-01

    Rice thaumatin-like protein (Rtlp1) is a high-molecular-weight antimicrobial pathogenesis-related protein that plays a role in plant stress response. This study examines transcriptional regulation of Rtlp1 using wild type and transgenic rice plants carrying a beta-glucuronidase (GUS) reporter gene driven by the Rtlp1 promoter (pRtlp1GUS). The Rtlp1 promoter is induced within 6 h after infection with rice blast fungus (Magnaporthe grisea). The Rtlp1 promoter is also induced by salicylic acid (SA), methyl jasmonate (MeJA), wounding or an elicitor from rice blast fungus. The function of the pRtlp1GUS reporter gene was analyzed by deletion mapping and transient expression assays in cell culture. A 120 bp truncated fusion construct with six W-boxes (5'-TGAC-3') demonstrated a strong dose-dependent elicitor-response. These results suggest that W-box elements are required for the response of the Rtlp1 promoter to fungal elicitors.

  13. 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

    SciTech Connect

    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.

  14. Endoplasmic Reticulum-Associated Degradation Is Required for Cold Adaptation and Regulation of Sterol Biosynthesis in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Loertscher, Jennifer; Larson, Lynnelle L.; Matson, Clinton K.; Parrish, Mark L.; Felthauser, Alicia; Sturm, Aaron; Tachibana, Christine; Bard, Martin; Wright, Robin

    2006-01-01

    Endoplasmic reticulum-associated degradation (ERAD) mediates the turnover of short-lived and misfolded proteins in the ER membrane or lumen. In spite of its important role, only subtle growth phenotypes have been associated with defects in ERAD. We have discovered that the ERAD proteins Ubc7 (Qri8), Cue1, and Doa10 (Ssm4) are required for growth of yeast that express high levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Interestingly, the observed growth defect was exacerbated at low temperatures, producing an HMGR-dependent cold sensitivity. Yeast strains lacking UBC7, CUE1, or DOA10 also assembled aberrant karmellae (ordered arrays of membranes surrounding the nucleus that assemble when HMGR is expressed at high levels). However, rather than reflecting the accumulation of abnormal karmellae, the cold sensitivity of these ERAD mutants was due to increased HMGR catalytic activity. Mutations that compromise proteasomal function also resulted in cold-sensitive growth of yeast with elevated HMGR, suggesting that improper degradation of ERAD targets might be responsible for the observed cold-sensitive phenotype. However, the essential ERAD targets were not the yeast HMGR enzymes themselves. The sterol metabolite profile of ubc7Δ cells was altered relative to that of wild-type cells. Since sterol levels are known to regulate membrane fluidity, the viability of ERAD mutants expressing normal levels of HMGR was examined at low temperatures. Cells lacking UBC7, CUE1, or DOA10 were cold sensitive, suggesting that these ERAD proteins have a role in cold adaptation, perhaps through effects on sterol biosynthesis. PMID:16607018

  15. FOXF1 Transcription Factor Is Required for Formation of Embryonic Vasculature by Regulating VEGF Signaling in Endothelial Cells

    PubMed Central

    Ren, Xiaomeng; Ustiyan, Vladimir; Pradhan, Arun; Cai, Yuqi; Havrilak, Jamie A.; Bolte, Craig S.; Shannon, John M.; Kalin, Tanya V.; Kalinichenko, Vladimir V.

    2016-01-01

    Rationale Inactivating mutations in the FOXF1 gene locus are frequently found in patients with Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACD/MPV), a lethal congenital disorder, which is characterized by severe abnormalities in the respiratory, cardio-vascular and gastro-intestinal systems. In mice, haploinsufficiency of the Foxf1 gene causes alveolar capillary dysplasia and developmental defects in lung, intestinal and gall bladder morphogenesis. Objective While FOXF1 is expressed in multiple mesenchyme-derived cell types, cellular origins and molecular mechanisms of developmental abnormalities in FOXF1-deficient mice and ACD/MPV patients remain uncharacterized due to lack of mouse models with cell-restricted inactivation of the Foxf1 gene. In the present study, the role of FOXF1 in endothelial cells was examined using a conditional knockout approach. Methods and Results A novel mouse line harboring Foxf1-floxed alleles was generated by homologous recombination. Tie2-Cre and Pdgfb-CreER transgenes were used to delete Foxf1 from endothelial cells. FOXF1-deficient embryos exhibited embryonic lethality, growth retardation, polyhydramnios, cardiac ventricular hypoplasia and vascular abnormalities in the lung, placenta, yolk sac and retina. Deletion of FOXF1 from endothelial cells reduced endothelial proliferation, increased apoptosis, inhibited VEGF signaling and decreased expression of endothelial genes critical for vascular development, including VEGF receptors Flt1 and Flk1, Pdgfb, Pecam1, CD34, integrin β3, ephrin B2, Tie2 and the non-coding RNA Fendrr. ChIP assay demonstrated that Flt1, Flk1, Pdgfb, Pecam1 and Tie2 genes are direct transcriptional targets of FOXF1. Conclusions FOXF1 is required for formation of embryonic vasculature by regulating endothelial genes critical for vascular development and VEGF signaling. PMID:25091710

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

    PubMed

    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. Copyright © 2016 the American Physiological Society.

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

    PubMed

    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.

  18. Eukaryotic translation initiation factor eIFiso4G is required to regulate violaxanthin De-epoxidase expression in Arabidopsis.

    PubMed

    Chen, Zhong; Jolley, Blair; Caldwell, Christian; Gallie, Daniel R

    2014-05-16

    The eukaryotic translation initiation factor (eIF) 4G is a scaffold protein that organizes the assembly of those initiation factors needed to recruit the 40 S ribosomal subunit to an mRNA. Plants, like many eukaryotes, express two eIF4G isoforms. eIFiso4G, one of the isoforms specific to plants, is unique among eukaryotic eIF4G proteins in that it is highly divergent and unusually small in size, raising the possibility of functional specialization. In this study, the role of eIFiso4G in plant growth was investigated using null mutants for the eIF4G isoforms in Arabidopsis. eIFiso4G loss of function mutants exhibited smaller cell, leaf, plant size, and biomass accumulation that correlated with its reduced photosynthetic activity, phenotypes not observed with the eIF4G loss of function mutant. Although no change in photorespiration or dark respiration was observed in the eIFiso4G loss of function mutant, a reduction in chlorophyll levels and an increase in the level of nonphotochemical quenching were observed. An increase in xanthophyll cycle activity and the generation of reactive oxygen species contributed to the qE and qI components of nonphotochemical quenching, respectively. An increase in the transcript and protein levels of violaxanthin de-epoxidase in the eIFiso4G loss of function mutant and an increase in its xanthophyll de-epoxidation state correlated with the higher qE associated with loss of eIFiso4G expression. These observations indicate that eIFiso4G expression is required to regulate violaxanthin de-epoxidase expression and to support photosynthetic activity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Functional characterization of a cyanobacterial OmpR/PhoB class transcription factor binding site controlling light color responses.

    PubMed

    Bezy, Ryan P; Kehoe, David M

    2010-11-01

    Complementary chromatic acclimation (CCA) allows many cyanobacteria to change the composition of their light-harvesting antennae for maximal absorption of different wavelengths of light. In the freshwater species Fremyella diplosiphon, this process is controlled by the ratio of red to green light and allows the differential regulation of two subsets of genes in the genome. This response to ambient light color is controlled in part by a two-component system that includes a phytochrome class photoreceptor and a response regulator with an OmpR/PhoB class DNA binding domain called RcaC. During growth in red light, RcaC is able to simultaneously activate expression of red light-induced genes and repress expression of green light-induced genes through binding to the L box promoter element. Here we investigate how the L box functions as both an activator and a repressor under the same physiological conditions by analyzing the effects of changing the position, orientation, and sequence of the L box. We demonstrate that changes in the local sequences surrounding the L box affect the strength of its activity and that the activating and repressing functions of the L box are orientation dependent. Also, the spacing between the L box and the transcription start site is critical for it to work as an activator, while its repressing role during light regulation requires additional upstream and downstream DNA sequence elements. The latter result suggests that the repressing function of RcaC requires it to operate in association with multiple additional DNA binding proteins, at least one of which is functioning as an activator.

  20. 41 CFR 101-1.4902-2053 - GSA Form 2053, Agency Consolidated Requirements for GSA Regulations and Other External Issuances.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...-2053 Public Contracts and Property Management Federal Property Management Regulations System FEDERAL PROPERTY MANAGEMENT REGULATIONS GENERAL 1-INTRODUCTION 1.49-Illustrations of Forms § 101-1.4902-2053 GSA... 41 Public Contracts and Property Management 2 2010-07-01 2010-07-01 true GSA Form 2053, Agency...

  1. Fusarium verticillioides SGE1 is required for full virulence and regulates expression of protein effector and secondary metabolite biosynthetic genes

    USDA-ARS?s Scientific Manuscript database

    The transition from one lifestyle to another in some fungi is initiated by a single orthologous gene, SGE1, that regulates markedly different genes in different fungi. Despite these differences, many of the regulated genes encode effector proteins or proteins involved in the synthesis of secondary m...

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  4. Fur Negatively Regulates hns and Is Required for the Expression of HilA and Virulence in Salmonella enterica Serovar Typhimurium▿ †

    PubMed Central

    Troxell, Bryan; Sikes, Michael L.; Fink, Ryan C.; Vazquez-Torres, Andres; Jones-Carson, Jessica; Hassan, Hosni M.

    2011-01-01

    Iron is an essential element for the survival of living cells. However, excess iron is toxic, and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In Salmonella, the Salmonella pathogenicity island 1 (SPI-1) encodes a type three secretion system, which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of hilA is Fur. The mechanism of hilA regulation by Fur is unknown. We report here that Fur is required for virulence in Salmonella enterica serovar Typhimurium and that Fur is required for the activation of hilA, as well as of other HilA-dependent genes, invF and sipC. The Fur-dependent regulation of hilA was independent of PhoP, a known repressor of hilA. Instead, the expression of the gene coding for the histone-like protein, hns, was significantly derepressed in the fur mutant. Indeed, the activation of hilA by Fur was dependent on 28 nucleotides located upstream of hns. Moreover, we used chromatin immunoprecipitation to show that Fur bound, in vivo, to the upstream region of hns in a metal-dependent fashion. Finally, deletion of fur in an hns mutant resulted in Fur-independent activation of hilA. In conclusion, Fur activates hilA by repressing the expression of hns. PMID:21075923

  5. Fur negatively regulates hns and is required for the expression of HilA and virulence in Salmonella enterica serovar Typhimurium.

    PubMed

    Troxell, Bryan; Sikes, Michael L; Fink, Ryan C; Vazquez-Torres, Andres; Jones-Carson, Jessica; Hassan, Hosni M

    2011-01-01

    Iron is an essential element for the survival of living cells. However, excess iron is toxic, and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In Salmonella, the Salmonella pathogenicity island 1 (SPI-1) encodes a type three secretion system, which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of hilA is Fur. The mechanism of hilA regulation by Fur is unknown. We report here that Fur is required for virulence in Salmonella enterica serovar Typhimurium and that Fur is required for the activation of hilA, as well as of other HilA-dependent genes, invF and sipC. The Fur-dependent regulation of hilA was independent of PhoP, a known repressor of hilA. Instead, the expression of the gene coding for the histone-like protein, hns, was significantly derepressed in the fur mutant. Indeed, the activation of hilA by Fur was dependent on 28 nucleotides located upstream of hns. Moreover, we used chromatin immunoprecipitation to show that Fur bound, in vivo, to the upstream region of hns in a metal-dependent fashion. Finally, deletion of fur in an hns mutant resulted in Fur-independent activation of hilA. In conclusion, Fur activates hilA by repressing the expression of hns.

  6. Regulation of cell reversal frequency in Myxococcus xanthus requires the balanced activity of CheY-like domains in FrzE and FrzZ.

    PubMed

    Kaimer, Christine; Zusman, David R

    2016-04-01

    The Frz pathway of Myxococcus xanthus controls cell reversal frequency to support directional motility during swarming and fruiting body formation. Previously, we showed that phosphorylation of the response regulator FrzZ correlates with reversal frequencies, suggesting that this activity represents the output of the Frz pathway. Here, we tested the effect of different expression levels of FrzZ and its cognate kinase FrzE on M. xanthus motility. FrzZ overexpression caused a slight increase in phosphorylation and reversals. By contrast, FrzE overexpression abolished phosphorylation of FrzZ; this inhibition required the response regulator domain of FrzE. FrzZ phosphorylation was restored when both FrzE and FrzZ were overexpressed together. Our results show that the response regulator domain of FrzE is a negative regulator of FrzE kinase activity. This inhibition can be modulated by FrzZ, which acts as a positive regulator. Interestingly, fluorescence microscopy revealed that FrzZ and FrzE localize differently: FrzE colocalizes with the FrzCD receptor and the nucleoid, while FrzZ shows dispersed and polar localization. However, FrzZ binds tightly to the truncated variant FrzEΔ(CheY) . This indicates that the response regulator domain of FrzE is required for the interaction between FrzE and FrzZ to be transient, providing an unexpected regulatory output to the Frz pathway.

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

    PubMed

    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.

  8. 40 CFR Appendix A to Part 282 - State Requirements Incorporated by Reference in Part 282 of the Code of Federal Regulations

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 27 2014-07-01 2014-07-01 false State Requirements Incorporated by Reference in Part 282 of the Code of Federal Regulations A Appendix A to Part 282 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) APPROVED UNDERGROUND STORAGE TANK PROGRAMS Pt. 282, App. A Appendix A...

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

    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

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

    PubMed

    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

  13. The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa.

    PubMed Central

    Whitchurch, C B; Alm, R A; Mattick, J S

    1996-01-01

    Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate. AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified. Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae. Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility. However, FimS does not appear to be required for alginate production in mucoid strains. FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems. The alternative sigma factor AlgU also affects both alginate production and twitching motility. Therefore, these two virulence determinants appear to be closely associated and coordinately regulated. Images Fig. 1 Fig. 2 Fig. 3 PMID:8790418

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

    PubMed Central

    Dressaire, Clémentine; Barahona, Susana; Galego, Lisete; Kaever, Volkhard; Jenal, Urs

    2017-01-01

    ABSTRACT 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. PMID:28928205

  15. Requirements for Defining Utility Drive Cycles: An Exploratory Analysis of Grid Frequency Regulation Data for Establishing Battery Performance Testing Standards

    SciTech Connect

    Hafen, Ryan P.; Vishwanathan, Vilanyur V.; Subbarao, Krishnappa; Kintner-Meyer, Michael CW

    2011-10-19

    Battery testing procedures are important for understanding battery performance, including degradation over the life of the battery. Standards are important to provide clear rules and uniformity to an industry. The work described in this report addresses the need for standard battery testing procedures that reflect real-world applications of energy storage systems to provide regulation services to grid operators. This work was motivated by the need to develop Vehicle-to-Grid (V2G) testing procedures, or V2G drive cycles. Likewise, the stationary energy storage community is equally interested in standardized testing protocols that reflect real-world grid applications for providing regulation services. As the first of several steps toward standardizing battery testing cycles, this work focused on a statistical analysis of frequency regulation signals from the Pennsylvania-New Jersey-Maryland Interconnect with the goal to identify patterns in the regulation signal that would be representative of the entire signal as a typical regulation data set. Results from an extensive time-series analysis are discussed, and the results are explained from both the statistical and the battery-testing perspectives. The results then are interpreted in the context of defining a small set of V2G drive cycles for standardization, offering some recommendations for the next steps toward standardizing testing protocols.

  16. DasR is a pleiotropic regulator required for antibiotic production, pigment biosynthesis, and morphological development in Saccharopolyspora erythraea.

    PubMed

    Liao, Cheng-Heng; Xu, Ya; Rigali, Sébastien; Ye, Bang-Ce

    2015-12-01

    The GntR-family transcription regulator, DasR, was previously identified as pleiotropic, controlling the primary amino sugar N-acetylglucosamine (GlcNAc) and chitin metabolism in Saccharopolyspora erythraea and Streptomyces coelicolor. Due to the remarkable regulatory impact of DasR on antibiotic production and development in the model strain of S. coelicolor, we here identified and characterized the role of DasR to secondary metabolite production and morphological development in industrial erythromycin-producing S. erythraea. The physiological studies have shown that a constructed deletion of dasR in S. erythraea resulted in antibiotic, pigment, and aerial hyphae production deficit in a nutrient-rich condition. DNA microarray assay, combined with quantitative real-time reverse transcription PCR (qRT-PCR), confirmed these results by showing the downregulation of the genes relating to secondary metabolite production in the dasR null mutant. Notably, electrophoretic mobility shift assays (EMSA) showed DasR as being the first identified regulator that directly regulates the pigment biosynthesis rpp gene cluster. In addition, further studies indicated that GlcNAc, the major nutrient signal of DasR-responsed regulation, blocked secondary metabolite production and morphological development. The effects of GlcNAc were shown to be caused by DasR mediation. These findings demonstrated that DasR is an important pleiotropic regulator for both secondary metabolism and morphological development in S. erythraea, providing new insights for the genetic engineering of S. erythraea with increased erythromycin production.

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

    PubMed

    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. Clavibacter michiganensis subsp. michiganensis Vatr1 and Vatr2 Transcriptional Regulators Are Required for Virulence in Tomato.

    PubMed

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

    2015-01-01

    The plant pathogen Clavibacter michiganensis subsp. michiganensis (Cmm) is a Gram-positive bacterium responsible for wilt and canker disease of tomato. While disease development is well characterized and diagnosed, molecular mechanisms of Cmm virulence are poorly understood. Here, we identified and characterized two Cmm transcriptional regulators, Vatr1 and Vatr2, that are involved in pathogenicity of Cmm. 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. While 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 Cmm and to reduced ethylene production in the plant. Transcriptomic analysis of wild-type Cmm 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 Cmm virulence.

  19. Regulation of the level of uncoupling protein in brown adipose tissue by insulin requires the mediation of the sympathetic nervous system.

    PubMed

    Géloën, A; Trayhurn, P

    1990-07-16

    The role of the sympathetic nervous system in the regulation by insulin of the level of uncoupling protein in brown adipose tissue has been examined. The amount of uncoupling protein was substantially reduced in streptozotocin-diabetic rats, while insulin replacement to diabetic animals induced a partial restoration. Unilateral denervation of the interscapular brown fat pads also lowered the amount of uncoupling protein, and in diabetic animals inhibited the stimulation of the level of the protein by insulin replacement. Maintenance of normal uncoupling protein levels requires both insulin and the sympathetic system; regulation of the protein by insulin involves sympathetic mediation.

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

    PubMed

    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

  1. Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton

    PubMed Central

    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.

    2013-01-01

    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

  2. A PmrB-Regulated Deacetylase Required for Lipid A Modification and Polymyxin Resistance in Acinetobacter baumannii.

    PubMed

    Chin, Chui-Yoke; Gregg, Kelsey A; Napier, Brooke A; Ernst, Robert K; Weiss, David S

    2015-12-01

    Emerging resistance to "last-resort" polymyxin antibiotics in Gram-negative bacteria is a significant threat to public health. We identified the Acinetobacter baumannii NaxD deacetylase as a critical mediator of lipid A modification resulting in polymyxin resistance and demonstrated that naxD is regulated by the sensor kinase PmrB. This represents the first description of a specific PmrB-regulated gene contributing to polymyxin resistance in A. baumannii and highlights NaxD as a putative drug target to reverse polymyxin resistance.

  3. A PmrB-Regulated Deacetylase Required for Lipid A Modification and Polymyxin Resistance in Acinetobacter baumannii

    PubMed Central

    Chin, Chui-Yoke; Gregg, Kelsey A.; Napier, Brooke A.; Ernst, Robert K.

    2015-01-01

    Emerging resistance to “last-resort” polymyxin antibiotics in Gram-negative bacteria is a significant threat to public health. We identified the Acinetobacter baumannii NaxD deacetylase as a critical mediator of lipid A modification resulting in polymyxin resistance and demonstrated that naxD is regulated by the sensor kinase PmrB. This represents the first description of a specific PmrB-regulated gene contributing to polymyxin resistance in A. baumannii and highlights NaxD as a putative drug target to reverse polymyxin resistance. PMID:26459891

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

    PubMed

    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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26294668

  6. Structural Requirements for Sorting Pro-Vasopressin to the Regulated Secretory Pathway in a Neuronal Cell Line

    PubMed Central

    Cool, David R.; Jackson, Steven B.; Waddell, Karen S.

    2009-01-01

    Vasopressin is a peptide hormone normally secreted via the regulated secretory pathway in neuro-endocrine cells. In an effort to determine which region of vasopressin contains sufficient information for sorting, we created five constructs with the cDNA for vasopressin or regions of vasopressin in frame with the gene for green fluorescent protein (GFP). Fluorescence microscopy of Neuro-2a cells expressing the constructs revealed full-length vasopressin-GFP (VP-GFP), neurophysin-GFP (NP-GFP) and arginine-vasopressin/neurophysin-GFP (AN-GFP), were localized to punctate granules in the neurites and accumulated at the tips of neurites, characteristic of regulated secretory granules. These fusion proteins were secreted in a regulated manner as determined by pulse-chase labeling experiments. Two other chimeric proteins, signalpeptide-GFP and AVP-GFP were localized to a perinuclear region, characteristic of the endoplasmic reticulum. Pulse/chase [35S]labeling followed by immunoprecipitation using anti-GFP antibody indicated that these two fusion proteins were constitutively secreted. We conclude that the neurophysin region of pro-vasopressin contains information that is both sufficient and necessary for sorting GFP into the regulated secretory pathway. PMID:19830265

  7. p73 is required for endothelial cell differentiation, migration and the formation of vascular networks regulating VEGF and TGFβ signaling

    PubMed Central

    Fernandez-Alonso, R; Martin-Lopez, M; Gonzalez-Cano, L; Garcia, S; Castrillo, F; Diez-Prieto, I; Fernandez-Corona, A; Lorenzo-Marcos, M E; Li, X; Claesson-Welsh, L; Marques, M M; Marin, M C

    2015-01-01

    Vasculogenesis, the establishment of the vascular plexus and angiogenesis, branching of new vessels from the preexisting vasculature, involves coordinated endothelial differentiation, proliferation and migration. Disturbances in these coordinated processes may accompany diseases such as cancer. We hypothesized that the p53 family member p73, which regulates cell differentiation in several contexts, may be important in vascular development. We demonstrate that p73 deficiency perturbed vascular development in the mouse retina, decreasing vascular branching, density and stability. Furthermore, p73 deficiency could affect non endothelial cells (ECs) resulting in reduced in vivo proangiogenic milieu. Moreover, p73 functional inhibition, as well as p73 deficiency, hindered vessel sprouting, tubulogenesis and the assembly of vascular structures in mouse embryonic stem cell and induced pluripotent stem cell cultures. Therefore, p73 is necessary for EC biology and vasculogenesis and, in particular, that DNp73 regulates EC migration and tube formation capacity by regulation of expression of pro-angiogenic factors such as transforming growth factor-β and vascular endothelial growth factors. DNp73 expression is upregulated in the tumor environment, resulting in enhanced angiogenic potential of B16-F10 melanoma cells. Our results demonstrate, by the first time, that differential p73-isoform regulation is necessary for physiological vasculogenesis and angiogenesis and DNp73 overexpression becomes a positive advantage for tumor progression due to its pro-angiogenic capacity. PMID:25571973

  8. p73 is required for endothelial cell differentiation, migration and the formation of vascular networks regulating VEGF and TGFβ signaling.

    PubMed

    Fernandez-Alonso, R; Martin-Lopez, M; Gonzalez-Cano, L; Garcia, S; Castrillo, F; Diez-Prieto, I; Fernandez-Corona, A; Lorenzo-Marcos, M E; Li, X; Claesson-Welsh, L; Marques, M M; Marin, M C

    2015-08-01

    Vasculogenesis, the establishment of the vascular plexus and angiogenesis, branching of new vessels from the preexisting vasculature, involves coordinated endothelial differentiation, proliferation and migration. Disturbances in these coordinated processes may accompany diseases such as cancer. We hypothesized that the p53 family member p73, which regulates cell differentiation in several contexts, may be important in vascular development. We demonstrate that p73 deficiency perturbed vascular development in the mouse retina, decreasing vascular branching, density and stability. Furthermore, p73 deficiency could affect non endothelial cells (ECs) resulting in reduced in vivo proangiogenic milieu. Moreover, p73 functional inhibition, as well as p73 deficiency, hindered vessel sprouting, tubulogenesis and the assembly of vascular structures in mouse embryonic stem cell and induced pluripotent stem cell cultures. Therefore, p73 is necessary for EC biology and vasculogenesis and, in particular, that DNp73 regulates EC migration and tube formation capacity by regulation of expression of pro-angiogenic factors such as transforming growth factor-β and vascular endothelial growth factors. DNp73 expression is upregulated in the tumor environment, resulting in enhanced angiogenic potential of B16-F10 melanoma cells. Our results demonstrate, by the first time, that differential p73-isoform regulation is necessary for physiological vasculogenesis and angiogenesis and DNp73 overexpression becomes a positive advantage for tumor progression due to its pro-angiogenic capacity.

  9. Down-Regulation of Type I Runx2 Mediated by Dexamethasone Is Required for 3T3-L1 Adipogenesis

    PubMed Central

    Zhang, You-you; Li, Xi; Qian, Shu-wen; Guo, Liang; Huang, Hai-yan; He, Qun; Liu, Yuan; Ma, Chun-gu

    2012-01-01

    Runx2, a runt-related transcriptional factor family member, is involved in the regulation of osteoblast differentiation. Interestingly, it is abundant in growth-arrested 3T3-L1 preadipocytes and was dramatically down-regulated during adipocyte differentiation. Knockdown of Runx2 expression promoted 3T3-L1 adipocyte differentiation, whereas overexpression inhibited adipocyte differentiation and promoted the trans-differentiation of 3T3-L1 preadipocytes to bone cells. Runx2 was down-regulated specifically by dexamethasone (DEX). Only type I Runx2 was expressed in 3T3-L1 preadipocytes. Using luciferase assay and chromatin immunoprecipitation-quantitative PCR analysis, it was found that DEX repressed this type of Runx2 at the transcriptional level through direct binding of the glucocorticoid receptor (GR) to a GR-binding element in the Runx2 P2 promoter. Further studies indicated that GR recruited histone deacetylase 1 to the Runx2 P2 promoter which then mediated the deacetylation of histone H4 and down-regulated Runx2 expression. Runx2 might play its repressive role through the induction of p27 expression, which blocked 3T3-L1 adipocyte differentiation by inhibiting mitotic clonal expansion. Taken together, we identified Runx2 as a new downstream target of DEX and explored a new pathway between DEX, Runx2, and p27 which contributed to the mechanism of the 3T3-L1 adipocyte differentiation. PMID:22422618

  10. Regulation of the L-type calcium channel by alpha 5beta 1 integrin requires signaling between focal adhesion proteins.

    PubMed

    Wu, X; Davis, G E; Meininger, G A; Wilson, E; Davis, M J

    2001-08-10

    The L-type calcium channel is the major calcium influx pathway in vascular smooth muscle and is regulated by integrin ligands, suggesting an important link between extracellular matrix and vascular tone regulation in tissue injury and remodeling. We examined the role of integrin-linked tyrosine kinases and focal adhesion proteins in regulation of L-type calcium current in single vascular myocytes. Soluble tyrosine kinase inhibitors blocked the increase in current produced by alpha(5) integrin antibody or fibronectin, whereas tyrosine phosphatase inhibition enhanced the effect. Cell dialysis with an antibody to focal adhesion kinase or with FRNK, the C-terminal noncatalytic domain of focal adhesion kinase, produced moderate (24 or 18%, respectively) inhibition of basal current but much greater inhibition (63 or 68%, respectively) of integrin-enhanced current. A c-Src antibody and peptide inhibitors of the Src homology-2 domain or a putative Src tyrosine phosphorylation site on the channel produced similar inhibition. Antibodies to the cytoskeletal proteins paxillin and vinculin, but not alpha-actinin, inhibited integrin-dependent current by 65-80%. Therefore, alpha(5)beta(1) integrin appears to regulate a tyrosine phosphorylation cascade involving Src and various focal adhesion proteins that control the function of the L-type calcium channel. This interaction may represent a novel mechanism for control of calcium influx in vascular smooth muscle and other cell types.

  11. Myosin IIb-dependent Regulation of Actin Dynamics Is Required for N-Methyl-D-aspartate Receptor Trafficking during Synaptic Plasticity.

    PubMed

    Bu, Yunfei; Wang, Ning; Wang, Shaoli; Sheng, Tao; Tian, Tian; Chen, Linlin; Pan, Weiwei; Zhu, Minsheng; Luo, Jianhong; Lu, Wei

    2015-10-16

    N-Methyl-d-aspartate receptor (NMDAR) synaptic incorporation changes the number of NMDARs at synapses and is thus critical to various NMDAR-dependent brain functions. To date, the molecules involved in NMDAR trafficking and the underlying mechanisms are poorly understood. Here, we report that myosin IIb is an essential molecule in NMDAR synaptic incorporation during PKC- or θ burst stimulation-induced synaptic plasticity. Moreover, we demonstrate that myosin light chain kinase (MLCK)-dependent actin reorganization contributes to NMDAR trafficking. The findings from additional mutual occlusion experiments demonstrate that PKC and MLCK share a common signaling pathway in NMDAR-mediated synaptic regulation. Because myosin IIb is the primary substrate of MLCK and can regulate actin dynamics during synaptic plasticity, we propose that the MLCK- and myosin IIb-dependent regulation of actin dynamics is required for NMDAR trafficking during synaptic plasticity. This study provides important insights into a mechanical framework for understanding NMDAR trafficking associated with synaptic plasticity.

  12. Light regulation of Fed-1 mRNA requires an element in the 5' untranslated region and correlates with differential polyribosome association.

    PubMed Central

    Dickey, L F; Petracek, M E; Nguyen, T T; Hansen, E R; Thompson, W F

    1998-01-01

    Light regulation of Fed-1 mRNA abundance in the leaves of green plants is primarily a post-transcriptional process. Previously, we have shown that the Fed-1 mRNA light response requires an open reading frame, indicating that the light regulation of the mRNA depends on its concurrent translation. We now show that light-induced increases in Fed-1 mRNA abundance are associated with increases in polyribosome association that require both a functional AUG and a normal Fed-1 translational start context. We also present evidence that light regulation of Fed-1 mRNA levels requires more than efficient translation per se. Substitution of the efficiently translated tobacco mosaic virus Omega 5' untranslated region resulted in a loss of Fed-1 light regulation. In addition, we identified a CAT T repeat element located near the 5' terminus of the Fed-1 5' untranslated region that is essential for light regulation. We introduced two different mutations in the CAT T repeat element, but only one of these substitutions blocked the normal light effect on polyribosome association, whereas both altered dark-induced Fed-1 mRNA disappearance. The element may thus be important for Fed-1 mRNA stability rather than polyribosome loading. We propose a model in which Fed-1 mRNA is relatively stable when it is associated with polyribosomes in illuminated plants but in darkness is not polyribosome associated and is thus rapidly degraded by a process involving the CAT T repeat element. PMID:9501119

  13. Communication between binding sites is required for YqjI regulation of target promoters within the yqjH-yqjI intergenic region.

    PubMed

    Wang, Suning; Blahut, Matthew; Wu, Yun; Philipkosky, Katherine E; Outten, F Wayne

    2014-09-01

    The nickel-responsive transcription factor YqjI represses its own transcription and transcription of the divergent yqjH gene, which encodes a novel ferric siderophore reductase. The intergenic region between the two promoters is complex, with multiple sequence features that may impact YqjI-dependent regulation of its two target promoters. We utilized mutagenesis and DNase I footprinting to characterize YqjI regulation of the yqjH-yqjI intergenic region. The results show that YqjI binding results in an extended footprint at the yqjI promoter (site II) compared to the yqjH promoter (site I). Mutagenesis of in vivo gene reporter constructs revealed that the two YqjI binding sites, while separated by nearly 200 bp, appear to communicate in order to provide full YqjI-dependent regulation at the two target promoters. Thus, YqjI binding at both promoters is required for full repression of either promoter, suggesting that the two YqjI binding sites cooperate to control transcription from the divergent promoters. Furthermore, internal deletions that shorten the total length of the intergenic region disrupt the ability of YqjI to regulate the yqjH promoter. Finally, mutagenesis of the repetitive extragenic palindromic (REP) elements within the yqjH-yqjI intergenic region shows that these sequences are not required for YqjI regulation. These studies provide a complex picture of novel YqjI transcriptional regulation within the yqjH-yqjI intergenic region and suggest a possible model for communication between the YqjI binding sites at each target promoter.

  14. The KLP-7 Residue S546 Is a Putative Aurora Kinase Site Required for Microtubule Regulation at the Centrosome in C. elegans.

    PubMed

    Han, Xue; Adames, Kelly; Sykes, Ellen M E; Srayko, Martin

    2015-01-01

    Regulation of microtubule dynamics is essential for many cellular processes, including proper assembly and function of the mitotic spindle. The kinesin-13 microtubule-depolymerizing enzymes provide one mechanism to regulate microtubule behaviour temporally and spatially. Vertebrate MCAK locates to chromatin, kinetochores, spindle poles, microtubule tips, and the cytoplasm, implying that the regulation of kinesin-13 activity and subcellular targeting is complex. Phosphorylation of kinesin-13 by Aurora kinase inhibits microtubule depolymerization activity and some Aurora phosphorylation sites on kinesin-13 are required for subcellular localization. Herein, we determine that a C. elegans deletion mutant klp-7(tm2143) causes meiotic and mitotic defects that are consistent with an increase in the amount of microtubules in the cytoplasmic and spindle regions of meiotic embryos, and an increase in microtubules emanating from centrosomes. We show that KLP-7 is phosphorylated by Aurora A and Aurora B kinases in vitro, and that the phosphorylation by Aurora A is stimulated by TPXL-1. Using a structure-function approach, we establish that one putative Aurora kinase site, S546, within the C-terminal part of the core domain is required for the function, but not subcellular localization, of KLP-7 in vivo. Furthermore, FRAP analysis reveals microtubule-dependent differences in the turnover of KLP-7(S546A) and KLP-7(S546E) mutant proteins at the centrosome, suggesting a possible mechanism for the regulation of KLP-7 by Aurora kinase.

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

    PubMed

    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.

  16. Distinct requirements of wls, wnt9a, wnt5b and gpc4 in regulating chondrocyte maturation and timing of endochondral ossification

    PubMed Central

    Ling, Irving TC; Rochard, Lucie; Liao, Eric C.

    2017-01-01

    Formation of the mandible requires progressive morphologic change, proliferation, differentiation and organization of chondrocytes preceding osteogenesis. The Wnt signaling pathway is involved in regulating bone development and maintenance. Chondrocytes that are fated to become bone require Wnt to polarize and orientate appropriately to initiate the endochondral ossification program. Although the canonical Wnt signaling has been well studied in the context of bone development, the effects of non-canonical Wnt signaling in regulating the timing of cartilage maturation and subsequent bone formation in shaping ventral craniofacial structure is not fully understood.. Here we examined the role of the non-canonical Wnt signaling pathway (wls, gpc4, wnt5b and wnt9a) in regulating zebrafish Meckel’s cartilage maturation to the onset of osteogenic differentiation. We found that disruption of wls resulted in a significant loss of craniofacial bone, whereas lack of gpc4, wnt5b and wnt9a resulted in severely delayed endochondral ossification. This study demonstrates the importance of the non-canonical Wnt pathway in regulating coordinated ventral cartilage morphogenesis and ossification. PMID:27908786

  17. The Stringent Response Regulator DksA Is Required for Salmonella enterica Serovar Typhimurium Growth in Minimal Medium, Motility, Biofilm Formation, and Intestinal Colonization

    PubMed Central

    Azriel, Shalhevet; Goren, Alina; Rahav, Galia

    2015-01-01

    Salmonella enterica serovar Typhimurium is a facultative intracellular human and animal bacterial pathogen posing a major threat to public health worldwide. Salmonella pathogenicity requires complex coordination of multiple physiological and virulence pathways. DksA is a conserved Gram-negative regulator that belongs to a distinct group of transcription factors that bind directly to the RNA polymerase secondary channel, potentiating the effect of the signaling molecule ppGpp during a stringent response. Here, we established that in S. Typhimurium, dksA is induced during the logarithmic phase and DksA is essential for growth in minimal defined medium and plays an important role in motility and biofilm formation. Furthermore, we determined that DksA positively regulates the Salmonella pathogenicity island 1 and motility-chemotaxis genes and is necessary for S. Typhimurium invasion of human epithelial cells and uptake by macrophages. In contrast, DksA was found to be dispensable for S. Typhimurium host cell adhesion. Finally, using the colitis mouse model, we found that dksA is spatially induced at the midcecum during the early stage of the infection and required for gastrointestinal colonization and systemic infection in vivo. Taken together, these data indicate that the ancestral stringent response regulator DksA coordinates various physiological and virulence S. Typhimurium programs and therefore is a key virulence regulator of Salmonella. PMID:26553464

  18. The KLP-7 Residue S546 Is a Putative Aurora Kinase Site Required for Microtubule Regulation at the Centrosome in C. elegans

    PubMed Central

    Han, Xue; Adames, Kelly; Sykes, Ellen M. E.; Srayko, Martin

    2015-01-01

    Regulation of microtubule dynamics is essential for many cellular processes, including proper assembly and function of the mitotic spindle. The kinesin-13 microtubule-depolymerizing enzymes provide one mechanism to regulate microtubule behaviour temporally and spatially. Vertebrate MCAK locates to chromatin, kinetochores, spindle poles, microtubule tips, and the cytoplasm, implying that the regulation of kinesin-13 activity and subcellular targeting is complex. Phosphorylation of kinesin-13 by Aurora kinase inhibits microtubule depolymerization activity and some Aurora phosphorylation sites on kinesin-13 are required for subcellular localization. Herein, we determine that a C. elegans deletion mutant klp-7(tm2143) causes meiotic and mitotic defects that are consistent with an increase in the amount of microtubules in the cytoplasmic and spindle regions of meiotic embryos, and an increase in microtubules emanating from centrosomes. We show that KLP-7 is phosphorylated by Aurora A and Aurora B kinases in vitro, and that the phosphorylation by Aurora A is stimulated by TPXL-1. Using a structure-function approach, we establish that one putative Aurora kinase site, S546, within the C-terminal part of the core domain is required for the function, but not subcellular localization, of KLP-7 in vivo. Furthermore, FRAP analysis reveals microtubule-dependent differences in the turnover of KLP-7(S546A) and KLP-7(S546E) mutant proteins at the centrosome, suggesting a possible mechanism for the regulation of KLP-7 by Aurora kinase. PMID:26168236

  19. Pak4 Is Required during Epithelial Polarity Remodeling through Regulating AJ Stability and Bazooka Retention at the ZA

    PubMed Central

    Walther, Rhian F.; Nunes de Almeida, Francisca; Vlassaks, Evi; Burden, Jemima J.; Pichaud, Franck

    2016-01-01

    Summary The ability of epithelial cells to assemble into sheets relies on their zonula adherens (ZA), a circumferential belt of adherens junction (AJ) material, which can be remodeled during development to shape organs. Here, we show that during ZA remodeling in a model neuroepithelial cell, the Cdc42 effector P21-activated kinase 4 (Pak4/Mbt) regulates AJ morphogenesis and stability through β-catenin (β-cat/Arm) phosphorylation. We find that β-catenin phosphorylation by Mbt, and associated AJ morphogenesis, is needed for the retention of the apical determinant Par3/Bazooka at the remodeling ZA. Importantly, this retention mechanism functions together with Par1-dependent lateral exclusion of Par3/Bazooka to regulate apical membrane differentiation. Our results reveal an important functional link between Pak4, AJ material morphogenesis, and polarity remodeling during organogenesis downstream of Par3. PMID:27052178

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

    PubMed Central

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

    2013-01-01

    Summary 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 Zn2+-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. PMID:23616927