Science.gov

Sample records for regulator rcac require

  1. Informational requirements for transcriptional regulation.

    PubMed

    O'Neill, Patrick K; Forder, Robert; Erill, Ivan

    2014-05-01

    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

  2. Regulations which require employee training: An overview

    SciTech Connect

    Gilfus, L.J.

    1995-11-01

    In order to identify the Occupational Safety and Health Administration (OSHA) regulations which apply to a certain plant or facility, one needs to find the reference materials that are needed and that can be trusted. An overview is presented of the following: Title 29 -- Labor, OSHA Training Requirements; Title 40, Environmental Protection Agency Training Requirements; and Title 49, Department of Transportation Training Regulations.

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

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-27

    ... minutes per submission assuming an offeror's use of electronic information tracking and retrieval... Regulation; Information Collection; Qualification Requirements AGENCY: Department of Defense (DOD), General... reinstatement request for an information collection requirement regarding an existing OMB clearance....

  5. 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... covering contractor legal management requirements and make conforming amendments to the Department of... rulemaking to revise existing regulations covering contractor legal management requirements and...

  6. 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.

  7. Melatonin is required for the circadian regulation of sleep

    PubMed Central

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

    2015-01-01

    Summary 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. PMID:25754820

  8. 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.

  9. 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...

  10. 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...

  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, 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 owner or operator shall comply with all applicable State implementation plan emission limits...

  13. 40 CFR 63.312 - Existing regulations and requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 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 owner or operator shall comply with all applicable State implementation plan emission limits...

  14. 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... correcting a final rule that appeared in the Federal Register of May 3, 2013 (78 FR 25795). In this...

  15. 78 FR 17186 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-20

    ... Regulation Supplement (DFARS); Notification Requirements for Critical Safety Items AGENCY: Defense... Acquisition Regulation Supplement (DFARS), Notification Requirements for Critical Safety Items; OMB...

  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

    2014-09-01

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

  18. 75 FR 6186 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 205...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-08

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 205, Publicizing Contract Actions AGENCY: Defense Acquisition Regulations System... approved information collection requirement. SUMMARY: In compliance with Section 3506(c)(2)(A) of...

  19. 75 FR 20825 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DoD Pilot...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-21

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DoD Pilot Mentor-Protege Program AGENCY: Defense Acquisition Regulations System... approved information collection requirement. ] SUMMARY: In compliance with section 3506(c)(2)(A) of...

  20. 76 FR 50461 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Types of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-15

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

  1. 78 FR 70025 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-22

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Subcontracting Policies and Procedures AGENCY: Defense Acquisition Regulations System... approved information collection requirement. SUMMARY: In compliance with section 3506(c)(2)(A) of...

  2. 78 FR 63461 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Foreign...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-24

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Foreign Acquisition AGENCY: Defense Acquisition Regulations System, Department of...) 372-6094. Mail: Defense Acquisition Regulations System, Attn: Amy Williams, OUSD(AT&L)DPAP(DARS),...

  3. GABAA receptor regulation of voluntary ethanol drinking requires PKCepsilon.

    PubMed

    Besheer, Joyce; Lepoutre, Veronique; Mole, Beth; Hodge, Clyde W

    2006-11-01

    Protein kinase C (PKC) regulates a variety of neural functions, including ion channel activity, neurotransmitter release, receptor desensitization and differentiation. We have shown previously that mice lacking the epsilon-isoform of PKC (PKCepsilon) self-administer 75% less ethanol and exhibit supersensitivity to acute ethanol and allosteric positive modulators of GABA(A) receptors when compared with wild-type controls. The purpose of the present study was to examine involvement of PKCepsilon in GABA(A) receptor regulation of voluntary ethanol drinking. To address this question, PKCepsilon null-mutant and wild-type control mice were allowed to drink ethanol (10% v/v) vs. water on a two-bottle continuous access protocol. The effects of diazepam (nonselective GABA(A) BZ positive modulator), zolpidem (GABA(A) alpha1 agonist), L-655,708 (BZ-sensitive GABA(A) alpha5 inverse agonist), and flumazenil (BZ antagonist) were then tested on ethanol drinking. Ethanol intake (grams/kg/day) by wild-type mice decreased significantly after diazepam or zolpidem but increased after L-655,708 administration. Flumazenil antagonized diazepam-induced reductions in ethanol drinking in wild-type mice. However, ethanol intake by PKCepsilon null mice was not altered by any of the GABAergic compounds even though effects were seen on water drinking in these mice. Increased acute sensitivity to ethanol and diazepam, which was previously reported, was confirmed in PKCepsilon null mice. Thus, results of the present study show that PKCepsilon null mice do not respond to doses of GABA(A) BZ receptor ligands that regulate ethanol drinking by wild-type control mice. This suggests that PKCepsilon may be required for GABA(A) receptor regulation of chronic ethanol drinking.

  4. 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 for a nationally recognized testing laboratory'' (The Regulation). The Regulation... 1910.7, ``definition and requirements for a nationally recognized testing laboratory'' (the Regulation... specified by the Regulation on the Definition and Requirements of a Nationally Recognized Testing...

  5. 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...

  6. 76 FR 72916 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ... comments, identified by OMB Control Number 0704-0454, using any of the following methods: Email: dfars@osd... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Administrative Matters AGENCY: Defense Acquisition Regulations System, Department...

  7. 78 FR 70294 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-25

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Administrative Matters AGENCY: Defense Acquisition Regulations System, Department of... technology. The Office of Management and Budget (OMB) has approved this information collection under...

  8. 77 FR 43076 - Federal Acquisition Regulation; Information Collection; Value Engineering Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-23

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

  9. 75 FR 45104 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Contract...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-02

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition... Regulations System, Department of Defense (DoD). ACTION: Notice and request for comments. SUMMARY: In... announces the proposed extension of a public information collection requirement and seeks public comment...

  10. 75 FR 68331 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DFARS Part...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-05

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition... Acquisition Regulations System, Department of Defense (DoD). ACTION: Notice and request for comments regarding a proposed extension of an approved information collection requirement. SUMMARY: In compliance...

  11. 75 FR 26739 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 244...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 244, Subcontracting Policies and Procedures (OMB Control Number 0704-0253) AGENCY... regarding a proposed extension of an approved information collection requirement. SUMMARY: In...

  12. 75 FR 10790 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-09

    ... Regulation Supplement; Notification Requirements for Critical Safety Items AGENCY: Defense Acquisition... Supplement (DFARS) Notification Requirements for Critical Safety Items; OMB Control Number 0704-0441....

  13. translin Is Required for Metabolic Regulation of Sleep.

    PubMed

    Murakami, Kazuma; Yurgel, Maria E; 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

    2016-04-01

    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

  14. Calcium-regulated exocytosis is required for cell membrane resealing

    PubMed Central

    1995-01-01

    Using confocal microscopy, we visualized exocytosis during membrane resealing in sea urchin eggs and embryos. Upon wounding by a laser beam, both eggs and embryos showed a rapid burst of localized Ca(2+)- regulated exocytosis. The rate of exocytosis was correlated quantitatively with successfully resealing. In embryos, whose activated surfaces must first dock vesicles before fusion, exocytosis and membrane resealing were inhibited by neurotoxins that selectively cleave the SNARE complex proteins, synaptobrevin, SNAP-25, and syntaxin. In eggs, whose cortical vesicles are already docked, vesicles could be reversibly undocked with externally applied stachyose. If cortical vesicles were undocked both exocytosis and plasma membrane resealing were completely inhibited. When cortical vesicles were transiently undocked, exposure to tetanus toxin and botulinum neurotoxin type C1 rendered them no longer competent for resealing, although botulinum neurotoxin type A was still ineffective. Cortical vesicles transiently undocked in the presence of tetanus toxin were subsequently fusion incompetent although to a large extent they retained their ability to redock when stachyose was diluted. We conclude that addition of internal membranes by exocytosis is required and that a SNARE-like complex plays differential roles in vesicle docking and fusion for the repair of disrupted plasma membrane. PMID:8557742

  15. 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... HEALTH CARE PREPAYMENT PLANS Medicare Contract Requirements § 417.478 Requirements of other laws and regulations. The contract must provide that the HMO or CMP agrees to comply with— (a) The requirements for...

  16. 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.

  17. 77 FR 67366 - Federal Acquisition Regulation; Information Collection; Quality Assurance Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-09

    ... Regulation; Information Collection; Quality Assurance Requirements AGENCY: Department of Defense (DOD... information collection requirement concerning quality assurance requirements. Public comments are particularly... and methodology; ways to enhance the quality, utility, and clarity of the information to be...

  18. 78 FR 19467 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-01

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS); DoD Pilot Mentor-Prot g Program AGENCY: Defense Acquisition Regulations System... approved information collection requirement. SUMMARY: In compliance with Section 3506(c)(2)(A) of...

  19. 75 FR 15388 - Amendment to the International Traffic in Arms Regulations: Removing Requirement for Prior...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-29

    ... Parts 124, 126, and 129 RIN 1400-AC62 Amendment to the International Traffic in Arms Regulations... amending the International Traffic in Arms Regulations (ITAR) to remove the requirements for prior approval... International Traffic in Arms Regulations (ITAR) at 22 CFR 123.16, to require Department of State...

  20. 21 CFR 16.24 - Regulatory hearing required by the act or a regulation.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... regulation. 16.24 Section 16.24 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN....24 Regulatory hearing required by the act or a regulation. (a) A regulatory hearing required by the act or a regulation under § 16.1(b) will be initiated in the same manner as other regulatory...

  1. 21 CFR 16.24 - Regulatory hearing required by the act or a regulation.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... regulation. 16.24 Section 16.24 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN....24 Regulatory hearing required by the act or a regulation. (a) A regulatory hearing required by the act or a regulation under § 16.1(b) will be initiated in the same manner as other regulatory...

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

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 5 2011-01-01 2011-01-01 false General requirements for all regulated articles. 318.13-3 Section 318.13-3 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND... NOTICES Regulated Articles From Hawaii and the Territories § 318.13-3 General requirements for...

  3. 49 CFR 171.26 - Additional requirements for the use of the IAEA Regulations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Additional requirements for the use of the IAEA Regulations. 171.26 Section 171.26 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS GENERAL INFORMATION, REGULATIONS,...

  4. 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...

  5. 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...

  6. 75 FR 68333 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-05

    ... Regulation Supplement; DFARS Appendix F, Material Inspection and Receiving Report (OMB Control Number 0704... Acquisition Regulation Supplement (DFARS) Appendix F, Material Inspection and Receiving Report; DD Form 250... Information Collection This information collection includes the requirements of DFARS Appendix F,...

  7. 78 FR 25795 - Contractor Legal Management Requirements; Acquisition Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-03

    ... Federal Register on August 31, 1994 (59 FR 44981). The interim Acquisition Letter was finalized as a Policy Statement on April 3, 1996 (61 FR 14763). This Policy Statement was followed by a formal... Regulations with an effective date of April 23, 2001 (66 FR 4616, 66 FR 19717). After a decade operating...

  8. 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),...

  9. 13 CFR 120.1511 - Certification and other reporting and notification requirements for Other Regulated SBLCs.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

  10. 77 FR 29983 - Federal Acquisition Regulation; Information Collection; Novation/Change of Name Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-21

    ... Regulation; Information Collection; Novation/ Change of Name Requirements AGENCY: Department of Defense (DOD... collection requirement concerning Novation/Change of Name Requirements. Public comments are particularly... comments identified by Information Collection 9000- 0076, Novation/Change of Name Requirements, by any...

  11. 76 FR 35218 - Federal Acquisition Regulation; Information Collection; Cost or Pricing Data Requirements and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-16

    ... Regulation; Information Collection; Cost or Pricing Data Requirements and Information Other Than Cost or... requirement concerning cost or pricing data requirements and information other than cost or pricing data... Information Collection 9000- 0013, Cost or Pricing Data Requirements and Information Other Than Cost...

  12. 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 .

  13. 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... Acquisition Regulations System, Department of Defense (DoD). ACTION: Notice and request for comments. SUMMARY...), DoD announces the proposed extension of a public information collection requirement and seeks...

  14. 76 FR 71707 - Revising Underground Storage Tank Regulations-Revisions to Existing Requirements and New...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-18

    ...EPA is proposing to make certain revisions to the 1988 underground storage tank (UST) technical, financial responsibility, and state program approval regulations. These changes establish federal requirements that are similar to key portions of the Energy Policy Act of 2005; they also update certain 1988 UST regulations. Proposed changes include: Adding secondary containment requirements for......

  15. 75 FR 964 - Acquisition Regulation: Subchapter E-General Contracting Requirements, Subchapter F-Special...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-07

    ... Regulation: Subchapter E--General Contracting Requirements, Subchapter F--Special Categories of Contracting... Acquisition Regulation (DEAR) Subchapters E-- General Contracting Requirements, F--Special Categories of...: Subchapters E, F, and G and RIN 1991-AB88, by any of the following methods: Federal eRulemaking Portal:...

  16. [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.

  17. 27 CFR 70.411 - Imposition of taxes, qualification requirements, and regulations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... operation of distilled spirits plants. Part 19 of title 27 CFR contains the regulations relating to the...) Miscellaneous liquor transactions. Part 29 of 27 CFR contains miscellaneous regulations relative to the... requirements therefor. (3) (4) Gauging of distilled spirits. Part 30 of title 27 CFR contains the...

  18. 27 CFR 70.411 - Imposition of taxes, qualification requirements, and regulations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... operation of distilled spirits plants. Part 19 of title 27 CFR contains the regulations relating to the...) Miscellaneous liquor transactions. Part 29 of 27 CFR contains miscellaneous regulations relative to the... requirements therefor. (3) (4) Gauging of distilled spirits. Part 30 of title 27 CFR contains the...

  19. 27 CFR 70.411 - Imposition of taxes, qualification requirements, and regulations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... operation of distilled spirits plants. Part 19 of title 27 CFR contains the regulations relating to the...) Miscellaneous liquor transactions. Part 29 of 27 CFR contains miscellaneous regulations relative to the... requirements therefor. (3) (4) Gauging of distilled spirits. Part 30 of title 27 CFR contains the...

  20. 27 CFR 70.411 - Imposition of taxes, qualification requirements, and regulations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... operation of distilled spirits plants. Part 19 of title 27 CFR contains the regulations relating to the...) Miscellaneous liquor transactions. Part 29 of 27 CFR contains miscellaneous regulations relative to the... requirements therefor. (3) (4) Gauging of distilled spirits. Part 30 of title 27 CFR contains the...

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

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  2. 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.

  3. 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... announcing a public workshop entitled ``FDA Clinical Trial Requirements, Regulations, Compliance, and Good... representatives. The program will focus on the relationships among the FDA and clinical trial staff,...

  4. 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...

  5. 75 FR 29456 - Acquisition Regulation: Subchapter E-General Contracting Requirements, Subchapter F-Special...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-26

    ... Acquisition Regulation: Subchapter E--General Contracting Requirements, Subchapter F--Special Categories of...) Subchapters E--General Contracting Requirements, F--Special Categories of Contracting, and G--Contract...: June 25, 2010. FOR FURTHER INFORMATION CONTACT: Barbara Binney at (202) 287-1340 or by e-mail,...

  6. 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...

  7. 12 CFR 350.12 - Disclosure required by applicable banking or securities law or regulations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 4 2011-01-01 2011-01-01 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...

  8. 12 CFR 350.12 - Disclosure required by applicable banking or securities law or regulations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 12 Banks and Banking 5 2013-01-01 2013-01-01 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...

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

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 5 2014-01-01 2014-01-01 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...

  10. 12 CFR 350.12 - Disclosure required by applicable banking or securities law or regulations.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 5 2012-01-01 2012-01-01 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...

  11. Pre-LTP requires extracellular signal-regulated kinase in the ACC

    PubMed Central

    Yamanaka, Manabu; Tian, Zhen; Darvish-Ghane, Soroush

    2016-01-01

    The extracellular signal-regulated kinase is an important protein kinase for cortical plasticity. Long-term potentiation in the anterior cingulate cortex is believed to play important roles in chronic pain, fear, and anxiety. Previous studies of extracellular signal-regulated kinase are mainly focused on postsynaptic form of long-term potentiation (post-long-term potentiation). Little is known about the relationship between extracellular signal-regulated kinase and presynaptic long-term potentiation (pre-long-term potentiation) in cortical synapses. In this study, we examined whether pre-long-term potentiation in the anterior cingulate cortex requires the activation of presynaptic extracellular signal-regulated kinase. We found that p42/p44 mitogen-activated protein kinase inhibitors, PD98059 and U0126, suppressed the induction of pre-long-term potentiation. By contrast, these inhibitors did not affect the maintenance of pre-long-term potentiation. Using pharmacological inhibitors, we found that pre-long-term potentiation recorded for 1 h did not require transcriptional or translational processes. Our results strongly indicate that the activation of presynaptic extracellular signal-regulated kinase is required for the induction of pre-long-term potentiation, and this involvement may explain the contribution of extracellular signal-regulated kinase to mood disorders. PMID:27178245

  12. 49 CFR 1013.3 - Review and reporting requirements for regulated carriers.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Exchange Board (17 CFR 240.13d-1) which reports the purchase of 5 percent or more of the registered... THE PROPER USE OF VOTING TRUSTS § 1013.3 Review and reporting requirements for regulated carriers. (a) Any carrier choosing to utilize a voting trust may voluntarily submit a copy of the voting trust...

  13. 49 CFR 1013.3 - Review and reporting requirements for regulated carriers.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Exchange Board (17 CFR 240.13d-1) which reports the purchase of 5 percent or more of the registered... THE PROPER USE OF VOTING TRUSTS § 1013.3 Review and reporting requirements for regulated carriers. (a) Any carrier choosing to utilize a voting trust may voluntarily submit a copy of the voting trust...

  14. 78 FR 38234 - Defense Federal Acquisition Regulation Supplement: Requirements for Acquisitions Pursuant to...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-26

    ... Acquisition Regulation Supplement: Requirements for Acquisitions Pursuant to Multiple Award Contracts (DFARS... INFORMATION: I. Background On October 25, 2002, a final DFARS rule was published (67 FR 65505) which... pursuant to multiple award contracts. Increasing savings in expenditures through competition is...

  15. 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, regulations, and application instructions. 25.200 Section 25.200 Grants and Agreements Office of Management and Budget Guidance for Grants and Agreements OFFICE OF MANAGEMENT AND BUDGET GOVERNMENTWIDE GUIDANCE FOR GRANTS AND AGREEMENTS Reserved...

  16. 77 FR 34927 - National Pollutant Discharge Elimination System-Proposed Regulations To Establish Requirements...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-12

    ... under section 316(b) of the Clean Water Act (CWA), at 76 FR 22174. EPA received voluminous comments and... on closed cycle cooling'' (76 FR 22174, Section VI.E). The four factors have not changed on the basis...--Proposed Regulations To Establish Requirements for Cooling Water Intake Structures at Existing...

  17. 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...

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

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 13 Business Credit and Assistance 1 2012-01-01 2012-01-01 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. 24 CFR 3280.3 - Manufactured home procedural and enforcement regulations and consumer manual requirements.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...-FEDERAL HOUSING COMMISSIONER, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT MANUFACTURED HOME CONSTRUCTION... 24 Housing and Urban Development 5 2014-04-01 2014-04-01 false Manufactured home procedural and enforcement regulations and consumer manual requirements. 3280.3 Section 3280.3 Housing and Urban...

  20. 26 CFR 56.6001-1 - Notice or regulations requiring records, statements, and special returns.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 17 2011-04-01 2011-04-01 false Notice or regulations requiring records, statements, and special returns. 56.6001-1 Section 56.6001-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) MISCELLANEOUS EXCISE TAXES (CONTINUED) PUBLIC CHARITY EXCISE...

  1. 26 CFR 56.6001-1 - Notice or regulations requiring records, statements, and special returns.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 17 2010-04-01 2010-04-01 false Notice or regulations requiring records, statements, and special returns. 56.6001-1 Section 56.6001-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) MISCELLANEOUS EXCISE TAXES (CONTINUED) PUBLIC CHARITY EXCISE...

  2. 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...

  3. 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...

  4. 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...

  5. 76 FR 68027 - Federal Acquisition Regulation; Certification Requirement and Procurement Prohibition Relating to...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-02

    ... RIN 9000-AL71 Federal Acquisition Regulation; Certification Requirement and Procurement Prohibition... Sanctions Act of 1996 (the Iran Sanctions Act). Section 106 imposes a procurement prohibition relating to..., Procurement Analyst, at (202) 219-0202, for clarification of content. For information pertaining to status...

  6. 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

    ... Defense Requirements for Ultra-Low Sulfur Diesel and Gasoline Benzene Technical Amendment AGENCY... gasoline benzene regulations to allow disqualified small refiners the same opportunity to generate gasoline benzene credits as that afforded to non-small refiners. DATES: Comments: Comments must be received on...

  7. 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

    ... adopting Sec. 1026.36(i). \\1\\ 78 FR 11279 (Feb. 15, 2013). A. Title XIV Rulemaking Effective Dates In... become effective sooner than January 2014.\\5\\ \\5\\ 77 FR 55272 (Sept. 7, 2012). The Bureau received very... Requirements Under the Truth In Lending Act (Regulation Z); Prohibition on Financing Credit Insurance...

  8. 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...

  9. 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....

  10. 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 PMID:25562896

  11. 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

  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. An essential single domain response regulator required for normal cell division and differentiation in Caulobacter crescentus.

    PubMed Central

    Hecht, G B; Lane, T; Ohta, N; Sommer, J M; Newton, A

    1995-01-01

    Signal transduction pathways mediated by sensor histidine kinases and cognate response regulators control a variety of physiological processes in response to environmental conditions. Here we show that in Caulobacter crescentus these systems also play essential roles in the regulation of polar morphogenesis and cell division. Previous studies have implicated histidine kinase genes pleC and divJ in the regulation of these developmental events. We now report that divK encodes an essential, cell cycle-regulated homolog of the CheY/Spo0F subfamily and present evidence that this protein is a cognate response regulator of the histidine kinase PleC. The purified kinase domain of PleC, like that of DivJ, can serve as an efficient phosphodonor to DivK and as a phospho-DivK phosphatase. Based on these and earlier genetic results we propose that PleC and DivK are members of a signal transduction pathway that couples motility and stalk formation to completion of a late cell division cycle event. Gene disruption experiments and the filamentous phenotype of the conditional divK341 mutant reveal that DivK also functions in an essential signal transduction pathway required for cell division, apparently in response to another histidine kinase. We suggest that phosphotransfer mediated by these two-component signal transduction systems may represent a general mechanism regulating cell differentiation and cell division in response to successive cell cycle checkpoints. Images PMID:7664732

  14. 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

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

    PubMed

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

    2015-08-01

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

  16. Dendritic cell maturation requires STAT1 and is under feedback regulation by suppressors of cytokine signaling.

    PubMed

    Jackson, Sharon H; Yu, Cheng-Rong; Mahdi, Rashid M; Ebong, Samuel; Egwuagu, Charles E

    2004-02-15

    In this study we show that activation of STAT pathways is developmentally regulated and plays a role in dendritic cell (DC) differentiation and maturation. The STAT6 signaling pathway is constitutively activated in immature DC (iDC) and declines as iDCs differentiate into mature DCs (mDCs). However, down-regulation of this pathway during DC differentiation is accompanied by dramatic induction of suppressors of cytokine signaling 1 (SOCS1), SOCS2, SOCS3, and cytokine-induced Src homology 2-containing protein expression, suggesting that inhibition of STAT6 signaling may be required for DC maturation. In contrast, STAT1 signaling is most robust in mDCs and is not inhibited by the up-regulated SOCS proteins, indicating that STAT1 and STAT6 pathways are distinctly regulated in maturing DC. Furthermore, optimal activation of STAT1 during DC maturation requires both IL-4 and GM-CSF, suggesting that synergistic effects of both cytokines may in part provide the requisite STAT1 signaling intensity for DC maturation. Analyses of STAT1(-/-) DCs reveal a role for STAT1 in repressing CD86 expression in precursor DCs and up-regulating CD40, CD11c, and SOCS1 expression in mDCs. We further show that SOCS proteins are differentially induced by IL-4 and GM-CSF in DCs. SOCS1 is primarily induced by IL-4 through a STAT1-dependent mechanism, whereas SOCS3 is induced mainly by GM-CSF. Taken together, these results suggest that cytokine-induced maturation of DCs is under feedback regulation by SOCS proteins and that the switch from constitutive activation of the STAT6 pathway in iDCs to predominant use of STAT1 signals in mDC is mediated in part by STAT1-induced SOCS expression.

  17. 33 CFR 165.830 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... regulations contained in 33 CFR 165.13 apply to this section. (e) Eighth Coast Guard District Inland River RNA...— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR 160.204. Certain... CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415...

  18. 33 CFR 165.830 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... regulations contained in 33 CFR 165.13 apply to this section. (e) Eighth Coast Guard District Inland River RNA...— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR 160.204. Certain... CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415...

  19. 33 CFR 165.830 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... regulations contained in 33 CFR 165.13 apply to this section. (e) Eighth Coast Guard District Inland River RNA...— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR 160.204. Certain... CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415...

  20. 33 CFR 165.830 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... regulations contained in 33 CFR 165.13 apply to this section. (e) Eighth Coast Guard District Inland River RNA...— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR 160.204. Certain... CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415...

  1. 33 CFR 165.830 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... regulations contained in 33 CFR 165.13 apply to this section. (e) Eighth Coast Guard District Inland River RNA...— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR 160.204. Certain... CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415...

  2. 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

    ... Commodity Futures Trading Commission. (3) Self-regulatory organization: (i) Shall have the same meaning as... futures commission merchants. A financial institution regulated by a self-regulatory organization shall be... requirements for financial institutions regulated by a Federal functional regulator or a...

  3. 31 CFR 1020.210 - Anti-money laundering program requirements for financial institutions regulated only by a Federal...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Relating to Money and Finance (Continued) FINANCIAL CRIMES ENFORCEMENT NETWORK, DEPARTMENT OF THE TREASURY RULE FOR BANKS Programs § 1020.210 Anti-money laundering program requirements for financial... requirements for financial institutions regulated only by a Federal functional regulator, including...

  4. 31 CFR 1020.210 - Anti-money laundering program requirements for financial institutions regulated only by a Federal...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Relating to Money and Finance (Continued) FINANCIAL CRIMES ENFORCEMENT NETWORK, DEPARTMENT OF THE TREASURY RULE FOR BANKS Programs § 1020.210 Anti-money laundering program requirements for financial... requirements for financial institutions regulated only by a Federal functional regulator, including...

  5. 31 CFR 1020.210 - Anti-money laundering program requirements for financial institutions regulated only by a Federal...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Relating to Money and Finance (Continued) FINANCIAL CRIMES ENFORCEMENT NETWORK, DEPARTMENT OF THE TREASURY RULE FOR BANKS Programs § 1020.210 Anti-money laundering program requirements for financial... requirements for financial institutions regulated only by a Federal functional regulator, including...

  6. 31 CFR 1020.210 - Anti-money laundering program requirements for financial institutions regulated only by a Federal...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Relating to Money and Finance (Continued) FINANCIAL CRIMES ENFORCEMENT NETWORK, DEPARTMENT OF THE TREASURY RULE FOR BANKS Programs § 1020.210 Anti-money laundering program requirements for financial... requirements for financial institutions regulated only by a Federal functional regulator, including...

  7. IL-12 is required for mTOR regulation of memory CTLs during viral infection

    PubMed Central

    Garcia, Karla; Sun, Zhifeng; Mattson, Elliot; Li, Lei; Smyth, Kendra; Xiao, Zhengguo

    2014-01-01

    The induction of functional memory CTLs is a major goal of vaccination against intracellular pathogens. IL-12 is critical for the generation of memory CTLs, and inhibition of mTOR by rapamycin can effectively enhance the memory CTL response. Yet, the role of IL-12 in mTOR’s regulation of memory CTL is unknown. Here, we hypothesized that the immunostimulatory effects of mTOR on memory CTLs requires IL-12 signaling. Our results revealed that rapamycin increased the generation of memory CTLs in vaccinia virus infection, and this enhancement was dependent upon the IL-12 signal. Furthermore, IL-12 receptor deficiency diminished the secondary expansion of rapamycin-regulated memory, and resultant secondary memory CTLs were abolished. Rapamycin enhanced IL-12 signaling by up regulating IL-12 receptor β2 expression and STAT4 phosphorylation in CTLs during early infection. In addition, rapamycin continually suppressed T-bet expression in both WT and IL-12 receptor knockout CTLs. These results indicate an essential role for IL-12 in the regulation of memory CTLs by mTOR, and highlight the importance of considering the interplay between cytokines and adjuvants during vaccine design. PMID:24898389

  8. TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis

    PubMed Central

    Shambharkar, Prashant B.; Bittinger, Mark; Latario, Brian; Xiong, ZhaoHui; Bandyopadhyay, Somnath; Davis, Vanessa; Lin, Victor; Yang, Yi; Valdez, Reginald; Labow, Mark A.

    2015-01-01

    Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis. PMID:25996873

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

    PubMed Central

    Chen, Ke-Xian

    2009-01-01

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

  10. Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles.

    PubMed

    Kong, Dong; Farmer, Veronica; Shukla, Anil; James, Jana; Gruskin, Richard; Kiriyama, Shigeo; Loncarek, Jadranka

    2014-09-29

    Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.

  11. Unusually long-lived pause required for regulation of a Rho-dependent transcription terminator

    PubMed Central

    Hollands, Kerry; Sevostiyanova, Anastasia; Groisman, Eduardo A.

    2014-01-01

    Up to half of all transcription termination events in bacteria rely on the RNA-dependent helicase Rho. However, the nucleic acid sequences that promote Rho-dependent termination remain poorly characterized. Defining the molecular determinants that confer Rho-dependent termination is especially important for understanding how such terminators can be regulated in response to specific signals. Here, we identify an extraordinarily long-lived pause at the site where Rho terminates transcription in the 5′-leader region of the Mg2+ transporter gene mgtA in Salmonella enterica. We dissect the sequence elements required for prolonged pausing in the mgtA leader and establish that the remarkable longevity of this pause is required for a riboswitch to stimulate Rho-dependent termination in the mgtA leader region in response to Mg2+ availability. Unlike Rho-dependent terminators described previously, where termination occurs at multiple pause sites, there is a single site of transcription termination directed by Rho in the mgtA leader. Our data suggest that Rho-dependent termination events that are subject to regulation may require elements distinct from those operating at constitutive Rho-dependent terminators. PMID:24778260

  12. 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.

  13. 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...

  14. 78 FR 68829 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Contract...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-15

    ... Regulation Supplement; Contract Financing AGENCY: Defense Acquisition Regulations System, Department of... Federal Acquisition Regulation Supplement (DFARS) Part 232, Contract Financing, and related clause...

  15. 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...

  16. Transcriptional regulation of Notch and Delta: requirement for neuroblast segregation in Drosophila.

    PubMed

    Seugnet, L; Simpson, P; Haenlin, M

    1997-05-01

    Segregation of a single neural precursor from each proneural cluster in Drosophila relies on Notch-mediated lateral signalling. Studies concerning the spacing of precursors for the microchaetes of the peripheral nervous system suggested the existence of a regulatory loop between Notch and its ligand Delta within each cell that is under transcriptional control. Activation of Notch leads to repression of the achaete-scute genes which themselves regulate transcription of Delta, perhaps directly. Here we have tested a requirement for transcriptional regulation of Notch and/or Delta during neuroblast segregation in embryos, by providing Notch and Delta ubiquitously at uniform levels. Neuroblast segregation occurs normally under conditions of uniform Notch expression. Under conditions of uniform Delta expression, a single neuroblast segregates from each proneural group in 80% of the cases, more than one in the remaining 20%. Thus transcriptional regulation of Delta is largely dispensable. We discuss the possibility that segregation of single precursors in the central nervous system may rely on a heterogeneous distribution of neural potential between different cells of the proneural group. Notch signalling would enable all cells to mutually repress each other and only a cell with an elevated neural potential could overcome this repression. PMID:9169848

  17. 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

    2014-01-01

    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

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

    PubMed

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

    2006-07-01

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

  19. 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.

  20. 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.

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

    PubMed

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

    2006-06-01

    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.

  2. 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.

  3. Proper gibberellin localization in vascular tissue is required to regulate adventitious root development in tobacco.

    PubMed

    Niu, Shihui; Li, Zhexin; Yuan, Huwei; Fang, Pan; Chen, Xiaoyang; Li, Wei

    2013-08-01

    Bioactive gibberellins (GAs) are involved in many developmental aspects of the life cycle of plants, acting either directly or through interaction with other hormones. Accumulating evidence suggests that GAs have an important effect on root growth; however, there is currently little information on the specific regulatory mechanism of GAs during adventitious root development. A study was conducted on tobacco (Nicotiana tabacum) plants for altered rates of biosynthesis, catabolism, and GA signalling constitutively or in specific tissues using a transgenic approach. In the present study, PtGA20ox, PtGA2ox1, and PtGAI were overexpressed under the control of the 35S promoter, vascular cambium-specific promoter (LMX5), or root meristem-specific promoter (TobRB7), respectively. Evidence is provided that the precise localization of bioactive GA in the stem but not in the roots is required to regulate adventitious root development in tobacco. High levels of GA negatively regulate the early initiation step of root formation through interactions with auxin, while a proper and mobile GA signal is required for the emergence and subsequent long-term elongation of established primordia. The results demonstrated that GAs have an inhibitory effect on adventitious root formation but a stimulatory effect on root elongation. PMID:23918971

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

    PubMed

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

    2016-04-01

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

  5. 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

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

    PubMed Central

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

    2016-01-01

    Landmark epigenetic events underlie early embryonic development, yet how epigenetic modifiers are regulated to achieve rapid epigenome re-patterning is not known. Uhrf1 and DNA methyltransferase 1 (Dnmt1) are known to largely mediate maintenance DNA methylation and Uhrf1 is also required for both Dnmt1 localization and stability. Here, we investigate how these two key epigenetic modifiers regulate early zebrafish development and characterize the developmental consequences of disrupting their homeostatic relationship. Unlike Uhrf1 knockdown, which causes developmental arrest and death prior to gastrulation, overexpression of human UHRF1 (WT-UHRF1) caused asymmetric epiboly, inefficient gastrulation and multi-systemic defects. UHRF1 phosphorylation was previously demonstrated as essential for zebrafish embryogenesis, and we found that penetrance of the asymmetric epiboly phenotype was significantly increased in embryos injected with mRNA encoding non-phosphorylatable UHRF1 (UHRF1S661A). Surprisingly, both WT-UHRF1 and UHRF1S661A overexpression caused DNA hypomethylation. However, since other approaches that caused an equivalent degree of DNA hypomethylation did not cause the asymmetric epiboly phenotype, we conclude that bulk DNA methylation is not the primary mechanism. Instead, UHRF1S661A overexpression resulted in accumulation of Dnmt1 protein and the overexpression of both WT and a catalytically inactive Dnmt1 phenocopied the WT-UHRF1 overexpressing embryos. Dnmt1 knockdown suppressed the phenotype caused by UHRF1S661A overexpression, and Uhrf1 knockdown suppressed the effect of Dnmt1 overexpression. Therefore, we conclude that the interaction between these two proteins is the mechanism underlying the observed developmental defects. This indicates that Dnmt1 stability requires UHRF1 phosphorylation and that crosstalk between the proteins is essential for the function of these two important epigenetic regulators during gastrulation. PMID:26851214

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

    PubMed

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

    2013-12-23

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

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. 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

  17. Homotypic vacuole fusion requires VTI11 and is regulated by phosphoinositides.

    PubMed

    Zheng, Jiameng; Han, Sang Won; Rodriguez-Welsh, Maria Fernanda; Rojas-Pierce, Marcela

    2014-06-01

    Most plant cells contain a large central vacuole that is essential to maintain cellular turgor. We report a new mutant allele of VTI11 that implicates the SNARE protein VTI11 in homotypic fusion of protein storage and lytic vacuoles. Fusion of the multiple vacuoles present in vti11 mutants could be induced by treatment with Wortmannin and LY294002, which are inhibitors of Phosphatidylinositol 3-Kinase (PI3K). We provide evidence that Phosphatidylinositol 3-Phosphate (PtdIns(3)P) regulates vacuole fusion in vti11 mutants, and that fusion of these vacuoles requires intact microtubules and actin filaments. Finally, we show that Wortmannin also induced the fusion of guard cell vacuoles in fava beans, where vacuoles are naturally fragmented after ABA-induced stomata closure. These results suggest a ubiquitous role of phosphoinositides in vacuole fusion, both during the development of the large central vacuole and during the dynamic vacuole remodeling that occurs as part of stomata movements.

  18. The Cryptococcus neoformans Rim101 Transcription Factor Directly Regulates Genes Required for Adaptation to the Host

    PubMed Central

    O'Meara, Teresa R.; Xu, Wenjie; Selvig, Kyla M.; O'Meara, Matthew J.; Mitchell, Aaron P.

    2014-01-01

    The Rim101 protein is a conserved pH-responsive transcription factor that mediates important interactions between several fungal pathogens and the infected host. In the human fungal pathogen Cryptococcus neoformans, the Rim101 protein retains conserved functions to allow the microorganism to respond to changes in pH and other host stresses. This coordinated cellular response enables this fungus to effectively evade the host immune response. Preliminary studies suggest that this conserved transcription factor is uniquely regulated in C. neoformans both by the canonical pH-sensing pathway and by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Here we present comparative transcriptional data that demonstrate a strong concordance between the downstream effectors of PKA and Rim101. To define Rim101-dependent gene expression during a murine lung infection, we used nanoString profiling of lung tissue infected with a wild-type or rim101Δ mutant strain. In this setting, we demonstrated that Rim101 controls the expression of multiple cell wall-biosynthetic genes, likely explaining the enhanced immunogenicity of the rim101Δ mutant. Despite its divergent upstream regulation, the C. neoformans Rim101 protein recognizes a conserved DNA binding motif. Using these data, we identified direct targets of this transcription factor, including genes involved in cell wall regulation. Therefore, the Rim101 protein directly controls cell wall changes required for the adaptation of C. neoformans to its host environment. Moreover, we propose that integration of the cAMP/PKA and pH-sensing pathways allows C. neoformans to respond to a broad range of host-specific signals. PMID:24324006

  19. Transcriptional regulation and locations of Agrobacterium tumefaciens genes required for complete catabolism of octopine.

    PubMed

    Cho, K; Fuqua, C; Winans, S C

    1997-01-01

    By screening for octopine-inducible gene expression, we previously identified all the genes required for utilization of octopine as a source of carbon, nitrogen, and energy. They are (i) octopine oxidase, which converts octopine to arginine and pyruvate and is encoded by the ooxAB operon, (ii) arginase, which converts arginine to ornithine and urea and is encoded by arcA, (iii) ornithine cyclodeaminase, which converts ornithine to proline and ammonia and is encoded by the homologous arcB and ocd genes, and (iv) proline dehydrogenase, which converts proline to glutamate and is encoded by putA. Here we describe the regulation and localization of each of these genes. The ooxA-ooxB-ocd operon was previously shown to reside on the Ti plasmid and to be directly inducible by octopine. The arcAB operon is directly inducible by arginine, while it is induced by octopine only in strains that can convert octopine to arginine. Ornithine may also be a direct inducer of arcAB. putA is directly inducible by proline, while induction by octopine and by arginine (and probably by ornithine) requires their conversion to proline. Genetic studies indicate that arcAB and putA are localized on a conjugal genetic element. This element can be transferred to other Agrobacterium tumefaciens strains by a mechanism that does not require recA-dependent homologous recombination. Transfer of this genetic element from A. tumefaciens R10 requires at least one tra gene found on its Ti plasmid, indicating that this element is not self-transmissible but is mobilizable by the Ti plasmid. The DNA containing the arcAB and putA genes comigrates with a 243-kb linear molecular weight standard on field inversion electrophoretic gels. PMID:8981973

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

    PubMed Central

    Christianson, John P.; Flyer-Adams, Johanna G.; Drugan, Robert C.; Amat, Jose; Daut, Rachel A.; Foilb, Allison R.; Watkins, Linda R.; Maier, Steven F.

    2014-01-01

    Behaviorally controllable stressors confer protection from the neurochemical and behavioral consequences of future uncontrollable stressors, a phenomenon termed “behavioral immunization”. Recent data implicate protein synthesis within the ventromedial prefrontal cortex (mPFC) as critical to behavioral immunization. Adult, male Sprague-Dawley rats were exposed to a series of controllable tailshocks and 1 week later to uncontrollable tailshocks, followed 24 h later by social exploration and shuttlebox escape tests. To test the involvement of N-methyl-D-aspartate receptors (NMDARs) and the extracellular signal-regulated kinase (ERK) cascade in behavioral immunization, either D-AP5 or the MEK inhibitor U0126 was injected to the prelimbic (PL) or infralimbic (IL) mPFC prior to controllable stress exposure. Phosphorylated ERK and P70S6K, regulators of transcription and translation, were quantified by Western blot or immunohistochemistry after controllable or uncontrollable tailshocks. Prior controllable stress prevented the social exploration and shuttlebox performance deficits caused by the later uncontrollable stressor, and this effect was blocked by injections of D-AP5 into mPFC. A significant increase in phosphorylated ERK1 and ERK2, but not P70S6K, occurred within the PL and IL in rats exposed to controllable stress, but not to uncontrollable stress. However, U0126 only prevented behavioral immunization when injected to the PL. We provide evidence that NMDAR and ERK dependent signaling within the PL region is required for behavioral immunization, a learned form of stressor resistance. PMID:25324750

  1. Nuclear-localized CTP:phosphocholine cytidylyltransferase α regulates phosphatidylcholine synthesis required for lipid droplet biogenesis

    PubMed Central

    Aitchison, Adam J.; Arsenault, Daniel J.; Ridgway, Neale D.

    2015-01-01

    The reversible association of CTP:phosphocholine cytidylyltransferase α (CCTα) with membranes regulates the synthesis of phosphatidylcholine (PC) by the CDP-choline (Kennedy) pathway. Based on results with insect CCT homologues, translocation of nuclear CCTα onto cytoplasmic lipid droplets (LDs) is proposed to stimulate the synthesis of PC that is required for LD biogenesis and triacylglycerol (TAG) storage. We examined whether this regulatory mechanism applied to LD biogenesis in mammalian cells. During 3T3-L1 and human preadipocyte differentiation, CCTα expression and PC synthesis was induced. In 3T3-L1 cells, CCTα translocated from the nucleoplasm to the nuclear envelope and cytosol but did not associate with LDs. The enzyme also remained in the nucleus during human adipocyte differentiation. RNAi silencing in 3T3-L1 cells showed that CCTα regulated LD size but did not affect TAG storage or adipogenesis. LD biogenesis in nonadipocyte cell lines treated with oleate also promoted CCTα translocation to the nuclear envelope and/or cytoplasm but not LDs. In rat intestinal epithelial cells, CCTα silencing increased LD size, but LD number and TAG deposition were decreased due to oleate-induced cytotoxicity. We conclude that CCTα increases PC synthesis for LD biogenesis by translocation to the nuclear envelope and not cytoplasmic LDs. PMID:26108622

  2. 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.

  3. The regulation of glial-specific splicing of Neurexin IV requires HOW and Cdk12 activity.

    PubMed

    Rodrigues, Floriano; Thuma, Leila; Klämbt, Christian

    2012-05-01

    The differentiation of the blood-brain barrier (BBB) is an essential process in the development of a complex nervous system and depends on alternative splicing. In the fly BBB, glial cells establish intensive septate junctions that require the cell-adhesion molecule Neurexin IV. Alternative splicing generates two different Neurexin IV isoforms: Neurexin IV(exon3), which is found in cells that form septate junctions, and Neurexin IV(exon4), which is found in neurons that form no septate junctions. Here, we show that the formation of the BBB depends on the RNA-binding protein HOW (Held out wings), which triggers glial specific splicing of Neurexin IV(exon3). Using a set of splice reporters, we show that one HOW-binding site is needed to include one of the two mutually exclusive exons 3 and 4, whereas binding at the three further motifs is needed to exclude exon 4. The differential splicing is controlled by nuclear access of HOW and can be induced in neurons following expression of nuclear HOW. Using a novel in vivo two-color splicing detector, we then screened for genes required for full HOW activity. This approach identified Cyclin-dependent kinase 12 (Cdk12) and the splicesosomal component Prp40 as major determinants in regulating HOW-dependent splicing of Neurexin IV. Thus, in addition to the control of nuclear localization of HOW, the phosphorylation of the C-terminal domain of the RNA polymerase II by Cdk12 provides an elegant mechanism in regulating timed splicing of newly synthesized mRNA molecules.

  4. 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...

  5. Nutritional requirements and nitrogen-dependent regulation of proteinase activity of Lactobacillus helveticus CRL 1062.

    PubMed

    Hebert, E M; Raya, R R; De Giori, G S

    2000-12-01

    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 beta-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

  6. Gravin regulates mesodermal cell behavior changes required for axis elongation during zebrafish gastrulation.

    PubMed

    Weiser, Douglas C; Pyati, Ujwal J; Kimelman, David

    2007-06-15

    Convergent extension of the mesoderm is the major driving force of vertebrate gastrulation. During this process, mesodermal cells move toward the future dorsal side of the embryo, then radically change behavior as they initiate extension of the body axis. How cells make this transition in behavior is unknown. We have identified the scaffolding protein and tumor suppressor Gravin as a key regulator of this process in zebrafish embryos. We show that Gravin is required for the conversion of mesodermal cells from a highly migratory behavior to the medio-laterally intercalative behavior required for body axis extension. In the absence of Gravin, paraxial mesodermal cells fail to shut down the protrusive activity mediated by the Rho/ROCK/Myosin II pathway, resulting in embryos with severe extension defects. We propose that Gravin functions as an essential scaffold for regulatory proteins that suppress the migratory behavior of the mesoderm during gastrulation, and suggest that this function also explains how Gravin inhibits invasive behaviors in metastatic cells.

  7. 76 FR 53886 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Special...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-30

    ... Regulation Supplement; Special Contracting Methods AGENCY: Defense Acquisition Regulations System, Department... following methods: Regulations.gov : http://www.regulations.gov . Follow the instructions for submitting... Contracting Methods, and related provisions and clauses at DFARS 252.217-7012, Liability and Insurance;...

  8. Temporal requirements of the fragile X mental retardation protein in the regulation of synaptic structure.

    PubMed

    Gatto, Cheryl L; Broadie, Kendal

    2008-08-01

    Fragile X syndrome (FraX), caused by the loss-of-function of one gene (FMR1), is the most common inherited form of both mental retardation and autism spectrum disorders. The FMR1 product (FMRP) is an mRNA-binding translation regulator that mediates activity-dependent control of synaptic structure and function. To develop any FraX intervention strategy, it is essential to define when and where FMRP loss causes the manifestation of synaptic defects, and whether the reintroduction of FMRP can restore normal synapse properties. In the Drosophila FraX model, dFMRP loss causes neuromuscular junction (NMJ) synapse over-elaboration (overgrowth, overbranching, excess synaptic boutons), accumulation of development-arrested satellite boutons, and altered neurotransmission. We used the Gene-Switch method to conditionally drive dFMRP expression to define the spatiotemporal requirements in synaptic mechanisms. Constitutive induction of targeted neuronal dFMRP at wild-type levels rescues all synaptic architectural defects in Drosophila Fmr1 (dfmr1)-null mutants, demonstrating a presynaptic requirement for synapse structuring. By contrast, presynaptic dFMRP expression does not ameliorate functional neurotransmission defects, indicating a postsynaptic dFMRP requirement. Strikingly, targeted early induction of dFMRP effects nearly complete rescue of synaptic structure defects, showing a primarily early-development role. In addition, acute dFMRP expression at maturity partially alleviates dfmr1-null defects, although rescue is not as complete as either early or constitutive dFMRP expression, showing a modest capacity for late-stage structural plasticity. We conclude that dFMRP predominantly acts early in synaptogenesis to modulate architecture, but that late dFMRP introduction at maturity can weakly compensate for early absence of dFMRP function. PMID:18579676

  9. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling

    PubMed Central

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  10. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling.

    PubMed

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  11. 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…

  12. RBPJ in mouse Sertoli cells is required for proper regulation of the testis stem cell niche.

    PubMed

    Garcia, Thomas Xavier; Farmaha, Jaspreet Kaur; Kow, Sean; Hofmann, Marie-Claude

    2014-12-01

    Stem cells are influenced by their surrounding microenvironment, or niche. In the testis, Sertoli cells are the key niche cells directing the population size and differentiation fate of spermatogonial stem cells (SSCs). Failure to properly regulate SSCs leads to infertility or germ cell hyperplasia. Several Sertoli cell-expressed genes, such as Gdnf and Cyp26b1, have been identified as being indispensable for the proper maintenance of SSCs in their niche, but the pathways that modulate their expression have not been identified. Although we have recently found that constitutively activating NOTCH signaling in Sertoli cells leads to premature differentiation of all prospermatogonia and sterility, suggesting that there is a crucial role for this pathway in the testis stem cell niche, a true physiological function of NOTCH signaling in Sertoli cells has not been demonstrated. To this end, we conditionally ablated recombination signal binding protein for immunoglobulin kappa J region (Rbpj), a crucial mediator of NOTCH signaling, in Sertoli cells using Amh-cre. Rbpj knockout mice had: significantly increased testis sizes; increased expression of niche factors, such as Gdnf and Cyp26b1; significant increases in the number of pre- and post-meiotic germ cells, including SSCs; and, in a significant proportion of mice, testicular failure and atrophy with tubule lithiasis, possibly due to these unsustainable increases in the number of germ cells. We also identified germ cells as the NOTCH ligand-expressing cells. We conclude that NOTCH signaling in Sertoli cells is required for proper regulation of the testis stem cell niche and is a potential feedback mechanism, based on germ cell input, that governs the expression of factors that control SSC proliferation and differentiation.

  13. 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.

  14. 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...

  15. 75 FR 54526 - Defense Federal Acquisition Regulation Supplement; Additional Requirements Applicable to...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-08

    ..., Defense Acquisition Regulations System. 0 Therefore, the interim rule published at 75 FR 9114 on March 1... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF DEFENSE Defense Acquisition Regulations System 48 CFR Part 217 Defense Federal Acquisition Regulation...

  16. The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation.

    PubMed

    Zhang, Jixiang; Xie, Shaojun; Cheng, Jinkui; Lai, Jinsheng; Zhu, Jian-Kang; Gong, Zhizhong

    2016-06-01

    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

  17. Multiple protein kinase A-regulated events are required for transcriptional induction by cAMP.

    PubMed Central

    Brindle, P; Nakajima, T; Montminy, M

    1995-01-01

    The second messenger cAMP stimulates the expression of numerous genes via the protein kinase A-mediated phosphorylation of the cAMP response element-binding protein (CREB) at Ser-133. Ser-133 phosphorylation, in turn, appears to induce target gene expression by promoting interaction between CREB and CBP, a 265-kDa nuclear phospho-CREB-binding protein. It is unclear, however, whether Ser-133 phosphorylation per se is sufficient for CREB-CBP complex formation and for target gene induction in vivo. Here we examine CREB activity in Jurkat T cells after stimulation of the T-cell receptor (TCR), an event that leads to calcium entry and diacylglycerol production. Triggering of the TCR stimulated Ser-133 phosphorylation of CREB with high stoichiometry, but TCR activation did not promote CREB-CBP complex formation or target gene induction unless suboptimal doses of cAMP agonist were provided as a costimulus. Our results demonstrate that, in addition to mediating Ser-133 phosphorylation of CREB, protein kinase A regulates additional proteins that are required for recruitment of the transcriptional apparatus to cAMP-responsive genes. Images Fig. 1 Fig. 2 Fig. 3 PMID:7479832

  18. Cell Type–dependent Requirement for PIP Box–regulated Cdt1 Destruction During S Phase

    PubMed Central

    Lee, Hyun O.; Zacharek, Sima J.; Xiong, Yue

    2010-01-01

    DNA synthesis–coupled proteolysis of the prereplicative complex component Cdt1 by the CRL4Cdt2 E3 ubiquitin ligase is thought to help prevent rereplication of the genome during S phase. To directly test whether CRL4Cdt2-triggered destruction of Cdt1 is required for normal cell cycle progression in vivo, we expressed a mutant version of Drosophila Cdt1 (Dup), which lacks the PCNA-binding PIP box (DupΔPIP) and which cannot be regulated by CRL4Cdt2. DupΔPIP is inappropriately stabilized during S phase and causes developmental defects when ectopically expressed. DupΔPIP restores DNA synthesis to dup null mutant embryonic epidermal cells, but S phase is abnormal, and these cells do not progress into mitosis. In contrast, DupΔPIP accumulation during S phase did not adversely affect progression through follicle cell endocycles in the ovary. In this tissue the combination of DupΔPIP expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration. We could not detect Dup hyperaccumulation using mutations in the CRL4Cdt2 components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation. These data indicate that PIP box–mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain DupΔPIP activity in some endocycling cell types. PMID:20826610

  19. Vernalization and the chilling requirement to exit bud dormancy: shared or separate regulation?

    PubMed Central

    Brunner, Amy M.; Evans, Luke M.; Hsu, Chuan-Yu; Sheng, Xiaoyan

    2014-01-01

    Similarities have long been recognized between vernalization, the prolonged exposure to cold temperatures that promotes the floral transition in many plants, and the chilling requirement to release bud dormancy in woody plants of temperate climates. In both cases the extended chilling period occurring during winter is used to coordinate developmental events to the appropriate seasonal time. However, whether or not these processes share common regulatory components and molecular mechanisms remain largely unknown. Both gene function and association genetics studies in Populus are beginning to answer this question. In Populus, studies have revealed that orthologs of the antagonistic flowering time genes FT and CEN/TFL1 might have central roles in both processes. We review Populus seasonal shoot development related to dormancy release and the floral transition and evidence for FT/TFL1-mediated regulation of these processes to consider the question of regulatory overlap. In addition, we discuss the potential for and challenges to integrating functional and population genomics studies to uncover the regulatory mechanisms underpinning these processes in woody plant systems. PMID:25566302

  20. 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

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

    NASA Astrophysics Data System (ADS)

    Wang, Jian; Wang, Wen-Xiong

    2016-05-01

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

  2. 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

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

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 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... 7 Agriculture 5 2013-01-01 2013-01-01 false Movement of plant pests regulated; permits...

  4. 75 FR 68330 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-05

    ... Regulation Supplement; Occupational Safety and Drug-Free Work Force (OMB Control Number 0704-0272) AGENCY... 223, Occupational Safety and Drug-Free Work Force, and related clauses in DFARS 252.223; OMB Control..., identified by OMB Control Number ] 0704-0272, using any of the following methods: o Regulations.gov :...

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

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-15

    ... Regulation Supplement; Part 251, Contractor Use of Government Supply Sources AGENCY: Defense Acquisition... Regulation Supplement (DFARS) Part 251, Contractor Use of Government Supply Sources, and the associated clauses at DFARS 252.251-7000, Ordering from Government Supply Sources; and 252.251-7001, Use...

  6. 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...

  7. Phospholipid transfer protein Sec14 is required for trafficking from endosomes and regulates distinct trans-Golgi export pathways.

    PubMed

    Curwin, Amy J; Fairn, Gregory D; McMaster, Christopher R

    2009-03-13

    A protein known to regulate both lipid metabolism and vesicular transport is the phosphatidylcholine/phosphatidylinositol transfer protein Sec14 of Saccharomyces cerevisiae. Sec14 is thought to globally affect secretion from the trans-Golgi. The results from a synthetic genetic array screen for genes whose inactivation impaired growth of cells with a temperature-sensitive SEC14 allele implied Sec14 regulates transport into and out of the Golgi. This prompted us to examine the role of Sec14 in various vesicular transport pathways. We determined that Sec14 function was required for the route followed by Bgl2, whereas trafficking of other secreted proteins, including Hsp150, Cts1, Scw4, Scw10, Exg1, Cis3, and Ygp1, still occurred, indicating Sec14 regulates specific trans-Golgi export pathways. Upon diminution of Sec14 function, the v-SNARE Snc1 accumulated in endosomes and the trans-Golgi. Its accumulation in endosomes is consistent with Sec14 being required for transport from endosomes to the trans-Golgi. Sec14 was also required for trafficking of Ste3 and the lipophilic dye FM4-64 from the plasma membrane to the vacuole at the level of the endosome. The combined genetic and cell biology data are consistent with regulation of endosome trafficking being a major role for Sec14. We further determined that lipid ligand occupancy differentially regulates Sec14 functions.

  8. 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

    ...\\ 66 FR 67680 (December 31, 2001), codified at 31 CFR 1010.330. \\7\\ 26 CFR 1.6050I-1. As amended... 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...

  9. Caveolin-1–dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo

    PubMed Central

    Marchiando, Amanda M.; Shen, Le; Graham, W. Vallen; Weber, Christopher R.; Schwarz, Brad T.; Austin, Jotham R.; Raleigh, David R.; Guan, Yanfang; Watson, Alastair J.M.; Montrose, Marshall H.

    2010-01-01

    Epithelial paracellular barrier function, determined primarily by tight junction permeability, is frequently disrupted in disease. In the intestine, barrier loss can be mediated by tumor necrosis factor (α) (TNF) signaling and epithelial myosin light chain kinase (MLCK) activation. However, TNF induces only limited alteration of tight junction morphology, and the events that couple structural reorganization to barrier regulation have not been defined. We have used in vivo imaging and transgenic mice expressing fluorescent-tagged occludin and ZO-1 fusion proteins to link occludin endocytosis to TNF-induced tight junction regulation. This endocytosis requires caveolin-1 and is essential for structural and functional tight junction regulation. These data demonstrate that MLCK activation triggers caveolin-1–dependent endocytosis of occludin to effect structural and functional tight junction regulation. PMID:20351069

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

    PubMed

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

    2013-04-01

    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

  11. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... with the requirements in appendix A of 10 CFR part 40 of this chapter to the maximum extent practicable... material, an Agreement State shall require: (1) Compliance with requirements in appendix A of 10 CFR part... in appendix A of 10 CFR part 40 of this chapter adopted and enforced by the Commission for the...

  12. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... with the requirements in appendix A of 10 CFR part 40 of this chapter to the maximum extent practicable... material, an Agreement State shall require: (1) Compliance with requirements in appendix A of 10 CFR part... in appendix A of 10 CFR part 40 of this chapter adopted and enforced by the Commission for the...

  13. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... with the requirements in appendix A of 10 CFR part 40 of this chapter to the maximum extent practicable... material, an Agreement State shall require: (1) Compliance with requirements in appendix A of 10 CFR part... in appendix A of 10 CFR part 40 of this chapter adopted and enforced by the Commission for the...

  14. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... with the requirements in appendix A of 10 CFR part 40 of this chapter to the maximum extent practicable... material, an Agreement State shall require: (1) Compliance with requirements in appendix A of 10 CFR part... in appendix A of 10 CFR part 40 of this chapter adopted and enforced by the Commission for the...

  15. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... with the requirements in appendix A of 10 CFR part 40 of this chapter to the maximum extent practicable... material, an Agreement State shall require: (1) Compliance with requirements in appendix A of 10 CFR part... in appendix A of 10 CFR part 40 of this chapter adopted and enforced by the Commission for the...

  16. Mycobacterium tuberculosis Phosphate Uptake System Component PstA2 Is Not Required for Gene Regulation or Virulence.

    PubMed

    Tischler, Anna D; Leistikow, Rachel L; Ramakrishnan, Pavithra; Voskuil, Martin I; McKinney, John D

    2016-01-01

    The Mycobacterium tuberculosis genome encodes two complete high-affinity Pst phosphate-specific transporters. We previously demonstrated that a membrane-spanning component of one Pst system, PstA1, was essential both for M. tuberculosis virulence and for regulation of gene expression in response to external phosphate availability. To determine if the alternative Pst system is similarly required for virulence or gene regulation, we constructed a deletion of pstA2. Transcriptome analysis revealed that PstA2 is not required for regulation of gene expression in phosphate-replete growth conditions. PstA2 was also dispensable for replication and virulence of M. tuberculosis in a mouse aerosol infection model. However, a ΔpstA1ΔpstA2 double mutant was attenuated in mice lacking the cytokine interferon-gamma, suggesting that M. tuberculosis requires high-affinity phosphate transport to survive phosphate limitation encountered in the host. Surprisingly, ΔpstA2 bacteria were more resistant to acid stress in vitro. This phenotype is intrinsic to the alternative Pst transporter since a ΔpstS1 mutant exhibited similar acid resistance. Our data indicate that the two M. tuberculosis Pst transporters have distinct physiological functions, with the PstA1 transporter being specifically involved in phosphate sensing and gene regulation while the PstA2 transporter influences survival in acidic conditions. PMID:27557082

  17. Mycobacterium tuberculosis Phosphate Uptake System Component PstA2 Is Not Required for Gene Regulation or Virulence

    PubMed Central

    Leistikow, Rachel L.; Ramakrishnan, Pavithra; Voskuil, Martin I.; McKinney, John D.

    2016-01-01

    The Mycobacterium tuberculosis genome encodes two complete high-affinity Pst phosphate-specific transporters. We previously demonstrated that a membrane-spanning component of one Pst system, PstA1, was essential both for M. tuberculosis virulence and for regulation of gene expression in response to external phosphate availability. To determine if the alternative Pst system is similarly required for virulence or gene regulation, we constructed a deletion of pstA2. Transcriptome analysis revealed that PstA2 is not required for regulation of gene expression in phosphate-replete growth conditions. PstA2 was also dispensable for replication and virulence of M. tuberculosis in a mouse aerosol infection model. However, a ΔpstA1ΔpstA2 double mutant was attenuated in mice lacking the cytokine interferon-gamma, suggesting that M. tuberculosis requires high-affinity phosphate transport to survive phosphate limitation encountered in the host. Surprisingly, ΔpstA2 bacteria were more resistant to acid stress in vitro. This phenotype is intrinsic to the alternative Pst transporter since a ΔpstS1 mutant exhibited similar acid resistance. Our data indicate that the two M. tuberculosis Pst transporters have distinct physiological functions, with the PstA1 transporter being specifically involved in phosphate sensing and gene regulation while the PstA2 transporter influences survival in acidic conditions. PMID:27557082

  18. Transcription factors of Lotus: regulation of isoflavonoid biosynthesis requires coordinated changes in transcription factor activity.

    PubMed

    Shelton, Dale; Stranne, Maria; Mikkelsen, Lisbeth; Pakseresht, Nima; Welham, Tracey; Hiraka, Hideki; Tabata, Satoshi; Sato, Shusei; Paquette, Suzanne; Wang, Trevor L; Martin, Cathie; Bailey, Paul

    2012-06-01

    Isoflavonoids are a class of phenylpropanoids made by legumes, and consumption of dietary isoflavonoids confers benefits to human health. Our aim is to understand the regulation of isoflavonoid biosynthesis. Many studies have shown the importance of transcription factors in regulating the transcription of one or more genes encoding enzymes in phenylpropanoid metabolism. In this study, we coupled bioinformatics and coexpression analysis to identify candidate genes encoding transcription factors involved in regulating isoflavonoid biosynthesis in Lotus (Lotus japonicus). Genes encoding proteins belonging to 39 of the main transcription factor families were examined by microarray analysis of RNA from leaf tissue that had been elicited with glutathione. Phylogenetic analyses of each transcription factor family were used to identify subgroups of proteins that were specific to L. japonicus or closely related to known regulators of the phenylpropanoid pathway in other species. R2R3MYB subgroup 2 genes showed increased expression after treatment with glutathione. One member of this subgroup, LjMYB14, was constitutively overexpressed in L. japonicus and induced the expression of at least 12 genes that encoded enzymes in the general phenylpropanoid and isoflavonoid pathways. A distinct set of six R2R3MYB subgroup 2-like genes was identified. We suggest that these subgroup 2 sister group proteins and those belonging to the main subgroup 2 have roles in inducing isoflavonoid biosynthesis. The induction of isoflavonoid production in L. japonicus also involves the coordinated down-regulation of competing biosynthetic pathways by changing the expression of other transcription factors. PMID:22529285

  19. 78 FR 15935 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-13

    ... Involving Aircraft, Missiles, and Space Launch Vehicles, requires the contractor to report promptly to the..., or space launch vehicle being manufactured, modified, repaired, or overhauled in connection with...

  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. 76 FR 24443 - Federal Acquisition Regulation; Service Contracts Reporting Requirements; Correction

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-02

    ... (202) 501-0044. Please cite FAR Case 2010-010. Correction In the proposed rule FR Doc. 2011-9515...., cost-reimbursement, time-and- materials, and labor-hour contracts) that already require contractors to... required to report on all cost-reimbursement, time-and-materials, and labor-hour contracts at or above...

  2. 76 FR 17138 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-28

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements... public workshop on FDA's clinical trial requirements is designed to aid the clinical research... interaction with FDA representatives. The program will focus on the relationships among FDA and clinical...

  3. 77 FR 8886 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-15

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements... public workshop on FDA's clinical trial requirements is designed to aid the clinical research... interaction with FDA representatives. The program will focus on the relationships among FDA and clinical...

  4. 76 FR 78933 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-20

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements... workshop. The public workshop on FDA's clinical trial requirements is designed to aid the clinical research... interaction with FDA representatives. The program will focus on the relationships among FDA and clinical...

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

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-17

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements... workshop. The public workshop on FDA's clinical trial requirements is designed to aid the clinical research... interaction with FDA representatives. The program will focus on the relationships among FDA and clinical...

  6. 75 FR 51824 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-23

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements... workshop. The public workshop on FDA's clinical trial requirements is designed to aid the clinical research... interaction with FDA representatives. The program will focus on the relationships among FDA and clinical...

  7. 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

    ... interest rate risk rule requirements. \\1\\ IRPS 03-2, 68 FR 31949 (May 29, 2003). B. Why is the Board... net worth and interest rate risk requirements. The amended IRPS would result in more robust... rule amends the asset range in 12 CFR 741.3(b)(5)(i), NCUA's interest rate risk rule. In 12 CFR...

  8. 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... Register on November 18, 2011 (76 FR 71708) (FRL-9485-5). These changes establish federal requirements...

  9. 77 FR 12927 - Federal Acquisition Regulation: Requirements for Acquisitions Pursuant to Multiple-Award Contracts

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-02

    .... Background DoD, GSA, and NASA published an interim rule in the Federal Register at 76 FR 14548 on March 16... Register at 76 FR 14548 on March 16, 2011, is adopted as final with the following changes: PART 8--REQUIRED...; (5) Competition requirements for establishing BPAs and allowing flexibility in establishing...

  10. 33 CFR 165.921 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in this section— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR... explosives as defined in 49 CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415 or, for which a permit is required as a condition of a Research and...

  11. 33 CFR 165.921 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... in this section— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR... explosives as defined in 49 CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415 or, for which a permit is required as a condition of a Research and...

  12. 33 CFR 165.921 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in this section— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR... explosives as defined in 49 CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415 or, for which a permit is required as a condition of a Research and...

  13. 33 CFR 165.921 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... in this section— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR... explosives as defined in 49 CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415 or, for which a permit is required as a condition of a Research and...

  14. 33 CFR 165.921 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... in this section— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR... explosives as defined in 49 CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415 or, for which a permit is required as a condition of a Research and...

  15. 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

    ... thresholds to grant relief from risk-based net worth and interest rate risk requirements. The amended IRPS... interest rate risk rule requirements. To cross-reference IRPS 13-1, the final rule makes a technical... used to define small entity under the RFA and to determine the applicability of interest rate risk...

  16. 77 FR 74177 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Production...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-13

    ... Regulation Supplement; Production Surveillance and Reporting (OMB Control Number 0704-0250) AGENCY: Defense... or other forms of information technology. The Office of Management and Budget (OMB) has approved this... contract administration personnel to perform production surveillance to monitor contractor progress...

  17. 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

    ... of Federal Regulations EPA U.S. Environmental Protection Agency FR Federal Register GHG greenhouse... industry sectors (74 FR 56260). Under 40 CFR part 98 of the GHG Reporting Rule (hereinafter referred to as... information that are not ] emissions data, and are CBI'' (74 FR 56287, October 30, 2009). On July 7, 2010,...

  18. 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

  19. A Comparison of the Mandatory Continuing Education (MCE) Requirements of the Regulated Health Occupations in Minnesota.

    ERIC Educational Resources Information Center

    Green-Eide, Beth

    A study reviewed and compared initial and renewal practices for licensure/registration of 13 health care occupations regulated in the state of Minnesota. It examined mandatory continuing education (MCE) documentation and the practices of licensing boards in their enforcement of the MCE legislation. The Minnesota Statutes and Rules for the…

  20. 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...

  1. 77 FR 51686 - Specialty Crops; Import Regulations; New Pistachio Import Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-27

    ... in the marketing order for domestic pistachios as established at 69 FR 17844 (April 5, 2004) and amended at 74 FR 56532 (November 2, 2009). Under the new regulation, ``pistachio'' means the nut of the... testing procedures that align with the Codex Alimentarius Commission's (Codex) sampling plan (75 FR...

  2. 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

    ... degrees longitude. \\1\\ The reader may refer to the Proposed Rulemaking, December 5, 1991 (56 FR 63774), and the preamble to the final rule promulgated September 4, 1992 (57 FR 40792) for further background and information on the OCS regulations. On October 2, 2009 (74 FR 50939), EPA proposed to...

  3. 17 CFR 230.508 - Insignificant deviations from a term, condition or requirement of Regulation D.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ..., Dec. 30, 1958, unless otherwise noted. Cross Reference: For regulations of Small Business Administration under the Small Business Investment Act of 1958, see 13 CFR, Chapter I. ... Small Business Investment Companies Authority: Sections 230.601 to 230.610a issued under sec. 19,...

  4. 17 CFR 230.508 - Insignificant deviations from a term, condition or requirement of Regulation D.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ..., Dec. 30, 1958, unless otherwise noted. Cross Reference: For regulations of Small Business Administration under the Small Business Investment Act of 1958, see 13 CFR, Chapter I. ... Small Business Investment Companies Authority: Sections 230.601 to 230.610a issued under sec. 19,...

  5. 17 CFR 230.508 - Insignificant deviations from a term, condition or requirement of Regulation D.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ..., Dec. 30, 1958, unless otherwise noted. Cross Reference: For regulations of Small Business Administration under the Small Business Investment Act of 1958, see 13 CFR, Chapter I. ... Small Business Investment Companies Authority: Sections 230.601 to 230.610a issued under sec. 19,...

  6. 77 FR 25611 - Endangered and Threatened Wildlife and Plants; Revised Implementing Regulations for Requirements...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-01

    ... Code of Federal Regulations. In the interest of making the process of designating critical habitat more... with what they learned. DATES: This rule becomes effective May 31, 2012. ADDRESSES: This final rule is..., jurisdictions, and interested parties for use in their computer databases and to make available to...

  7. 76 FR 28405 - Endangered and Threatened Wildlife and Plants; Revised Implementing Regulations for Requirements...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-17

    ... Federal Register for codification in the Code of Federal Regulations. In the interest of making the... alia, to modify or streamline them in accordance with what has been learned. DATES: We will accept comments from all interested parties until July 18, 2011. Please note that if you are using the Federal...

  8. 27 CFR 70.411 - Imposition of taxes, qualification requirements, and regulations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...: (1) Establishment and operation of distilled spirits plants. Part 19 of title 27 CFR contains the... vaporizing process. (2) Miscellaneous liquor transactions. Part 29 of 27 CFR contains miscellaneous... 27 CFR contains the regulations that. prescribe the gauging instruments, and methods or techniques...

  9. 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

    ... to be discussed are FDA regulations with respect to importing pharmaceutical products, medical... meeting. SUMMARY: The Food and Drug Administration (FDA) is announcing the following meeting..., Chicago, IL 60661; 312-596-4217; email: lisa.misevicz@fda.hhs.gov . Registration: Send...

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

    PubMed

    Lord, Caleb C; Ferguson, Daniel; Thomas, Gwynneth; Brown, Amanda L; Schugar, Rebecca C; Burrows, Amy; Gromovsky, Anthony D; Betters, Jenna; Neumann, Chase; Sacks, Jessica; Marshall, Stephanie; Watts, Russell; Schweiger, Martina; Lee, Richard G; Crooke, Rosanne M; Graham, Mark J; Lathia, Justin D; Sakaguchi, Takuya F; Lehner, Richard; Haemmerle, Guenter; Zechner, Rudolf; Brown, J Mark

    2016-07-26

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

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

    PubMed

    Lord, Caleb C; Ferguson, Daniel; Thomas, Gwynneth; Brown, Amanda L; Schugar, Rebecca C; Burrows, Amy; Gromovsky, Anthony D; Betters, Jenna; Neumann, Chase; Sacks, Jessica; Marshall, Stephanie; Watts, Russell; Schweiger, Martina; Lee, Richard G; Crooke, Rosanne M; Graham, Mark J; Lathia, Justin D; Sakaguchi, Takuya F; Lehner, Richard; Haemmerle, Guenter; Zechner, Rudolf; Brown, J Mark

    2016-07-26

    Adipose triglyceride lipase (ATGL) and comparative gene identification 58 (CGI-58) are critical regulators of triacylglycerol (TAG) turnover. CGI-58 is thought to regulate TAG mobilization by stimulating the enzymatic activity of ATGL. However, it is not known whether this coactivation function of CGI-58 occurs in vivo. Moreover, the phenotype of human CGI-58 mutations suggests ATGL-independent functions. Through direct comparison of mice with single or double deficiency of CGI-58 and ATGL, we show here that CGI-58 knockdown causes hepatic steatosis in both the presence and absence of ATGL. CGI-58 also regulates hepatic diacylglycerol (DAG) and inflammation in an ATGL-independent manner. Interestingly, ATGL deficiency, but not CGI-58 deficiency, results in suppression of the hepatic and adipose de novo lipogenic program. Collectively, these findings show that CGI-58 regulates hepatic neutral lipid storage and inflammation in the genetic absence of ATGL, demonstrating that mechanisms driving TAG lipolysis in hepatocytes differ significantly from those in adipocytes. PMID:27396333

  12. 21 CFR 369.21 - Drugs; warning and caution statements required by regulations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... children under 12 years of age consult a physician immediately. ALCOHOL RUBBING COMPOUND. (See 26 CFR 182... Commissioner may establish by regulation an acceptable alternative method, e.g., a type size smaller than 1/16... time of use; (c) Products of a net quantity of contents of less than 2 ozs. that are designed...

  13. 21 CFR 369.21 - Drugs; warning and caution statements required by regulations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... children under 12 years of age consult a physician immediately. ALCOHOL RUBBING COMPOUND. (See 26 CFR 182... Commissioner may establish by regulation an acceptable alternative method, e.g., a type size smaller than 1/16... time of use; (c) Products of a net quantity of contents of less than 2 ozs. that are designed...

  14. ECM-Regulator timp Is Required for Stem Cell Niche Organization and Cyst Production in the Drosophila Ovary

    PubMed Central

    Pearson, John R.; Zurita, Federico; Tomás-Gallardo, Laura; Díaz-Torres, Alfonsa; Díaz de la Loza, María del Carmen; Franze, Kristian; Martín-Bermudo, María D.; González-Reyes, Acaimo

    2016-01-01

    The extracellular matrix (ECM) is a pivotal component adult tissues and of many tissue-specific stem cell niches. It provides structural support and regulates niche signaling during tissue maintenance and regeneration. In many tissues, ECM remodeling depends on the regulation of MMP (matrix metalloproteinase) activity by inhibitory TIMP (tissue inhibitors of metalloproteinases) proteins. Here, we report that the only Drosophila timp gene is required for maintaining the normal organization and function of the germline stem cell niche in adult females. timp mutant ovaries show reduced levels of both Drosophila Collagen IV α chains. In addition, tissue stiffness and the cellular organization of the ovarian niche are affected in timp mutants. Finally, loss of timp impairs the ability of the germline stem cell niche to generate new cysts. Our results demonstrating a crucial role for timp in tissue organization and gamete production thus provide a link between the regulation of ECM metabolism and tissue homeostasis. PMID:26808525

  15. 78 FR 68830 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Service...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-15

    ... requirements addressed in this notice are available on the World Wide Web at: http://www.acq.osd.mil/dpap/dars....237-7011, Preparation History, and DD Form 2063, Record of Preparation and Disposition of...

  16. 75 FR 12518 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 237...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-16

    ... collection requirements addressed in this notice are available electronically on the World Wide Web at: http... Responsibility, and 252.237-7011, Preparation History, and DD Form 2063, Record of Preparation and Disposition...

  17. 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

  18. The cytoskeletal regulator Genghis khan is required for columnar target specificity in the Drosophila visual system

    PubMed Central

    Gontang, Allison C.; Hwa, Jennifer J.; Mast, Joshua D.; Schwabe, Tina; Clandinin, Thomas R.

    2011-01-01

    A defining characteristic of neuronal cell type is the growth of axons and dendrites into specific layers and columns of the brain. Although differences in cell surface receptors and adhesion molecules are known to cause differences in synaptic specificity, differences in downstream signaling mechanisms that determine cell type-appropriate targeting patterns are unknown. Using a forward genetic screen in Drosophila, we identify the GTPase effector Genghis khan (Gek) as playing a crucial role in the ability of a subset of photoreceptor (R cell) axons to innervate appropriate target columns. In particular, single-cell mosaic analyses demonstrate that R cell growth cones lacking Gek function grow to the appropriate ganglion, but frequently fail to innervate the correct target column. Further studies reveal that R cell axons lacking the activity of the small GTPase Cdc42 display similar defects, providing evidence that these proteins regulate a common set of processes. Gek is expressed in all R cells, and a detailed structure-function analysis reveals a set of regulatory domains with activities that restrict Gek function to the growth cone. Although Gek does not normally regulate layer-specific targeting, ectopic expression of Gek is sufficient to alter the targeting choices made by another R cell type, the targeting of which is normally Gek independent. Thus, specific regulation of cytoskeletal responses to targeting cues is necessary for cell type-appropriate synaptic specificity. PMID:22007130

  19. The cytoskeletal regulator Genghis khan is required for columnar target specificity in the Drosophila visual system.

    PubMed

    Gontang, Allison C; Hwa, Jennifer J; Mast, Joshua D; Schwabe, Tina; Clandinin, Thomas R

    2011-11-01

    A defining characteristic of neuronal cell type is the growth of axons and dendrites into specific layers and columns of the brain. Although differences in cell surface receptors and adhesion molecules are known to cause differences in synaptic specificity, differences in downstream signaling mechanisms that determine cell type-appropriate targeting patterns are unknown. Using a forward genetic screen in Drosophila, we identify the GTPase effector Genghis khan (Gek) as playing a crucial role in the ability of a subset of photoreceptor (R cell) axons to innervate appropriate target columns. In particular, single-cell mosaic analyses demonstrate that R cell growth cones lacking Gek function grow to the appropriate ganglion, but frequently fail to innervate the correct target column. Further studies reveal that R cell axons lacking the activity of the small GTPase Cdc42 display similar defects, providing evidence that these proteins regulate a common set of processes. Gek is expressed in all R cells, and a detailed structure-function analysis reveals a set of regulatory domains with activities that restrict Gek function to the growth cone. Although Gek does not normally regulate layer-specific targeting, ectopic expression of Gek is sufficient to alter the targeting choices made by another R cell type, the targeting of which is normally Gek independent. Thus, specific regulation of cytoskeletal responses to targeting cues is necessary for cell type-appropriate synaptic specificity.

  20. [Regulation requirements for the protection of workers against electromagnetic fields occurring in the work environment].

    PubMed

    Aniołczyk, Halina; Zmyślony, Marek

    2006-01-01

    In Poland, electromagnetic fields (EMF), one of potentially hazardous physical factors occurring in the work environment, are subjected to compulsory surveillance. In 2001, the Directive issued by the Minister of Labor and Social Policy substantially changed the approach towards the protection of workers against EMF. The Directive regulates the whole range of EMF frequencies and electromagnetic radiation, namely from 0 Hz to 300 GHz, which means the possibility of assessing worker's EMF exposure, determined by exposure index, along with the hygiene assessment of EMF sources, defined by protection zones. In 2003-2005, a number of amended executive and supplementary regulations were issued. However, it should be emphasized that in the process of their elaboration, striving after perfection, numerous incoherent and ambiguous provisions were adopted, which finally created difficulties in the interpretation of individual regulations. This is also linked with doubts and discussions on their practical application by services responsible for control, measurements and monitoring of working conditions under the exposure to EMF. In this work an attempt was made to clarify all issues and arrange them according to the faced problems. The authors also present proposals how to solve all these problems.

  1. 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.

  2. 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

  3. A Mechanism Regulating G Protein-coupled Receptor Signaling That Requires Cycles of Protein Palmitoylation and Depalmitoylation* ♦

    PubMed Central

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

    2014-01-01

    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. PMID:24385443

  4. 49 CFR 192.13 - What general requirements apply to pipelines regulated under this part?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 3 2013-10-01 2013-10-01 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...

  5. 49 CFR 192.13 - What general requirements apply to pipelines regulated under this part?

    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...

  6. 49 CFR 192.13 - What general requirements apply to pipelines regulated under this part?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 3 2011-10-01 2011-10-01 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...

  7. 49 CFR 192.13 - What general requirements apply to pipelines regulated under this part?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 3 2014-10-01 2014-10-01 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...

  8. 49 CFR 192.13 - What general requirements apply to pipelines regulated under this part?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 3 2010-10-01 2010-10-01 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...

  9. 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…

  10. 42 CFR 51b.107 - Is participation in preventive health service programs required by these regulations?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 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...

  11. 78 FR 27908 - Federal Management Regulation (FMR); Mail Management; Financial Requirements for All Agencies

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-13

    .... Background On June 6, 2002, GSA published an interim rule in the Federal Register (67 FR 38899) that required..., 2003 (68 FR 56112). Many agencies unfortunately were not able to meet this goal. If agencies did not..., published in the Federal Register on May 13, 2008 (73 FR 27540), granted a 12 month deviation from April...

  12. 78 FR 25 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-02

    ... stay was previously published at 76 Federal Register (FR) 1360 (January 10, 2011), which expires... Acronyms CDC Certain Dangerous Cargo DHS Department of Homeland Security FR Federal Register NPRM Notice of... requirements for a two-year period. This suspension was published in the Federal Register at 76 FR...

  13. 78 FR 60216 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-01

    ... DHS Department of Homeland Security FR Federal Register NPRM Notice of Proposed Rulemaking A... requirements for a two-year period. This suspension was published in the Federal Register at 76 FR 1360... this suspension until midnight, September 30, 2013 (78 FR 25). At this time, there is no plan to...

  14. 78 FR 67132 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Contract...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-08

    ... 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...

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

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-05

    ... published in the Federal Register at 77 FR 43076, on July 23, 2012. No comments were received. Public... Register at 77 FR 43076, on July 23, 2012. Respondents: 1,934. Responses per Respondent: 2. Annual...., Washington, DC 20417. ATTN: Hada Flowers/IC 9000-0027, Value Engineering Requirements. Instructions:...

  16. 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.

  17. 77 FR 58817 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-24

    ... technology. The Office of Management and Budget (OMB) has approved this information collection for use... Financing, and the clause at 252.232-7002, Progress Payments for Foreign Military Sales Acquisitions; OMB... military equipment for foreign governments. To comply with this requirement, the Government needs to...

  18. 76 FR 14548 - Federal Acquisition Regulation; Requirements for Acquisitions Pursuant to Multiple-Award Contracts

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-16

    ... documented in the acquisition plan and contract file, (ii) the estimated value of the BPA does not exceed... the ordering activity to (i) provide a Request for Quotation (RFQ) to all BPA holders offering the required supplies or services under the BPA for orders over the SAT that includes a description of...

  19. 49 CFR 195.11 - What is a regulated rural gathering line and what requirements apply?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... subpart H of this part, except corrosion control is not required for pipelines existing on July 3, 2008... to continuously identify operating conditions that could contribute to internal corrosion. The program must include measures to prevent and mitigate internal corrosion, such as cleaning the...

  20. 75 FR 52622 - Amendment to the International Traffic in Arms Regulations: Removing Requirement for Prior...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-27

    ...'' through arms deals furthering the persecution of individuals, denial of human rights, terrorism, and... armed forces of a foreign country (42 FR 41631, dated August 18, 1977). Also, Sec. 124.06, entitled... effective January 1, 1985 (49 FR 47682, dated December 6, 1984). Section 126.8 did not require...

  1. 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...

  2. 78 FR 54403 - Removal of Transferred OTS Regulations Regarding Recordkeeping and Confirmation Requirements for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-04

    ...\\ \\2\\ 76 FR 39247 (July 6, 2011). Although section 312(b)(2)(B)(i)(II) of the Dodd-Frank Act, codified... them, as appropriate. \\3\\ 76 FR 47652 (Aug. 5, 2011). One of the OTS's rules transferred to the FDIC... confirmation requirements in appropriate circumstance.\\6\\ \\4\\ 44 FR 43260, 43261 (July 24, 1979). \\5\\ See 44...

  3. Endogenous purinergic signaling is required for osmotic volume regulation of retinal glial cells.

    PubMed

    Wurm, Antje; Lipp, Stephan; Pannicke, Thomas; Linnertz, Regina; Krügel, Ute; Schulz, Angela; Färber, Katrin; Zahn, Dirk; Grosse, Johannes; Wiedemann, Peter; Chen, Ju; Schöneberg, Torsten; Illes, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2010-03-01

    Intense neuronal activity in the sensory retina is associated with a volume increase of neuronal cells (Uckermann et al., J. Neurosci. 2004, 24:10149) and a decrease in the osmolarity of the extracellular space fluid (Dmitriev et al., Vis. Neurosci. 1999, 16:1157). Here, we show the existence of an endogenous purinergic mechanism that prevents hypoosmotic swelling of retinal glial (Müller) cells in mice. In contrast to the cells from wild-type mice, hypoosmotic stress induced rapid swelling of glial cell somata in retinal slices from mice deficient in P2Y(1), adenosine A(1) receptors, or ecto-5'-nucleotidase (CD73). Consistently, glial cell bodies in retinal slices from wild-type mice displayed osmotic swelling when P2Y(1) or A(1) receptors, or CD73, were pharmacologically blocked. Exogenous ATP, UTP, and UDP inhibited glial swelling in retinal slices, while the swelling of isolated glial cells was prevented by ATP but not by UTP or UDP, suggesting that uracil nucleotides indirectly regulate the glial cell volume via activation of neuronal P2Y(4/6) and neuron-to-glia signaling. It is suggested that autocrine/paracrine activation of purinergic receptors and enzymes is crucially involved in the regulation of the glial cell volume. PMID:20002522

  4. 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. PMID:16682458

  5. Homologues of Genetic Transformation DNA Import Genes Are Required for Rhodobacter capsulatus Gene Transfer Agent Recipient Capability Regulated by the Response Regulator CtrA

    PubMed Central

    Brimacombe, Cedric A.; Ding, Hao; Johnson, Jeanette A.

    2015-01-01

    ABSTRACT Gene transfer agents (GTAs) morphologically resemble small, double-stranded DNA (dsDNA) bacteriophages; however, their only known role is to package and transfer random pieces of the producing cell genome to recipient cells. The best understood GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that homologues of three genes involved in natural transformation in other bacteria, comEC, comF, and comM, are essential for RcGTA-mediated gene acquisition. This paper gives genetic and biochemical evidence that RcGTA-borne DNA entry into cells requires the ComEC and ComF putative DNA transport proteins and genetic evidence that putative cytoplasmic ComM protein of unknown function is required for recipient capability. Furthermore, the master regulator of RcGTA production in <1% of a cell population, CtrA, which is also required for gene acquisition in recipient cells, is expressed in the vast majority of the population. Our results indicate that RcGTA-mediated gene transfer combines key aspects of two bacterial horizontal gene transfer mechanisms, where donor DNA is packaged in transducing phage-like particles and recipient cells take up DNA using natural transformation-related machinery. Both of these differentiated subsets of a culture population, donors and recipients, are dependent on the same response regulator, CtrA. IMPORTANCE Horizontal gene transfer (HGT) is a major driver of bacterial evolution and adaptation to environmental stresses. Traits such as antibiotic resistance or metabolic properties can be transferred between bacteria via HGT; thus, HGT can have a tremendous effect on the fitness of a bacterial population. The three classically described HGT mechanisms are conjugation, transformation, and phage-mediated transduction. More recently, the HGT factor GTA was described, where random pieces of producing cell genome are packaged into phage-like particles that deliver DNA to recipient cells. In this report, we show that transport of

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

    PubMed

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

    2012-03-01

    Nitrogen catabolite repression (NCR) is a regulatory strategy found in microorganisms that restricts the utilization of complex and unfavored nitrogen sources in the presence of favored nitrogen sources. In fungi, this concept has been best studied in yeasts and filamentous ascomycetes, where the GATA transcription factors Gln3p and Gat1p (in yeasts) and Nit2/AreA (in ascomycetes) constitute the main positive regulators of NCR. The reason why functional Nit2 homologs of some phytopathogenic fungi are required for full virulence in their hosts has remained elusive. We have identified the Nit2 homolog in the basidiomycetous phytopathogen Ustilago maydis and show that it is a major, but not the exclusive, positive regulator of nitrogen utilization. By transcriptome analysis of sporidia grown on artificial media devoid of favored nitrogen sources, we show that only a subset of nitrogen-responsive genes are regulated by Nit2, including the Gal4-like transcription factor Ton1 (a target of Nit2). Ustilagic acid biosynthesis is not under the control of Nit2, while nitrogen starvation-induced filamentous growth is largely dependent on functional Nit2. nit2 deletion mutants show the delayed initiation of filamentous growth on maize leaves and exhibit strongly compromised virulence, demonstrating that Nit2 is required to efficiently initiate the pathogenicity program of U. maydis. PMID:22247264

  7. 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

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

    PubMed

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

    2012-03-01

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

  9. The Arabidopsis Endosomal Sorting Complex Required for Transport III Regulates Internal Vesicle Formation of the Prevacuolar Compartment and Is Required for Plant Development1[C][W][OPEN

    PubMed Central

    Cai, Yi; Zhuang, Xiaohong; Gao, Caiji; Wang, Xiangfeng; Jiang, Liwen

    2014-01-01

    We have established an efficient transient expression system with several vacuolar reporters to study the roles of endosomal sorting complex required for transport (ESCRT)-III subunits in regulating the formation of intraluminal vesicles of prevacuolar compartments (PVCs)/multivesicular bodies (MVBs) in plant cells. By measuring the distributions of reporters on/within the membrane of PVC/MVB or tonoplast, we have identified dominant negative mutants of ESCRT-III subunits that affect membrane protein degradation from both secretory and endocytic pathways. In addition, induced expression of these mutants resulted in reduction in luminal vesicles of PVC/MVB, along with increased detection of membrane-attaching vesicles inside the PVC/MVB. Transgenic Arabidopsis (Arabidopsis thaliana) plants with induced expression of ESCRT-III dominant negative mutants also displayed severe cotyledon developmental defects with reduced cell size, loss of the central vacuole, and abnormal chloroplast development in mesophyll cells, pointing out an essential role of the ESCRT-III complex in postembryonic development in plants. Finally, membrane dissociation of ESCRT-III components is important for their biological functions and is regulated by direct interaction among Vacuolar Protein Sorting-Associated Protein20-1 (VPS20.1), Sucrose Nonfermenting7-1, VPS2.1, and the adenosine triphosphatase VPS4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1. PMID:24812106

  10. Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution

    PubMed Central

    Lee, Wook-Bin; Kang, Ji-Seon; Choi, Won Young; Zhang, Quanri; Kim, Chul Han; Choi, Un Yung; Kim-Ha, Jeongsil; Kim, Young-Joon

    2016-01-01

    In response to persistent mycobacteria infection, the host induces a granuloma, which often fails to eradicate bacteria and results in tissue damage. Diverse host receptors are required to control the formation and resolution of granuloma, but little is known concerning their regulatory interactions. Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production. In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution. Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls. PMID:27089465

  11. 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

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

    PubMed Central

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

    2012-01-01

    Short hairpin RNAs (shRNAs) are versatile tools for analyzing loss-of-function phenotypes in vitro and in vivo1. 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. PMID:21131983

  13. Mitogen-Activated Protein Kinase Kinase 3 Is Required for Regulation during Dark-Light Transition.

    PubMed

    Lee, Horim

    2015-07-01

    Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition. PMID:26082029

  14. Rbfox proteins regulate tissue-specific alternative splicing of Mef2D required for muscle differentiation.

    PubMed

    Runfola, Valeria; Sebastian, Soji; Dilworth, F Jeffrey; Gabellini, Davide

    2015-02-15

    Among the Mef2 family of transcription factors, Mef2D is unique in that it undergoes tissue-specific splicing to generate an isoform that is essential for muscle differentiation. However, the mechanisms mediating this muscle-specific processing of Mef2D remain unknown. Using bioinformatics, we identified Rbfox proteins as putative modulators of Mef2D muscle-specific splicing. Accordingly, we found direct and specific Rbfox1 and Rbfox2 binding to Mef2D pre-mRNA in vivo. Gain- and loss-of-function experiments demonstrated that Rbfox1 and Rbfox2 cooperate in promoting Mef2D splicing and subsequent myogenesis. Thus, our findings reveal a new role for Rbfox proteins in regulating myogenesis through activation of essential muscle-specific splicing events.

  15. SUMOylation is developmentally regulated and required for cell pairing during conjugation in Tetrahymena thermophila.

    PubMed

    Nasir, Amjad M; Yang, Qianyi; Chalker, Douglas L; Forney, James D

    2015-02-01

    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.

  16. Regulated Catalysis of Extracellular Nucleotides by Vascular CD39/ENTPD1 Is Required for Liver Regeneration

    PubMed Central

    BELDI, GUIDO; WU, YAN; SUN, XIAOFENG; IMAI, MASATO; ENJYOJI, KEIICHI; CSIZMADIA, EVA; CANDINAS, DANIEL; ERB, LAURIE; ROBSON, SIMON C.

    2010-01-01

    Background & Aims Little is known about how endothelial cells respond to injury, regulate hepatocyte turnover and reconstitute the hepatic vasculature. We aimed to determine the effects of the vascular ectonucleotidase CD39 on sinusoidal endothelial cell responses following partial hepatectomy and to dissect purinergic and growth factor interactions in this model. Methods Parameters of liver injury and regeneration, as well as the kinetics of hepatocellular and sinusoidal endothelial cell proliferation, were assessed following partial hepatectomy in mice that do not express CD39, that do not express ATP/UTP receptor P2Y2, and in controls. The effects of extracellular ATP on vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-6 responses were determined in vivo and in vitro. Phosphorylation of the endothelial VEGF receptor in response to extracellular nucleotides and growth factors was assessed in vitro. Results After partial hepatectomy, expression of the vascular ectonucleotidase CD39 increased on sinusoidal endothelial cells. Targeted disruption of CD39 impaired hepatocellular regeneration, reduced angiogenesis, and increased hepatic injury, resulting in pronounced vascular endothelial apoptosis, and decreased survival. Decreased HGF release by sinusoidal endothelial cells, despite high levels of VEGF, reduced paracrine stimulation of hepatocytes. Failure of VEGF receptor-2/KDR transactivation by extracellular nucleotides on CD39-null endothelial cells was associated with P2Y2 receptor desensitization. Conclusions Regulated phosphohydrolysis of extracellular nucleotides by CD39 coordinates both hepatocyte and endothelial cell proliferation following partial hepatectomy. Lack of CD39 activity is associated with decreased hepatic regeneration and failure of vascular reconstitution. PMID:18804472

  17. 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

  18. CDK2-dependent activation of PARP-1 is required for hormonal gene regulation in breast cancer cells

    PubMed Central

    Wright, Roni H.G.; Castellano, Giancarlo; Bonet, Jaume; Le Dily, Francois; Font-Mateu, Jofre; Ballaré, Cecilia; Nacht, A. Silvina; Soronellas, Daniel; Oliva, Baldo; Beato, Miguel

    2012-01-01

    Eukaryotic gene regulation implies that transcription factors gain access to genomic information via poorly understood processes involving activation and targeting of kinases, histone-modifying enzymes, and chromatin remodelers to chromatin. Here we report that progestin gene regulation in breast cancer cells requires a rapid and transient increase in poly-(ADP)-ribose (PAR), accompanied by a dramatic decrease of cellular NAD that could have broad implications in cell physiology. This rapid increase in nuclear PARylation is mediated by activation of PAR polymerase PARP-1 as a result of phosphorylation by cyclin-dependent kinase CDK2. Hormone-dependent phosphorylation of PARP-1 by CDK2, within the catalytic domain, enhances its enzymatic capabilities. Activated PARP-1 contributes to the displacement of histone H1 and is essential for regulation of the majority of hormone-responsive genes and for the effect of progestins on cell cycle progression. Both global chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) and gene expression analysis show a strong overlap between PARP-1 and CDK2. Thus, progestin gene regulation involves a novel signaling pathway that connects CDK2-dependent activation of PARP-1 with histone H1 displacement. Given the multiplicity of PARP targets, this new pathway could be used for the pharmacological management of breast cancer. PMID:22948662

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

    PubMed

    Chen, Ke-Xian; Li, Zu-Guang

    2009-11-01

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

  20. Mycobacterium tuberculosis Lsr2 Is a Global Transcriptional Regulator Required for Adaptation to Changing Oxygen Levels and Virulence

    PubMed Central

    Bartek, I. L.; Woolhiser, L. K.; Baughn, A. D.; Basaraba, R. J.; Jacobs, W. R.; Lenaerts, A. J.

    2014-01-01

    ABSTRACT To survive a dynamic host environment, Mycobacterium tuberculosis must endure a series of challenges, from reactive oxygen and nitrogen stress to drastic shifts in oxygen availability. The mycobacterial Lsr2 protein has been implicated in reactive oxygen defense via direct protection of DNA. To examine the role of Lsr2 in pathogenesis and physiology of M. tuberculosis, we generated a strain deleted for lsr2. Analysis of the M. tuberculosis Δlsr2 strain demonstrated that Lsr2 is not required for DNA protection, as this strain was equally susceptible as the wild type to DNA-damaging agents. The lsr2 mutant did display severe growth defects under normoxic and hyperoxic conditions, but it was not required for growth under low-oxygen conditions. However, it was also required for adaptation to anaerobiosis. The defect in anaerobic adaptation led to a marked decrease in viability during anaerobiosis, as well as a lag in recovery from it. Gene expression profiling of the Δlsr2 mutant under aerobic and anaerobic conditions in conjunction with published DNA binding-site data indicates that Lsr2 is a global transcriptional regulator controlling adaptation to changing oxygen levels. The Δlsr2 strain was capable of establishing an early infection in the BALB/c mouse model; however, it was severely defective in persisting in the lungs and caused no discernible lung pathology. These findings demonstrate M. tuberculosis Lsr2 is a global transcriptional regulator required for control of genes involved in adaptation to extremes in oxygen availability and is required for persistent infection. PMID:24895305

  1. The Orphan Response Regulator DigR Is Required for Synthesis of Extracellular Matrix Fibrils in Myxococcus xanthus†

    PubMed Central

    Overgaard, Martin; Wegener-Feldbrügge, Sigrun; Søgaard-Andersen, Lotte

    2006-01-01

    In Myxococcus xanthus, two-component systems have crucial roles in regulating motility behavior and development. Here we describe an orphan response regulator, consisting of an N-terminal receiver domain and a C-terminal DNA binding domain, which is required for A and type IV pilus-dependent gliding motility. Genetic evidence suggests that phosphorylation of the conserved, phosphorylatable aspartate residue in the receiver domain is required for DigR activity. Consistent with the defect in type IV pilus-dependent motility, a digR mutant is slightly reduced in type IV pilus biosynthesis, and the composition of the extracellular matrix fibrils is abnormal, with an increased content of polysaccharides and decreased accumulation of the FibA metalloprotease. By using genome-wide transcriptional profiling, 118 genes were identified that are directly or indirectly regulated by DigR. These 118 genes include only 2, agmQ and cheY4, previously implicated in A and type IV pilus-dependent motility, respectively. In silico analyses showed that 36% of the differentially expressed genes are likely to encode exported proteins. Moreover, four genes encoding homologs of extracytoplasmic function (ECF) sigma factors, which typically control aspects of cell envelope homeostasis, are differentially expressed in a digR mutant. We suggest that the DigR response regulator has an important function in cell envelope homeostasis and that the motility defects in a digR mutant are instigated by the abnormal cell envelope and abnormal expression of agmQ and cheY4. PMID:16740945

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

    PubMed Central

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

    2008-01-01

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

  3. 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.

  4. 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

  5. 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.

  6. A neurofibromatosis-1-regulated pathway is required for learning in Drosophila.

    PubMed

    Guo, H F; Tong, J; Hannan, F; Luo, L; Zhong, Y

    2000-02-24

    The tumour-suppressor gene Neurofibromatosis 1 (Nf1) encodes a Ras-specific GTPase activating protein (Ras-GAP). In addition to being involved in tumour formation, NF1 has been reported to cause learning defects in humans and Nf1 knockout mice. However, it remains to be determined whether the observed learning defect is secondary to abnormal development. The Drosophila NF1 protein is highly conserved, showing 60% identity of its 2,803 amino acids with human NF1 (ref. 12). Previous studies have suggested that Drosophila NF1 acts not only as a Ras-GAP but also as a possible regulator of the cAMP pathway that involves the rutabaga (rut)-encoded adenylyl cyclase. Because rut was isolated as a learning and short-term memory mutant, we have pursued the hypothesis that NF1 may affect learning through its control of the Rut-adenylyl cyclase/cAMP pathway. Here we show that NF1 affects learning and short-term memory independently of its developmental effects. We show that G-protein-activated adenylyl cyclase activity consists of NF1-independent and NF1-dependent components, and that the mechanism of the NF1-dependent activation of the Rut-adenylyl cyclase pathway is essential for mediating Drosophila learning and memory. PMID:10706287

  7. 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

  8. 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

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

    PubMed

    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

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

    PubMed

    Sun, Xin; Fu, Kai; Hodgson, Andrea; Wier, Eric M; Wen, Matthew G; Kamenyeva, Olena; Xia, Xue; Koo, Lily Y; Wan, Fengyi

    2016-09-01

    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

  11. Fgf is required to regulate anterior-posterior patterning in the Xenopus lateral plate mesoderm.

    PubMed

    Deimling, Steven J; Drysdale, Thomas A

    2011-01-01

    Given that the lateral plate mesoderm (LPM) gives rise to the cardiovascular system, identifying the cascade of signalling events that subdivides the LPM into distinct regions during development is an important question. Retinoic acid (RA) is known to be necessary for establishing the expression boundaries of important transcription factors that demarcate distinct regions along the anterior posterior axis of the LPM. Here, we demonstrate that fibroblast growth factor (Fgf) signalling is also necessary for regulating the expression domains of the same transcription factors (nkx2.5, foxf1, hand1 and sall3) by restricting the RA responsive LPM domains. When Fgf signalling is inhibited in neurula stage embryos, the more posterior LPM expression domains are lost, while the more anterior domains are extended further posterior. The domain changes are maintained throughout development as Fgf inhibition results in similar domain changes in late stage embryos. We also demonstrate that Fgf signalling is necessary for both the initiation of heart specification, and for maintaining heart specification until overt differentiation occurs. Fgf signalling is also necessary to restrict vascular patterning and create a vascular free domain in the posterior end of the LPM that correlates with the expression of hand1. Finally, we show cross talk between the RA and Fgf signalling pathways in the patterning of the LPM. We suggest that this tissue wide patterning event, active during the neurula stage, is an initial step in regional specification of the LPM, and this process is an essential early event in LPM patterning.

  12. 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

  13. 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. PMID:25037837

  14. Heterochromatic Genome Stability Requires Regulators of Histone H3 K9 Methylation

    PubMed Central

    Peng, Jamy C.; Karpen, Gary H.

    2009-01-01

    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. PMID:19325889

  15. 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.

  16. JunB is required for endothelial cell morphogenesis by regulating core-binding factor β

    PubMed Central

    Licht, Alexander H.; Pein, Oliver T.; Florin, Lore; Hartenstein, Bettina; Reuter, Hendrik; Arnold, Bernd; Lichter, Peter; Angel, Peter; Schorpp-Kistner, Marina

    2006-01-01

    The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis. PMID:17158955

  17. 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

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

    PubMed

    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

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

    PubMed

    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.

  20. SRC-3 is required for CAR-regulated hepatocyte proliferation and drug metabolism

    PubMed Central

    Chen, Tenghui; Chen, Qiang; Xu, Yixiang; Zhou, Qiling; Zhu, Jingwei; Zhang, Hao; Wu, Qiao; Xu, Jianming; Yu, Chundong

    2011-01-01

    Background & Aims Nuclear receptors such as pregnane X receptor and constitutive androstane receptor (CAR) are important regulators of drug-metabolizing systems such as P450s enzymes and modulate xenobiotic metabolism as well as hepatocellular proliferation. Binding of CAR to NR response elements alone is not sufficient to activate gene expression. Here we investigate the role of steroid receptor coactivator (SRC) family members in CAR-mediated hepatocyte proliferation and drug metabolism. Methods The role of SRCs in CAR activation was assessed in cell-based transfection assays and protein-protein interaction assays. The in vivo role of SRCs in CAR-mediated hepatocyte proliferation and drug metabolism was examined by using mice deficient in SRCs. Results SRC-3 displayed the highest coactivating activity to CAR compared with SRC-1 and SRC-2 in a cell-based reporter assay. Knockout of SRC-3 in mice attenuated hepatic hyperplasia induced by a CAR agonist 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), which was associated with a reduced expression of c-Myc and Foxm-1. In contrast, knockout of SRC-1 or SRC-2 in mice did not affect TCPOBOP-induced hepatic hyperplasia. SRC-3-deficient mice were hypersensitive to zoxazolamine-induced paralysis, but were resistant to acetaminophen hepatotoxicity induced by TCPOBOP, whereas mutant mice deficient in SRC-1 or SRC-2 exhibited severe acetaminophen hepatotoxicity similar to wild-type controls. Accordingly, deficiency in SRC-3, but not SRC-1 or SRC-2, resulted in a reduced CAR-mediated expression of drug metabolism-related genes in the liver. Conclusions Our study demonstrates that SRC-3 is the predominant transcriptional coactivator among the three SRC family members for CAR activation to promote hepatocyte proliferation and drug metabolism. PMID:21827731

  1. 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

  2. The CovS/CovR Acid Response Regulator Is Required for Intracellular Survival of Group B Streptococcus in Macrophages

    PubMed Central

    Cumley, Nicola J.; Smith, Leanne M.; Anthony, Mark

    2012-01-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  3. Nucleotide sequences required for the regulation of a rat alpha 2u-globulin gene by glucocorticoids.

    PubMed Central

    Addison, W R; Kurtz, D T

    1986-01-01

    alpha 2u-Globulin is a rat protein of as yet unknown function whose synthesis can be induced by glucocorticoids and several other hormones. Induction by glucocorticoids is a secondary response to the hormone: protein synthesis is required before the hormone can exert its stimulatory effect on alpha 2u-globulin transcription. We have used the linker-scanning mutagenesis procedure, followed by transfer of the mutant genes into mouse L-cells for analysis of their phenotype, to determine sequences within a cloned alpha 2u-globulin promoter that are required for its regulation by glucocorticoids. Mutations between positions -115 and -160 abolish or greatly reduce the inducibility of alpha 2u-globulin by the hormone. Mutations just upstream from this region, between positions -177 and -220, have an opposite effect; they increase induction two- to fourfold. Images PMID:2431290

  4. Sec22 regulates endoplasmic reticulum morphology but not autophagy and is required for eye development in Drosophila.

    PubMed

    Zhao, Xiaocui; Yang, Huan; Liu, Wei; Duan, Xiuying; Shang, Weina; Xia, Dajing; Tong, Chao

    2015-03-20

    The endoplasmic reticulum (ER) is a highly dynamic organelle that plays a critical role in many cellular processes. Abnormal ER morphology is associated with some human diseases, although little is known regarding how ER morphology is regulated. Using a forward genetic screen to identify genes that regulated ER morphology in Drosophila, we identified a mutant of Sec22, the orthologs of which in yeast, plants, and humans are required for ER to Golgi trafficking. However, the physiological function of Sec22 has not been previously investigated in animal development. A loss of Sec22 resulted in ER proliferation and expansion, enlargement of late endosomes, and abnormal Golgi morphology in mutant larvae fat body cells. However, starvation-induced autophagy was not affected by a loss of Sec22. Mosaic analysis of the eye revealed that Sec22 was required for photoreceptor morphogenesis. In Sec22 mutant photoreceptor cells, the ER was highly expanded and gradually lost normal morphology with aging. The rhabdomeres in mutants were small and sometimes fused with each other. The morphology of Sec22 mutant eyes resembled the eye morphology of flies with overexpressed eyc (eyes closed). eyc encodes for a Drosophila p47 protein that is required for membrane fusion. A loss of Syntaxin5 (Syx5), encoding for a t-SNARE on Golgi, also phenocopied the Sec22 mutant. Sec22 formed complexes with Syx5 and Eyc. Thus, we propose that appropriate trafficking between the ER and Golgi is required for maintaining ER morphology and for Drosophila eye morphogenesis. PMID:25670863

  5. Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation.

    PubMed

    Garcia, J A; Harrich, D; Soultanakis, E; Wu, F; Mitsuyasu, R; Gaynor, R B

    1989-03-01

    The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted

  6. 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

    2015-10-01

    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

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

    PubMed

    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

    2011-04-15

    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

  8. PPARα Is Required for PPARδ Action in Regulation of Body Weight and Hepatic Steatosis in Mice

    PubMed Central

    Garbacz, Wojciech G.; Huang, Jeffrey T. J.; Higgins, Larry G.; Wahli, Walter; Palmer, Colin N. A.

    2015-01-01

    Peroxisome proliferator activated receptors alpha (PPARα) and delta (PPARδ) belong to the nuclear receptor superfamily. PPARα is a target of well established lipid-lowering drugs. PPARδ (also known as PPARβ/δ) has been investigated as a promising antidiabetic drug target; however, the evidence in the literature on PPARδ effect on hepatic lipid metabolism is inconsistent. Mice conditionally expressing human PPARδ demonstrated pronounced weight loss and promoted hepatic steatosis when treated with GW501516 (PPARδ-agonist) when compared to wild type mice. This effect was completely absent in mice with either a dominant negative form of PPARδ or deletion of the DNA binding domain of PPARδ. This confirmed the absolute requirement for PPARδ in the physiological actions of GW501516 and confirmed the potential utility against the human form of this receptor. Surprisingly the genetic deletion of PPARα also abrogated the effect of GW501516 in terms of both weight loss and hepatic lipid accumulation. Also the levels of the PPARα endogenous agonist 16:0/18:1-GPC were shown to be modulated by PPARδ in wild type mice. Our results show that both PPARδ and PPARα receptors are essential for GW501516-driven adipose tissue reduction and subsequently hepatic steatosis, with PPARα working downstream of PPARδ. PMID:26604919

  9. 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

  10. 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

  11. 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.

  12. Cell cycle-regulated degradation of Xenopus cyclin B2 requires binding to p34cdc2.

    PubMed Central

    van der Velden, H M; Lohka, M J

    1994-01-01

    The protein kinase activity of the cell cycle regulator p34cdc2 is inactivated when the mitotic cyclin to which it is bound is degraded. The amino (N)-terminus of mitotic cyclins includes a conserved "destruction box" sequence that is essential for degradation. Although the N-terminus of sea urchin cyclin B confer cell cycle-regulated degradation to a fusion protein, a truncated protein containing only the N-terminus of Xenopus cyclin B2, including the destruction box, is stable under conditions where full length molecules are degraded. In an attempt to identify regions of cyclin B2, other than the destruction box, involved in degradation, the stability of proteins encoded by C-terminal deletion mutants of cyclin B2 was examined in Xenopus egg extracts. Truncated cyclin with only the first 90 amino acids was stable, but other C-terminal deletions lacking between 14 and 187 amino acids were unstable and were degraded by a mechanism that was neither cell cycle regulated nor dependent upon the destruction box. None of the C-terminal deletion mutants bound p34cdc2. To investigate whether the binding of p34cdc2 is required for cell cycle-regulated degradation, the behavior of proteins encoded by a series of full length Xenopus cyclin B2 cDNA with point mutations in conserved amino acids in the p34cdc2-binding domain was examined. All of the point mutants failed to form stable complexes with p34cdc, and their degradation was markedly reduced compared to wild-type cyclin. Similar results were obtained when the mutant cyclins were synthesized in reticulocyte lysates and when cyclin mRNA was translated directly in a Xenopus egg extract. These results indicate that mutations that interfere with p34cdc2 binding also interfere with cyclin destruction, suggesting that p34cdc2 binding is required for the cell cycle-regulated destruction of Xenopus cyclin B2. Images PMID:7812041

  13. 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

  14. 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

  15. 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.

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

    DOE PAGESBeta

    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 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

  17. Spatial regulation of Aurora A activity during mitotic spindle assembly requires RHAMM to correctly localize TPX2

    PubMed Central

    Chen, Helen; Mohan, Pooja; Jiang, Jihong; Nemirovsky, Oksana; He, Daniel; Fleisch, Markus C; Niederacher, Dieter; Pilarski, Linda M; Lim, C James; Maxwell, Christopher A

    2014-01-01

    Construction of a mitotic spindle requires biochemical pathways to assemble spindle microtubules and structural proteins to organize these microtubules into a bipolar array. Through a complex with dynein, the receptor for hyaluronan-mediated motility (RHAMM) cross-links mitotic microtubules to provide structural support, maintain spindle integrity, and correctly orient the mitotic spindle. Here, we locate RHAMM to sites of microtubule assembly at centrosomes and non-centrosome sites near kinetochores and demonstrate that RHAMM is required for the activation of Aurora kinase A. Silencing of RHAMM delays the kinetics of spindle assembly, mislocalizes targeting protein for XKlp2 (TPX2), and attenuates the localized activation of Aurora kinase A with a consequent reduction in mitotic spindle length. The RHAMM–TPX2 complex requires a C-terminal basic leucine zipper in RHAMM and a domain that includes the nuclear localization signal in TPX2. Together, our findings identify RHAMM as a critical regulator for Aurora kinase A signaling and suggest that RHAMM ensures bipolar spindle assembly and mitotic progression through the integration of biochemical and structural pathways. PMID:24875404

  18. 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.

  19. 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

  20. 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...

  1. 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

    ....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...

  2. Cofilin regulator 14-3-3zeta is an evolutionarily conserved protein required for phagocytosis and microbial resistance.

    PubMed

    Ulvila, Johanna; Vanha-aho, Leena-Maija; Kleino, Anni; Vähä-Mäkilä, Mari; Vuoksio, Milka; Eskelinen, Sinikka; Hultmark, Dan; Kocks, Christine; Hallman, Mikko; Parikka, Mataleena; Rämet, Mika

    2011-05-01

    Phagocytosis is an ancient cellular process that plays an important role in host defense. In Drosophila melanogaster phagocytic, macrophage-like hemocytes recognize and ingest microbes. We performed an RNAi-based in vitro screen in the Drosophila hemocyte cell line S2 and identified Abi, cpa, cofilin regulator 14-3-3ζ, tlk, CG2765, and CG15609 as mediators of bacterial phagocytosis. Of these identified genes, 14-3-3ζ had an evolutionarily conserved role in phagocytosis: bacterial phagocytosis was compromised when 14-3-3ζ was targeted with RNAi in primary Drosophila hemocytes and when the orthologous genes Ywhab and Ywhaz were silenced in zebrafish and mouse RAW 264.7 cells, respectively. In Drosophila and zebrafish infection models, 14-3-3ζ was required for resistance against Staphylococcus aureus. We conclude that 14-3-3ζ is essential for phagocytosis and microbial resistance in insects and vertebrates. PMID:21208897

  3. Myxococcus xanthus mokA Encodes a Histidine Kinase-Response Regulator Hybrid Sensor Required for Development and Osmotic Tolerance

    PubMed Central

    Kimura, Yoshio; Nakano, Hiromi; Terasaka, Hideaki; Takegawa, Kaoru

    2001-01-01

    A gene, mokA, encoding a protein with similarities to histidine kinase-response regulator hybrid sensor, was cloned from a Myxococcus xanthus genomic library. The predicted mokA gene product was found to contain three domains: an amino-terminal input domain, a central transmitter domain, and a carboxy-terminal receiver domain. mokA mutants placed under starvation conditions exhibited reduced sporulation. Mutation of mokA also caused marked growth retardation at high osmolarity. These results indicated that M. xanthus MokA is likely a transmembrane sensor that is required for development and osmotic tolerance. The putative function of MokA is similar to that of the hybrid histidine kinase, DokA, of the eukaryotic slime mold Dictyostelium discoideum. PMID:11157925

  4. 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.

  5. YfbA, a Yersinia pestis Regulator Required for Colonization and Biofilm Formation in the Gut of Cat Fleas

    PubMed Central

    Tam, Christina; Demke, Owen; Hermanas, Timothy; Mitchell, Anthony; Hendrickx, Antoni P. A.

    2014-01-01

    For transmission to new hosts, Yersinia pestis, the causative agent of plague, replicates as biofilm in the foregut of fleas that feed on plague-infected animals or humans. Y. pestis biofilm formation has been studied in the rat flea; however, little is known about the cat flea, a species that may bridge zoonotic and anthroponotic plague cycles. Here, we show that Y. pestis infects and replicates as a biofilm in the foregut of cat fleas in a manner requiring hmsFR, two determinants for extracellular biofilm matrix. Examining a library of transposon insertion mutants, we identified the LysR-type transcriptional regulator YfbA, which is essential for Y. pestis colonization and biofilm formation in cat fleas. PMID:24391055

  6. 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.

  7. 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

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

    PubMed Central

    Bilder, Patrick; Sun, Meihao; Lim, Jihyeon; Bielefeldt-Ohmann, Helle; Basaraba, Randall; So, Melvin; Zhu, Guofeng; Tufariello, JoAnn M.; Izzo, Angelo A.; Orme, Ian M.; Almo, Steve C.; Leyh, Thomas S.; Chan, John

    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 tuberculosis universal 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-Å-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. PMID:19478878

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

    PubMed

    Brown, David A; Yule, David I

    2010-02-01

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

  10. 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. PMID:26896396

  11. 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

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

    PubMed

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

    2014-10-13

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

  13. The Drosophila MAST kinase Drop out is required to initiate membrane compartmentalisation during cellularisation and regulates dynein-based transport.

    PubMed

    Hain, Daniel; Langlands, Alistair; Sonnenberg, Hannah C; Bailey, Charlotte; Bullock, Simon L; Müller, H-Arno J

    2014-05-01

    Cellularisation of the Drosophila syncytial blastoderm embryo into the polarised blastoderm epithelium provides an excellent model with which to determine how cortical plasma membrane asymmetry is generated during development. Many components of the molecular machinery driving cellularisation have been identified, but cell signalling events acting at the onset of membrane asymmetry are poorly understood. Here we show that mutations in drop out (dop) disturb the segregation of membrane cortical compartments and the clustering of E-cadherin into basal adherens junctions in early cellularisation. dop is required for normal furrow formation and controls the tight localisation of furrow canal proteins and the formation of F-actin foci at the incipient furrows. We show that dop encodes the single Drosophila homologue of microtubule-associated Ser/Thr (MAST) kinases. dop interacts genetically with components of the dynein/dynactin complex and promotes dynein-dependent transport in the embryo. Loss of dop function reduces phosphorylation of Dynein intermediate chain, suggesting that dop is involved in regulating cytoplasmic dynein activity through direct or indirect mechanisms. These data suggest that Dop impinges upon the initiation of furrow formation through developmental regulation of cytoplasmic dynein.

  14. The Drosophila MAST kinase Drop out is required to initiate membrane compartmentalisation during cellularisation and regulates dynein-based transport

    PubMed Central

    Hain, Daniel; Langlands, Alistair; Sonnenberg, Hannah C.; Bailey, Charlotte; Bullock, Simon L.; Müller, H.-Arno J.

    2014-01-01

    Cellularisation of the Drosophila syncytial blastoderm embryo into the polarised blastoderm epithelium provides an excellent model with which to determine how cortical plasma membrane asymmetry is generated during development. Many components of the molecular machinery driving cellularisation have been identified, but cell signalling events acting at the onset of membrane asymmetry are poorly understood. Here we show that mutations in drop out (dop) disturb the segregation of membrane cortical compartments and the clustering of E-cadherin into basal adherens junctions in early cellularisation. dop is required for normal furrow formation and controls the tight localisation of furrow canal proteins and the formation of F-actin foci at the incipient furrows. We show that dop encodes the single Drosophila homologue of microtubule-associated Ser/Thr (MAST) kinases. dop interacts genetically with components of the dynein/dynactin complex and promotes dynein-dependent transport in the embryo. Loss of dop function reduces phosphorylation of Dynein intermediate chain, suggesting that dop is involved in regulating cytoplasmic dynein activity through direct or indirect mechanisms. These data suggest that Dop impinges upon the initiation of furrow formation through developmental regulation of cytoplasmic dynein. PMID:24803657

  15. Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly.

    PubMed

    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

  16. 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.

  17. Identification of TSG101 functional domains and p21 loci required for TSG101-mediated p21 gene regulation.

    PubMed

    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 p21 (CIP1/WAF1) tumor 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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. PMID:23880637

  3. Development of guidance on applications of regulatory requirements for regulating large, contaminated equipment and large decommissioning and decontamination (D and D) components

    SciTech Connect

    Pope, R.B.; Easton, E.P.; Cook, J.R.; Boyle, R.W.

    1997-10-01

    In 1985, the International Atomic Energy Agency issued revised regulations for the safe transport of radioactive material. Significant were major changes to requirements for Low Specific Activity material and Surface Contaminated Objects. As these requirements were adopted into regulations in the US, it was recognized that guidance on how to apply these requirements to large, contaminated/activated pieces of equipment and decommissioning and decontamination objects would be needed both by the regulators and those regulated to clarify technical uncertainties and ensure implementation. Thus, the US Department of Transportation and the US Nuclear Regulatory Commission, with assistance of staff from Oak Ridge National Laboratory, are preparing regulatory guidance which will present examples of acceptable methods for demonstrating compliance with the revised rules for large items. Concepts being investigated for inclusion in the pending guidance are discussed in this paper. Under current plans, the guidance will be issued for public comment before final issuance in 1997.

  4. VdNUC-2, the Key Regulator of Phosphate Responsive Signaling Pathway, Is Required for Verticillium dahliae Infection.

    PubMed

    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.

  5. 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

  6. VdNUC-2, the Key Regulator of Phosphate Responsive Signaling Pathway, Is Required for Verticillium dahliae Infection.

    PubMed

    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

  7. 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.

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

    PubMed

    Hand, Laurence H; Moreland, Harriet J

    2014-03-01

    A surface water mineralization study (according to the Organisation for Economic Co-operation and Development [OECD] guideline OECD 309) is a new requirement in European Union agrochemical regulations; therefore, industry has little experience with this test. The guideline allows for a number of options within the test design, notably the options to conduct the study under diffuse light and to include an inoculum of suspended sediment. The present study was designed to investigate the potential impact of these options on the degradation rate of a representative compound. The fungicide, isopyrazam, was chosen as it was previously shown to be susceptible to metabolism by phototrophic organisms under a fluorescent light-dark cycle. The impact of diffuse light was investigated at light intensities representative of those at depth in large, open water bodies (<7% of the incident intensity), and it was demonstrated that metabolism of isopyrazam by phototrophic microorganisms was rapid (median degradation time for 50% of the test compound [DT50] < 50 d), whereas degradation in continuous darkness was negligible. Furthermore, investigation at 2 different light intensities resulted in similar degradation rates, indicating that this transformation mechanism was not proportional to light intensity, provided that there was sufficient light for photosynthesis to occur. Inclusion of suspended sediment did not have a significant impact on the degradation rate of isopyrazam, except at extremely high sediment concentrations, which were not considered representative of conditions in large, open water bodies.

  9. BDNF-mediated regulation of ethanol consumption requires the activation of the MAP kinase pathway and protein synthesis.

    PubMed

    Jeanblanc, Jerome; Logrip, Marian L; Janak, Patricia H; Ron, Dorit

    2013-02-01

    We previously found that the brain-derived neurotrophic factor (BDNF) in the dorsolateral striatum (DLS) is part of a homeostatic pathway that gates ethanol self-administration [Jeanblanc et al. (2009). J Neurosci, 29, 13494-13502)]. Specifically, we showed that moderate levels (10%) of ethanol consumption increase BDNF expression within the DLS, and that direct infusion of BDNF into the DLS decreases operant self-administration of a 10% ethanol solution. BDNF binding to its receptor, TrkB, activates the mitogen-activated protein kinase (MAPK), phospholipase C-γ (PLC-γ) and phosphatidylinositol 3-kinase (PI3K) pathways. Thus, here, we set out to identify which of these intracellular pathway(s) plays a role in the regulation of ethanol consumption by BDNF. We found that inhibition of the MAPK, but not PLC-γ or PI3K, activity blocks the BDNF-mediated reduction of ethanol consumption. As activation of the MAPK pathway leads to the initiation of transcription and/or translation events, we tested whether the BDNF-mediated reduction of ethanol self-administration requires de novo protein synthesis. We found that the inhibitory effect of BDNF on ethanol intake is blocked by the protein synthesis inhibitor cycloheximide. Together, our results show that BDNF attenuates ethanol drinking via activation of the MAPK pathway in a protein synthesis-dependent manner within the DLS.

  10. BDNF-mediated regulation of ethanol consumption requires the activation of the MAP kinase pathway and protein synthesis

    PubMed Central

    Jeanblanc, Jerome; Logrip, Marian L.; Janak, Patricia H.; Ron, Dorit

    2013-01-01

    We previously found that the brain-derived neurotrophic factor (BDNF) in the dorsolateral striatum (DLS) is part of a homeostatic pathway that gates ethanol self-administration [Jeanblanc et al. (2009). J Neurosci, 29, 13494–13502)]. Specifically, we showed that moderate levels (10%) of ethanol consumption increase BDNF expression within the DLS, and that direct infusion of BDNF into the DLS decreases operant self-administration of a 10% ethanol solution. BDNF binding to its receptor, TrkB, activates the mitogen-activated protein kinase (MAPK), phospholipase C-γ (PLC-γ) and phosphatidylinositol 3-kinase (PI3K) pathways. Thus, here, we set out to identify which of these intracellular pathway(s) plays a role in the regulation of ethanol consumption by BDNF. We found that inhibition of the MAPK, but not PLC-γ or PI3K, activity blocks the BDNF-mediated reduction of ethanol consumption. As activation of the MAPK pathway leads to the initiation of transcription and/or translation events, we tested whether the BDNF-mediated reduction of ethanol self-administration requires de novo protein synthesis. We found that the inhibitory effect of BDNF on ethanol intake is blocked by the protein synthesis inhibitor cycloheximide. Together, our results show that BDNF attenuates ethanol drinking via activation of the MAPK pathway in a protein synthesis-dependent manner within the DLS. PMID:23189980

  11. 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

  12. 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. PMID:25944094

  13. 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

  14. Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis

    PubMed Central

    Mancias, Joseph D; Pontano Vaites, Laura; Nissim, Sahar; Biancur, Douglas E; Kim, Andrew J; Wang, Xiaoxu; Liu, Yu; Goessling, Wolfram; Kimmelman, Alec C; Harper, J Wade

    2015-01-01

    NCOA4 is a selective cargo receptor for the autophagic turnover of ferritin, a process critical for regulation of intracellular iron bioavailability. However, how ferritinophagy flux is controlled and the roles of NCOA4 in iron-dependent processes are poorly understood. Through analysis of the NCOA4-FTH1 interaction, we demonstrate that direct association via a key surface arginine in FTH1 and a C-terminal element in NCOA4 is required for delivery of ferritin to the lysosome via autophagosomes. Moreover, NCOA4 abundance is under dual control via autophagy and the ubiquitin proteasome system. Ubiquitin-dependent NCOA4 turnover is promoted by excess iron and involves an iron-dependent interaction between NCOA4 and the HERC2 ubiquitin ligase. In zebrafish and cultured cells, NCOA4 plays an essential role in erythroid differentiation. This work reveals the molecular nature of the NCOA4-ferritin complex and explains how intracellular iron levels modulate NCOA4-mediated ferritinophagy in cells and in an iron-dependent physiological setting. DOI: http://dx.doi.org/10.7554/eLife.10308.001 PMID:26436293

  15. 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.

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

    PubMed

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

    2012-08-01

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

  17. 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.

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

    PubMed

    Hand, Laurence H; Moreland, Harriet J

    2014-03-01

    A surface water mineralization study (according to the Organisation for Economic Co-operation and Development [OECD] guideline OECD 309) is a new requirement in European Union agrochemical regulations; therefore, industry has little experience with this test. The guideline allows for a number of options within the test design, notably the options to conduct the study under diffuse light and to include an inoculum of suspended sediment. The present study was designed to investigate the potential impact of these options on the degradation rate of a representative compound. The fungicide, isopyrazam, was chosen as it was previously shown to be susceptible to metabolism by phototrophic organisms under a fluorescent light-dark cycle. The impact of diffuse light was investigated at light intensities representative of those at depth in large, open water bodies (<7% of the incident intensity), and it was demonstrated that metabolism of isopyrazam by phototrophic microorganisms was rapid (median degradation time for 50% of the test compound [DT50] < 50 d), whereas degradation in continuous darkness was negligible. Furthermore, investigation at 2 different light intensities resulted in similar degradation rates, indicating that this transformation mechanism was not proportional to light intensity, provided that there was sufficient light for photosynthesis to occur. Inclusion of suspended sediment did not have a significant impact on the degradation rate of isopyrazam, except at extremely high sediment concentrations, which were not considered representative of conditions in large, open water bodies. PMID:24318627

  19. 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.

  20. 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.

  1. Regulation of Myogenesis by Fibroblast Growth Factors Requires Beta-Gamma Subunits of Pertussis Toxin-Sensitive G Proteins

    PubMed Central

    Fedorov, Yuri V.; Jones, Nathan C.; Olwin, Bradley B.

    1998-01-01

    Terminal differentiation of skeletal muscle cells in culture is inhibited by a number of different growth factors whose subsequent intracellular signaling events are poorly understood. In this study, we have investigated the role of heterotrimeric G proteins in mediating fibroblast growth factor (FGF)-dependent signals that regulate myogenic differentiation. Pertussis toxin, which ADP-ribosylates and inactivates susceptible G proteins, promotes terminal differentiation in the presence of FGF-2, suggesting that Gα or Gβγ subunits or both are involved in transducing the FGF-dependent signal(s) that inhibits myogenesis. We found that Gβγ subunits are likely to be involved since the expression of the C terminus of β-adrenergic receptor kinase 1, a Gβγ subunit-sequestering agent, promotes differentiation in the presence of FGF-2, and expression of the free Gβγ dimer can replace FGF-2, rescuing cells from pertussis toxin-induced differentiation. Addition of pertussis toxin also blocked FGF-2-mediated activation of mitogen-activated protein kinases (MAPKs). Ectopic expression of dominant active mutants in the Ras/MAPK pathway rescued cells from pertussis toxin-induced terminal differentiation, suggesting that the Gβγ subunits act upstream of the Ras/MAPK pathway. It is unlikely that the pertussis toxin-sensitive pathway is activated by other, as yet unidentified FGF receptors since PDGF (platelet-derived growth factor)-stimulated MM14 cells expressing a chimeric receptor containing the FGF receptor-1 intracellular domain and the PDGF receptor extracellular domain were sensitive to pertussis toxin. Our data suggest that FGF-mediated signals involved in repression of myogenic differentiation are transduced by a pertussis toxin-sensitive G-protein-coupled mechanism. This signaling pathway requires the action of Gβγ subunits and activation of MAPKs to repress skeletal muscle differentiation. PMID:9742095

  2. 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. PMID:27151944

  3. 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,...

  4. 7 CFR 319.5 - Requirements for submitting requests to change the regulations in 7 CFR part 319.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... regulations in 7 CFR part 319. 319.5 Section 319.5 Agriculture Regulations of the Department of Agriculture... 7 CFR part 319. (a) Definitions. Commodity. A plant, plant product, or other agricultural product... year); (iv) Economic losses associated with pests of concern in the country; (v) Pest biology...

  5. 7 CFR 319.5 - Requirements for submitting requests to change the regulations in 7 CFR part 319.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... regulations in 7 CFR part 319. 319.5 Section 319.5 Agriculture Regulations of the Department of Agriculture... 7 CFR part 319. (a) Definitions. Commodity. A plant, plant product, or other agricultural product... year); (iv) Economic losses associated with pests of concern in the country; (v) Pest biology...

  6. 7 CFR 319.5 - Requirements for submitting requests to change the regulations in 7 CFR part 319.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... regulations in 7 CFR part 319. 319.5 Section 319.5 Agriculture Regulations of the Department of Agriculture... 7 CFR part 319. (a) Definitions. Commodity. A plant, plant product, or other agricultural product... year); (iv) Economic losses associated with pests of concern in the country; (v) Pest biology...

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

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... regulations in 7 CFR part 319. 319.5 Section 319.5 Agriculture Regulations of the Department of Agriculture... 7 CFR part 319. (a) Definitions. Commodity. A plant, plant product, or other agricultural product... year); (iv) Economic losses associated with pests of concern in the country; (v) Pest biology...

  8. 7 CFR 319.5 - Requirements for submitting requests to change the regulations in 7 CFR part 319.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... regulations in 7 CFR part 319. 319.5 Section 319.5 Agriculture Regulations of the Department of Agriculture... 7 CFR part 319. (a) Definitions. Commodity. A plant, plant product, or other agricultural product... year); (iv) Economic losses associated with pests of concern in the country; (v) Pest biology...

  9. Fusarium verticillioides SGE1 is required for full virulence and regulates expression of protein effector and secondary metabolite biosynthetic genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Fusarium verticillioides SGE1 is required for full virulence and regulates expression of protein effector and secondary metabolite biosynthetic genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The transition from one lifestyle to another in some fungi is initiated by a single orthologous gene, SGE1 in Fusarium oxysporum, that regulates markedly different gene sets in different fungi. Despite these differences, many of the regulated genes affect pathogenicity as they encode effector protei...

  11. Fibroblast Growth Factor (FGF) Signaling during Gastrulation Negatively Modulates the Abundance of MicroRNAs That Regulate Proteins Required for Cell Migration and Embryo Patterning*

    PubMed Central

    Bobbs, Alexander S.; Saarela, Aleksi V.; Yatskievych, Tatiana A.; Antin, Parker B.

    2012-01-01

    FGF signaling plays a pivotal role in regulating cell movements and lineage induction during gastrulation. Here we identify 44 microRNAs that are expressed in the primitive streak region of gastrula stage chicken embryos. We show that the primary effect of FGF signaling on microRNA abundance is to negatively regulate the levels of miR-let-7b, -9, -19b, -107, -130b, and -218. LIN28B inhibits microRNA processing and is positively regulated by FGF signaling. Gain- and loss-of-function experiments show that LIN28B negatively regulates the expression of miR-19b, -130b, and let-7b, whereas negative modulation of miR-9, -107, and -218 appears to be independent of LIN28B function. Predicted mRNA targets of the FGF-regulated microRNAs are over-represented in serine/threonine and tyrosine kinase receptors, including ACVR1, ACVR2B, PDGFRA, TGFBR1, and TGFBR3. Luciferase assays show that these and other candidates are targeted by FGF-regulated microRNAs. PDGFRA, a receptor whose activity is required for cell migration through the primitive streak, is a target of miR-130b and -218 in vivo. These results identify a novel mechanism by which FGF signaling regulates gene expression by negatively modulating microRNA abundance through both LIN28B-dependent and LIN28B-independent pathways. PMID:22995917

  12. Group A streptococcal growth phase-associated virulence factor regulation by a novel operon (Fas) with homologies to two-component-type regulators requires a small RNA molecule.

    PubMed

    Kreikemeyer, B; Boyle, M D; Buttaro, B A; Heinemann, M; Podbielski, A

    2001-01-01

    A novel growth phase-associated two-component-type regulator, Fas (fibronectin/fibrinogen binding/haemolytic activity/streptokinase regulator), of Streptococcus pyogenes was identified in the M1 genome sequence, based on homologies to the histidine protein kinase (HPK) and response regulator (RR) part of the Staphylococcus aureus Agr and Streptococcus pneumoniae Com quorum-sensing systems. The fas operon, present in all 12 tested M serotypes, was transcribed as polycystronic message (fasBCA) and contained genes encoding two potential HPKs (FasB and FasC) and one RR (FasA). Downstream of fasBCA, we identified a small 300 nucleotide monocistronic transcript, designated fasX, that did not appear to encode true peptide sequences. Measurements of luciferase promoter fusions revealed a growth phase-associated transcription of fasBCA and fasX, with peak activities during the late exponential phase. Insertional mutagenesis disrupting fasBCA and fasA led to a phenotype similar to agr-null mutations in S. aureus, with prolonged expression of extracellular matrix protein-binding adhesins and reduced expression of secreted virulence factors such as streptokinase and streptolysin S. In addition, fasX transcription was dependent on the RR FasA; however, deletion mutagenesis of fasX resulted in a similar phenotype to that of the fasBCA or fasA mutants. Complementation of the fasX deletion mutant, with the fasX gene expressed in trans from a plasmid, restored the wild-type fasBCA regulation pattern. This strongly suggested that fasX, a putative non-translated RNA, is the main effector molecule of the fas regulon. However, using spent culture supernatants from wild-type and fas mutant strains, we were not able to show an influence on the logarithmic growth phase expression of fas and dependent genes. Thus, despite structural and functional similarities between fas and agr, to date the fas operon appears not to be involved in group A streptococcal (GAS) quorum-sensing regulation

  13. 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

  14. 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

  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. The alternative splicing regulator Tra2b is required for somitogenesis and regulates splicing of an inhibitory Wnt11b isoform

    PubMed Central

    Dichmann, Darwin S; Walentek, Peter; Harland, Richard M

    2014-01-01

    SUMMARY Alternative splicing is pervasive in vertebrates, yet little is known about most isoforms or their regulation. transformer-2b (tra2b) encodes a splicing regulator whose endogenous function is poorly understood. Tra2b knockdown in Xenopus results in embryos with multiple defects, including defective somitogenesis. Using RNA-seq, we identify 142 splice changes, mostly intron retention and exon skipping, of which 89% are not in current annotations. A previously not described isoform of wnt11b retains the last intron, resulting in a truncated ligand (Wnt11b-short). We show that this isoform acts as a dominant-negative in cardiac gene induction and pronephric tubule formation. To determine the contribution of Wnt11b-short to the tra2b phenotype, we induce retention of intron4 in wnt11b, which recapitulates the failure to form somites but not other tra2b morphant defects. This alternative splicing of a Wnt ligand adds intricacy to a complex signaling pathway and highlights intron retention as a regulatory mechanism. PMID:25620705

  17. The regulation of corneal hydration by a salt pump requiring the presence of sodium and bicarbonate ions

    PubMed Central

    Hodson, Stuart

    1974-01-01

    endothelium; the passive salt flux would travel, in the opposite direction, into the stroma across the endothelium. 13. The kinetics of the passive water efflux, as a swollen cornea reverts to physiological hydration (the temperature reversal phenomenon) are predicted extremely well if the `active' salt flux is chosen at 3·3 × 10-7 m-mole. cm-2 sec-1. 14. The value of the active salt flux which cannot be measured directly is extrapolated to be somewhat greater than 2·8 × 10-7 m-moles. cm-2 sec-1; in good agreement with that required by the model to explain the temperature reversal phenomenon. 15. The model is further used to calculate the salt concentration difference across the endothelium (which drives salt passively into the stroma) at various stromal hydrations. 16. When an appropriate salt concentration is applied across the endothelium of de-epithelialized cornea, it generates a potential of the same polarity and similar magnitude to that found across the endothelium of equilibrated whole cornea. The endothelium acts like a cation exchange membrane. 17. Additionally the calculated salt concentration difference across the endothelium correlates well with the measured transendothelial potentials in whole cornea as the corneal hydration varies. 18. It is concluded that the model of an endothelial neutral salt `pump' regulating corneal hydration is self consistent. The spontaneous potential found across the endothelium could be caused by the consequential passive flux of salt in the opposite direction. PMID:16992435

  18. The Redox Regulator Fnr Is Required for Fermentative Growth and Enterotoxin Synthesis in Bacillus cereus F4430/73▿

    PubMed Central

    Zigha, Assia; Rosenfeld, Eric; Schmitt, Philippe; Duport, Catherine

    2007-01-01

    Glucose-grown cells of Bacillus cereus respond to anaerobiosis and low extracellular oxidoreduction potentials (ORP), notably by enhancing enterotoxin production. This response involves the ResDE two-component system. We searched the B. cereus genome for other redox response regulators potentially involved in this adaptive process, and we identified one gene encoding a protein predicted to have an amino acid sequence 58% identical (80% similar) to that of the Bacillus subtilis Fnr redox regulator. The fnr gene of the food-borne pathogen B. cereus F4430/73 has been cloned and partially characterized. We showed that fnr was up-regulated during anaerobic fermentation, especially when fermentation occurred at low ORP (under highly reducing conditions). The expression of fnr was down-regulated in the presence of O2 and nitrate which, unlike fumarate, stimulated the respiratory pathways. The inactivation of B. cereus fnr abolished fermentative growth but only moderately affected aerobic and anaerobic nitrate respiratory growth. Analyses of glucose by-products and the transcription profiles of key catabolic genes confirmed the strong regulatory impact of Fnr on B. cereus fermentative pathways. More importantly, the fnr mutation strongly decreased the expression of PlcR-dependent hbl and nhe genes, leading to the absence of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) secretion by the mutant. These data indicate that fnr is essential for both fermentation and toxinogenesis. The results also suggest that both Fnr and the ResDE two-component system belong to a redox regulatory pathway that functions at least partially independently of the pleiotropic virulence gene regulator PlcR to regulate enterotoxin gene expression. PMID:17259311

  19. 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

  20. 77 FR 72970 - Revisions to Stormwater Regulations To Clarify That an NPDES Permit Is Not Required for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-07

    ... industrial activity,'' the EPA promulgated the Phase I stormwater regulations. (55 FR 47990, November 16... to roads which are exclusively or primarily dedicated for use by the industrial facility. See 55 FR... that disturb one to five acres. (64 FR 68722, December 8, 1999). The EPA retains the authority...

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

    PubMed

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

    2015-10-01

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

  2. 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

  3. 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.

  4. Requirement of ArcA for redox regulation in Escherichia coli under microaerobic but not anaerobic or aerobic conditions.

    PubMed

    Alexeeva, Svetlana; Hellingwerf, Klaas J; Teixeira de Mattos, M Joost

    2003-01-01

    In Escherichia coli, the two-component regulatory ArcAB system functions as a major control system for the regulation of expression of genes encoding enzymes involved in both aerobic and anaerobic catabolic pathways. Previously, we have described the physiological response of wild-type E. coli to changes in oxygen availability through the complete range from anaerobiosis to full aerobiosis (S. Alexeeva, B. de Kort, G. Sawers, K. J. Hellingwerf, and M. J. Teixeira de Mattos, J. Bacteriol. 182:4934-4940, 2000, and S. Alexeeva, K. J. Hellingwerf, and M. J. Teixeira de Mattos, J. Bacteriol. 184:1402-1406, 2002). Here, we address the question of the contribution of the ArcAB-dependent transcriptional regulation to this response. Wild-type E. coli and a mutant lacking the ArcA regulator were grown in glucose-limited chemostat cultures at controlled levels of oxygen availability ranging from full aerobiosis to complete anaerobiosis. A flux analysis of the distribution of catabolic fluxes over parallel pathways was carried out, and the intracellular redox state (as reflected by the NADH/NAD ratio) was monitored for all steady states. Deletion of ArcA neither significantly altered the in vivo activity of the pyruvate dehydrogenase complex and pyruvate formate lyase nor significantly affected catabolism under fully aerobic and fully anaerobic conditions. In contrast, profound effects of the absence of ArcA were seen under conditions of oxygen-restricted growth: increased respiration, an altered electron flux distribution over the cytochrome o- and d-terminal oxidases, and a significant change in the intracellular redox state were observed. Thus, the ArcA regulator was found to exert major control on flux distribution, and it is concluded that the ArcAB system should be considered a microaerobic redox regulator.

  5. cis-Acting Sequences Required for NtcB-Dependent, Nitrite-Responsive Positive Regulation of the Nitrate Assimilation Operon in the Cyanobacterium Synechococcus sp. Strain PCC 7942

    PubMed Central

    Maeda, Shin-Ichi; Kawaguchi, Yuriko; Ohe, Taka-Aki; Omata, Tatsuo

    1998-01-01

    There are three binding sites for NtcA (nirI, nirII, and nirIII), the global nitrogen regulator of cyanobacteria, in the DNA region between the two divergently transcribed operons (nirA and nirB operons) involved in nitrate assimilation in Synechococcus sp. strain PCC 7942. Using the luxAB reporter system, we showed that nirI and nirIII, which are located 23 bp upstream from the −10 promoter element of nirA and nirB, respectively, are required for induction by nitrogen depletion of the nirA and nirB operons, respectively. The induction of nirA operon transcription was a prerequisite for the nitrite-responsive positive regulation of the transcription by NtcB, a LysR-type protein. The NtcA-binding site nirII, located in the middle of the nirA-nirB intergenic region, and a potential binding site for a LysR-type protein (TGCAN5TGCA; designated L1), located between nirI and nirII, were required for the nitrite-responsive, NtcB-dependent enhancement of nirA operon transcription. Although the requirement for the L1 site was consistent with the involvement of the LysR family protein NtcB in transcriptional regulation, NtcB did not bind to the nirA regulatory region in vitro in the presence of nitrite and NtcA, suggesting the involvement of some additional factor(s) in the regulation. An L1-like inverted repeat with the consensus sequence TGCN7GCA was conserved in the nirA promoter region of cyanobacteria, being centered at position −23 with respect to the NtcA-binding site corresponding to nirI, which suggested the common occurrence of nitrite-responsive regulation of the nitrate assimilation operon among cyanobacteria. PMID:9696753

  6. The Stringent Response Regulator DksA Is Required for Salmonella enterica Serovar Typhimurium Growth in Minimal Medium, Motility, Biofilm Formation, and Intestinal Colonization.

    PubMed

    Azriel, Shalhevet; Goren, Alina; Rahav, Galia; Gal-Mor, Ohad

    2015-11-09

    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.

  7. 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

  8. 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

  9. 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

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

    PubMed

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

    2015-12-10

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

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

    PubMed Central

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

    2015-01-01

    Estrogen-related receptor alpha (ESRRa) regulates a number of cellular processes including development of bone and muscles. However, direct evidence regarding its involvement in cartilage development remains elusive. In this report, we establish an in vivo role of Esrra in cartilage development during embryogenesis in zebrafish. Gene expression analysis indicates that esrra is expressed in developing pharyngeal arches where genes necessary for cartilage development are also expressed. Loss of function analysis shows that knockdown of esrra impairs expression of genes including sox9, col2a1, sox5, sox6, runx2 and col10a1 thus induces abnormally formed cartilage in pharyngeal arches. Importantly, we identify putative ESRRa binding elements in upstream regions of sox9 to which ESRRa can directly bind, indicating that Esrra may directly regulate sox9 expression. Accordingly, ectopic expression of sox9 rescues defective formation of cartilage induced by the knockdown of esrra. Taken together, our results indicate for the first time that ESRRa is essential for cartilage development by regulating sox9 expression during vertebrate development. PMID:26657540

  12. The INO80 Complex Requires the Arp5-Ies6 Subcomplex for Chromatin Remodeling and Metabolic Regulation

    PubMed Central

    Yao, Wei; King, Devin A.; Beckwith, Sean L.; Gowans, Graeme J.; Yen, Kuangyu; Zhou, Coral

    2016-01-01

    ATP-dependent chromatin remodeling complexes are essential for transcription regulation, and yet it is unclear how these multisubunit complexes coordinate their activities to facilitate diverse transcriptional responses. In this study, we found that the conserved Arp5 and Ies6 subunits of the Saccharomyces cerevisiae INO80 chromatin-remodeler form an abundant and distinct subcomplex in vivo and stimulate INO80-mediated activity in vitro. Moreover, our genomic studies reveal that the relative occupancy of Arp5-Ies6 correlates with nucleosome positioning at transcriptional start sites and expression levels of >1,000 INO80-regulated genes. Notably, these genes are significantly enriched in energy metabolism pathways. Specifically, arp5Δ, ies6Δ, and ino80Δ mutants demonstrate decreased expression of genes involved in glycolysis and increased expression of genes in the oxidative phosphorylation pathway. Deregulation of these metabolic pathways results in constitutively elevated mitochondrial potential and oxygen consumption. Our results illustrate the dynamic nature of the INO80 complex assembly and demonstrate for the first time that a chromatin remodeler regulates glycolytic and respiratory capacity, thereby maintaining metabolic stability. PMID:26755556

  13. Kindlin-3 interacts with the ribosome and regulates c-Myc expression required for proliferation of chronic myeloid leukemia cells

    PubMed Central

    Qu, Jing; Ero, Rya; Feng, Chen; Ong, Li-Teng; Tan, Hui-Foon; Lee, Hui-Shan; Ismail, Muhammad HB; Bu, Wen-Ting; Nama, Srikanth; Sampath, Prabha; Gao, Yong-Gui; Tan, Suet-Mien

    2015-01-01

    Kindlins are FERM-containing cytoplasmic proteins that regulate integrin-mediated cell-cell and cell-extracellular matrix (ECM) attachments. Kindlin-3 is expressed in hematopoietic cells, platelets, and endothelial cells. Studies have shown that kindlin-3 stabilizes cell adhesion mediated by ß1, ß2, and ß3 integrins. Apart from integrin cytoplasmic tails, kindlins are known to interact with other cytoplasmic proteins. Here we demonstrate that kindlin-3 can associate with ribosome via the receptor for activated-C kinase 1 (RACK1) scaffold protein based on immunoprecipitation, ribosome binding, and proximity ligation assays. We show that kindlin-3 regulates c-Myc protein expression in the human chronic myeloid leukemia cell line K562. Cell proliferation was reduced following siRNA reduction of kindlin-3 expression and a significant reduction in tumor mass was observed in xenograft experiments. Mechanistically, kindlin-3 is involved in integrin α5ß1-Akt-mTOR-p70S6K signaling; however, its regulation of c-Myc protein expression could be independent of this signaling axis. PMID:26677948

  14. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis

    PubMed Central

    Zhao, Xu; Yang, Ying; Sun, Bao-Fa; Shi, Yue; Yang, Xin; Xiao, Wen; Hao, Ya-Juan; Ping, Xiao-Li; Chen, Yu-Sheng; Wang, Wen-Jia; Jin, Kang-Xuan; Wang, Xing; Huang, Chun-Min; Fu, Yu; Ge, Xiao-Meng; Song, Shu-Hui; Jeong, Hyun Seok; Yanagisawa, Hiroyuki; Niu, Yamei; Jia, Gui-Fang; Wu, Wei; Tong, Wei-Min; Okamoto, Akimitsu; He, Chuan; Danielsen, Jannie M Rendtlew; Wang, Xiu-Jie; Yang, Yun-Gui

    2014-01-01

    The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5′- and 3′-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis. PMID:25412662

  15. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis.

    PubMed

    Zhao, Xu; Yang, Ying; Sun, Bao-Fa; Shi, Yue; Yang, Xin; Xiao, Wen; Hao, Ya-Juan; Ping, Xiao-Li; Chen, Yu-Sheng; Wang, Wen-Jia; Jin, Kang-Xuan; Wang, Xing; Huang, Chun-Min; Fu, Yu; Ge, Xiao-Meng; Song, Shu-Hui; Jeong, Hyun Seok; Yanagisawa, Hiroyuki; Niu, Yamei; Jia, Gui-Fang; Wu, Wei; Tong, Wei-Min; Okamoto, Akimitsu; He, Chuan; Rendtlew Danielsen, Jannie M; Wang, Xiu-Jie; Yang, Yun-Gui

    2014-12-01

    The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5'- and 3'-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis. PMID:25412662

  16. The yeast prion [SWI+] abolishes multicellular growth by triggering conformational changes of multiple regulators required for flocculin gene expression

    PubMed Central

    Du, Zhiqiang; Zhang, Ying; Li, Liming

    2016-01-01

    Summary While transcription factors are prevalent among yeast prion proteins, the role of prion-mediated transcriptional regulation remains elusive. We show here that the yeast prion [SWI+] abolishes flocculin (FLO) gene expression and results in a complete loss of multicellularity. Further investigation demonstrates that besides Swi1, multiple other proteins essential for FLO expression, including Mss11, Sap30, and Msn1 also undergo conformational changes, and become inactivated in [SWI+] cells. Moreover, the asparagine-rich region of Mss11 can exist as prion-like aggregates specifically in [SWI+] cells, which are SDS-resistant, heritable, and curable, but become metastable after separation from [SWI+]. Our findings thus reveal a prion-mediated mechanism through which multiple regulators in a biological pathway can be inactivated. In combination with the partial loss-of-function phenotypes of [SWI+] cells on non-glucose sugar utilization, our data therefore demonstrate that a prion can influence differently on distinct traits through multi-level regulations, providing insights into the biological roles of prions. PMID:26711350

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

    PubMed

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

    2007-04-01

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

  18. 76 FR 14812 - Final Regulation Extending the Reporting Deadline for Year 2010 Data Elements Required Under the...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-18

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  19. 77 FR 53870 - Availability of a Legal Entity Identifier Meeting the Requirements of the Regulations of the...

    Federal Register 2010, 2011, 2012, 2013, 2014

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  20. 75 FR 35338 - Implementation of Regulations Required Under Title XI of the Food, Conservation and Energy Act of...

    Federal Register 2010, 2011, 2012, 2013, 2014

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    ...; ] DEPARTMENT OF AGRICULTURE Grain Inspection, Packers and Stockyards Administration 9 CFR Part 201 RIN 0580..., USDA. ACTION: Proposed rule. SUMMARY: The Department of Agriculture (USDA), Grain Inspection, Packers... in the livestock and poultry industries and required the Secretary of Agriculture (Secretary)...

  1. A peroxiredoxin, PRDX-2, is required for insulin secretion and insulin/IIS-dependent regulation of stress resistance and longevity.

    PubMed

    Oláhová, Monika; Veal, Elizabeth A

    2015-08-01

    Peroxiredoxins (Prx) are abundant thiol peroxidases with a conserved anti-ageing role. In contrast to most animals, the nematode worm, Caenorhabditis elegans, encodes a single cytosolic 2-Cys Prx, PRDX-2, rendering it an excellent model for examining how peroxiredoxins affect animal physiology and ageing. Our previous work revealed that, although PRDX-2 protects against the toxicity of peroxides, enigmatically, prdx-2-mutant animals are hyper-resistant to other forms of oxidative stress. Here, we have investigated the basis for this increased resistance. Mammalian FOXO and Nrf2 transcription factors directly promote the expression of a range of detoxification enzymes. We show that the FOXO orthologue, DAF-16, and the Nrf2 orthologue, SKN-1, are required for the increased stress resistance of prdx-2-mutant worms. Our data suggest that PRDX-2 is required for normal levels of insulin secretion and hence the inhibition of DAF-16 and SKN-1 by insulin/IGF-1-like signalling (IIS) under nutrient-rich conditions. Intriguingly, loss of PRDX-2 increases DAF-16 and SKN-1 activities sufficiently to increase arsenite resistance without initiating other IIS-inhibited processes. Together, these data suggest that loss of peroxiredoxin function may increase stress resistance by reducing insulin secretion, but that further changes in insulin signalling are required for the reprogramming of development and fat metabolism. In addition, we reveal that the temperature-dependent prolongevity function of PRDX-2 is required for the extended lifespan associated with several pathways, including further reductions in IIS.

  2. A peroxiredoxin, PRDX-2, is required for insulin secretion and insulin/IIS-dependent regulation of stress resistance and longevity

    PubMed Central

    Oláhová, Monika; Veal, Elizabeth A

    2015-01-01

    Peroxiredoxins (Prx) are abundant thiol peroxidases with a conserved anti-ageing role. In contrast to most animals, the nematode worm, Caenorhabditis elegans, encodes a single cytosolic 2-Cys Prx, PRDX-2, rendering it an excellent model for examining how peroxiredoxins affect animal physiology and ageing. Our previous work revealed that, although PRDX-2 protects against the toxicity of peroxides, enigmatically, prdx-2-mutant animals are hyper-resistant to other forms of oxidative stress. Here, we have investigated the basis for this increased resistance. Mammalian FOXO and Nrf2 transcription factors directly promote the expression of a range of detoxification enzymes. We show that the FOXO orthologue, DAF-16, and the Nrf2 orthologue, SKN-1, are required for the increased stress resistance of prdx-2-mutant worms. Our data suggest that PRDX-2 is required for normal levels of insulin secretion and hence the inhibition of DAF-16 and SKN-1 by insulin/IGF-1-like signalling (IIS) under nutrient-rich conditions. Intriguingly, loss of PRDX-2 increases DAF-16 and SKN-1 activities sufficiently to increase arsenite resistance without initiating other IIS-inhibited processes. Together, these data suggest that loss of peroxiredoxin function may increase stress resistance by reducing insulin secretion, but that further changes in insulin signalling are required for the reprogramming of development and fat metabolism. In addition, we reveal that the temperature-dependent prolongevity function of PRDX-2 is required for the extended lifespan associated with several pathways, including further reductions in IIS. PMID:25808059

  3. 76 FR 76874 - Implementation of Regulations Required Under Title XI of the Food, Conservation and Energy Act of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-09

    ... on these comments. Section V provides the revised impact analyses including those required by..., (75 FR 35338) provided a 60-day comment period to end on August 23, 2010. In response to requests for... November 22, 2010 (75 FR 44163). GIPSA considered all comments postmarked or electronically submitted...

  4. Regulatory Requirements and Technical Analysis for Department of Energy Regulated Performance Assessments of Shallow-Trench Disposal of Low-Level Radioactive Waste at the Nevada Test Site

    NASA Astrophysics Data System (ADS)

    Crowe, B.; Black, P.; Tauxe, J.; Yucel, V.; Rawlinson, S.; Colarusso, A.; DiSanza, F.

    2001-12-01

    The National Nuclear Security Administration, Nevada Operations Office (NNSA/NV) operates and maintains two active facilities on the Nevada Test Site (NTS) that dispose Department of Energy (DOE) defense-generated low-level radioactive (LLW), mixed radioactive, and classified waste in shallow trenches, pits and large-diameter boreholes. The operation and maintenance of the LLW disposal sites are self-regulated under DOE Order 435.1, which requires review of a Performance Assessment for four performance objectives: 1) all pathways 25 mrem/yr limit; 2) atmospheric pathways 10 mrem/yr limit; 3) radon flux density of 20 pCi/m2/s; and 4) groundwater resource protection (Safe Drinking Water Act; 4 mrem/yr limit). The inadvertent human intruder is protected under a dual 500- and 100-mrem limit (acute and chronic exposure). In response to the Defense Nuclear Facilities Safety Board Recommendation 92 2, a composite analysis is required that must examine all interacting sources for compliance against both 30 and 100 mrem/yr limits. A small component of classified transuranic waste is buried at intermediate depths in 3-meter diameter boreholes at the Area 5 LLW disposal facility and is assessed through DOE-agreement against the requirements of the Environmental Protection Agency (EPA)'s 40 CFR 191. The hazardous components of mixed LLW are assessed against RCRA requirements. The NTS LLW sites fall directly under three sets of federal regulations and the regulatory differences result not only in organizational challenges, but also in different decision objectives and technical paths to completion. The DOE regulations require deterministic analysis for a 1,000-year compliance assessment supplemented by probabilistic analysis under a long-term maintenance program. The EPA regulations for TRU waste are probabilistically based for a compliance interval of 10,000 years. Multiple steps in the assessments are strongly dependent on assumptions for long-term land use policies

  5. Expression atlas of the multivalent epigenetic regulator Brpf1 and its requirement for survival of mouse embryos

    PubMed Central

    You, Linya; Chen, Lulu; Penney, Janice; Miao, Dengshun; Yang, Xiang-Jiao

    2014-01-01

    Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a unique epigenetic regulator that contains multiple structural domains for recognizing different chromatin modifications. In addition, it possesses sequence motifs for forming multiple complexes with three different histone acetyltransferases, MOZ, MORF, and HBO1. Within these complexes, BRPF1 serves as a scaffold for bridging subunit interaction, stimulating acetyltransferase activity, governing substrate specificity and stimulating gene expression. To investigate how these molecular interactions are extrapolated to biological functions of BRPF1, we utilized a mouse strain containing a knock-in reporter and analyzed the spatiotemporal expression from embryos to adults. The analysis revealed dynamic expression in the extraembryonic, embryonic, and fetal tissues, suggesting important roles of Brpf1 in prenatal development. In support of this, inactivation of the mouse Brpf1 gene causes lethality around embryonic day 9.5. After birth, high expression is present in the testis and specific regions of the brain. The 4-dimensional expression atlas of mouse Brpf1 should serve as a valuable guide for analyzing its interaction with Moz, Morf, and Hbo1 in vivo, as well as for investigating whether Brpf1 functions independently of these three enzymatic epigenetic regulators. PMID:24646517

  6. The Balanced Regulation of Hsc70 by DNJ-13 and UNC-23 Is Required for Muscle Functionality*

    PubMed Central

    Papsdorf, Katharina; Sacherl, Julia; Richter, Klaus

    2014-01-01

    The molecular chaperone Hsc70 assists in the folding of non-native proteins together with its J domain- and BAG domain-containing cofactors. In Caenorhabditis elegans, two BAG domain-containing proteins can be identified, one of them being UNC-23, whose mutation induces severe motility dysfunctions. Using reporter strains, we find that the full-length UNC-23, in contrast to C-terminal fragments, localizes specifically to the muscular attachment sites. C-terminal fragments of UNC-23 instead perform all Hsc70-related functions, like ATPase stimulation and regulation of folding activity, albeit with lower affinity than BAG-1. Interestingly, overexpression of CFP-Hsc70 can induce muscular defects in wild-type nematodes that phenocopy the knockout of its cofactor UNC-23. Strikingly, the motility dysfunction in the unc-23 mutated strain can be cured specifically by down-regulation of the antagonistic Hsc70 cochaperone DNJ-13, implying that the severe phenotype is caused by misregulation of the Hsc70 cycle. These findings point out that the balanced action of cofactors in the ATP-driven cycle of Hsc70 is crucial for the contribution of Hsc70 to muscle functionality. PMID:25053410

  7. Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.

    PubMed Central

    Baum, G; Lev-Yadun, S; Fridmann, Y; Arazi, T; Katsnelson, H; Zik, M; Fromm, H

    1996-01-01

    Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants. Images PMID:8670800

  8. Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.

    PubMed

    Baum, G; Lev-Yadun, S; Fridmann, Y; Arazi, T; Katsnelson, H; Zik, M; Fromm, H

    1996-06-17

    Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.

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

    PubMed Central

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

    2016-01-01

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

  10. An Intronic Enhancer Is Required for Deflagellation-induced Transcriptional Regulation of a Chlamydomonas reinhardtii Dynein Gene

    PubMed Central

    Kang, Yong; Mitchell, David R.

    1998-01-01

    Chlamydomonas reinhardtii flagellar regeneration is accompanied by rapid induction of genes encoding a large set of flagellar structural components and provides a model system to study coordinate gene regulation and organelle assembly. After deflagellation, the abundance of a 70-kDa flagellar dynein intermediate chain (IC70, encoded by ODA6) mRNA increases approximately fourfold within 40 min and returns to predeflagellation levels by ∼90 min. We show by nuclear run-on that this increase results, in part, from increased rates of transcription. To localize cis induction elements, we created an IC70 minigene and measured accumulation, in C. reinhardtii, of transcripts from the endogenous gene and from introduced promoter deletion constructs. Clones containing 416 base pairs (bp) of 5′- and 2 kilobases (kb) of 3′-flanking region retained all sequences necessary for a normal pattern of mRNA abundance change after deflagellation. Extensive 5′- and 3′- flanking region deletions, which removed multiple copies of a proposed deflagellation-response element (the tub box), did not eliminate induction, and the IC70 5′-flanking region alone did not confer deflagellation responsiveness to a promoterless arylsulfatase (ARS) gene. Instead, an intron in the IC70 gene 5′-untranslated region was found to contain the deflagellation response element. These results suggest that the tub box does not play an essential role in deflagellation-induced transcriptional regulation of this dynein gene. PMID:9802898

  11. A DNA2 Homolog Is Required for DNA Damage Repair, Cell Cycle Regulation, and Meristem Maintenance in Plants1[OPEN

    PubMed Central

    Jia, Ning; Liu, Xiaomin; Gao, Hongbo

    2016-01-01

    Plant meristem cells divide and differentiate in a spatially and temporally regulated manner, ultimately giving rise to organs. In this study, we isolated the Arabidopsis jing he sheng 1 (jhs1) mutant, which exhibited retarded growth, an abnormal pattern of meristem cell division and differentiation, and morphological defects such as fasciation, an irregular arrangement of siliques, and short roots. We identified JHS1 as a homolog of human and yeast DNA Replication Helicase/Nuclease2, which is known to be involved in DNA replication and damage repair. JHS1 is strongly expressed in the meristem of Arabidopsis. The jhs1 mutant was sensitive to DNA damage stress and had an increased DNA damage response, including increased expression of genes involved in DNA damage repair and cell cycle regulation, and a higher frequency of homologous recombination. In the meristem of the mutant plants, cell cycle progression was delayed at the G2 or late S phase and genes essential for meristem maintenance were misregulated. These results suggest that JHS1 plays an important role in DNA replication and damage repair, meristem maintenance, and development in plants. PMID:26951435

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

    PubMed Central

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

    2014-01-01

    Summary Male fertility in flowering plants relies on proper division and differentiation of cells in the anther, a process that gives rise to four somatic layers surrounding central germinal cells. The maize gene male sterility32 (ms32) encodes a basic helix–loop–helix (bHLH) transcription factor, which functions as an important regulator of both division and differentiation during anther development. After the four somatic cell layers are generated properly through successive periclinal divisions, in the ms32 mutant, tapetal precursor cells fail to differentiate, and, instead, undergo additional periclinal divisions to form extra layers of cells. These cells become vacuolated and expand, and lead to failure in pollen mother cell development. ms32 expression is specific to the pre-meiotic anthers and is distributed initially broadly in the four lobes, but as the anther develops, its expression becomes restricted to the innermost somatic layer, the tapetum. The ms32-ref mac1-1 double mutant is unable to form tapetal precursors and also exhibits excessive somatic proliferation leading to numerous, disorganized cell layers, suggesting a synergistic interaction between ms32 and mac1. Altogether, our results show that MS32 is a major regulator in maize anther development that promotes tapetum differentiation and inhibits periclinal division once a tapetal cell is specified. PMID:24033746

  13. Na/sup +/ requirement for growth, photosynthesis, and pH regulation of the alkalotolerant cyanobacterium Synechococcus leopoliensis

    SciTech Connect

    Miller, A.G.; Turpin, D.H.; Canvin, D.T.

    1984-07-01

    It was found that Na/sup +/ is required for all the alkalotolerance of the cyanobacterium Synechococcus leopoliensis. Cell division did not occur at any pH in the absence of Na/sup +/, but cells inoculated into Na/sup +/-free growth medium at pH 6.8 did continue metabolic activity, and over a period of 48 h, the cells became twice their normal size. Many of these cells remained viable for at least 59 h and formed colonies on Na/sup +/-containing medium. Cells grown in the presence of Na/sup +/ and inoculated into Na/sup +/-free growth medium at pH 9.6 rapidly lost viability. An Na/sup +/ concentration of ca. 0.5 milliequivalents x liter/sup -1/ was required for sustained growth above pH 9.0. The Na/sup +/ requirement could be only partially met by Li/sup +/ and not at all by K/sup +/ or Rb/sup +/. Cells incubated in darkness in growth medium at pH 6.8 had an intracellular pH near neutrality in the presence or absence of Na/sup +/. When the external pH was shifted to 9.6, only cells in the presence of Na/sup +/ were able to maintain an intracellular pH near 7.0. The membrane potential, however, remained high (-120mV) in the absence or presence of Na/sup +/ unless collapsed by the addition of gramicidin. Thus, the inability to maintain a neutral intracellular pH at pH 9.6 in the absence of Na/sup +/ was not due to a generalized disruption of membrane integrity. Even cells containing Na/sup +/ still required added Na/sup +/ to restore photosynthetic rates to normal after the cells had been washed in Na/sup +/-free buffer at pH 9.6. This requirement was only partially met by Li/sup +/ and was not met at all by K/sup +/, Rb/sup +/, Cs/sup +/, Mg/sup 2 +/, or Ca/sup 2 +/. The restoration of photosynthesis by added Na/sup +/ occurred within 30 s and suggests a role for extracellular Na/sup +/. Part of our results can be explained in terms of the operation of an Na/sup +//H/sup +/ antiporter activity in the plasma membrane, but some results would seem to require other

  14. Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

    PubMed Central

    2010-01-01

    Background Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains. Results We examined the localization of Crb2a constructs and their effects on rod morphology. We found that only the full-length Crb2a protein approximated the normal localization of Crb2a protein apical to adherens junctions in the photoreceptor inner segment. Several Crb2a construct proteins localized abnormally to the outer segment and one construct localized abnormally to the cell body. Overexpression of full-length Crb2a greatly increased inner segment size while expression of several other constructs increased outer segment size. Conclusions Our observations suggest that particular domains in Crb2a regulate its localization and thus may regulate its regionalized function. Our results also suggest that the PDZ-binding domain in Crb2a might bring a protein(s) into

  15. The Burkholderia cenocepacia K56-2 pleiotropic regulator Pbr, is required for stress resistance and virulence.

    PubMed

    Ramos, Christian G; Sousa, Silvia A; Grilo, André M; Eberl, Leo; Leitão, Jorge H

    2010-05-01

    Burkholderia cenocepacia is one of the most virulent species of the Burkholderia cepacia complex, a group of bacteria that emerged as important pathogens, especially to cystic fibrosis (CF) patients. In this study, we report the identification and characterization of a mutant strain derived form the CF isolate Burkholderia cenocepacia K56-2, carrying a plasposon insertion in a gene, located in a 3516 bp chromosomal region with an atypical G+C content, encoding a 80 amino acid putative regulatory protein named Pbr. Besides its inability to produce phenazines, the B. cenocepacia K56-2 pbr mutant exhibited a pleiotropic phenotype, including impaired survival to oxidative and osmotic stress, aromatic amino acid and prolonged nutrient starvation periods. In addition, the pbr mutant exhibited decreased virulence the nematode Caenorhabditis elegans. Altogether, our results demonstrate the involvement of Pbr on the regulation of phenazine biosynthesis, and an important role for this regulatory protein on several cellular processes related to stress resistance and virulence.

  16. Upstream Stimulatory Factor is Required for Human Angiotensinogen Expression and Differential Regulation by the A-20C Polymorphism

    PubMed Central

    Dickson, Matthew E.; Tian, Xin; Liu, Xuebo; Davis, Deborah R.; Sigmund, Curt D.

    2008-01-01

    Among naturally-occurring polymorphisms in the 5’ flanking region of the human angiotensinogen (AGT) gene, the −20 and −217 polymorphisms have the strongest effects on AGT regulation in AGT-expressing cells derived from liver, kidney, brain, and fat. These polymorphisms may affect allele-specific transcription factor binding, and the high-expressing alleles are both relatively common. We show herein that the −20C allele has higher transcriptional activity than −20A, and the −20A allele confers no additional transactivation potential beyond that of a mutated vector. Gel shift assays show that USF1 and USF2 preferentially bind the −20C allele whereas the −20A allele retains a low affinity USF binding site. Plasmid immunoprecipitation assays confirmed preferential association of USF1 with the −20C allele in transfected HepG2 cells. Chromatin immunoprecipitation confirmed that USF1 binds to the endogenous AGT −20C allele in CCF cells, the only cell line tested that carries the −20C allele, and to the human AGT promoter in liver and adipose tissue from transgenic mice. Transduction of AGT-expressing cells with shRNAs specifically targeting USF1 or USF2, resulted in cell- and allele-specific attenuation of AGT promoter activity. In vivo, knockdown of USF expression in the liver of transgenic mice expressing the −20C allele of AGT resulted in lower AGT expression, a decrease in circulating human AGT protein, but no change in expression of GAPDH or HNF-4α. We conclude that USF1 functionally and differentially regulates AGT expression via the −20 polymorphism and that the differential expression exhibited by −20 can be accounted for by differential association with USF1. PMID:18802024

  17. Convergence of multiple signaling pathways is required to coordinately up-regulate mtDNA and mitochondrial biogenesis during T cell activation.

    PubMed

    D'Souza, Anthony D; Parikh, Neal; Kaech, Susan M; Shadel, Gerald S

    2007-12-01

    The quantity and activity of mitochondria vary dramatically in tissues and are modulated in response to changing cellular energy demands and environmental factors. The amount of mitochondrial DNA (mtDNA), which encodes essential subunits of the oxidative phosphorylation complexes required for cellular ATP production, is also tightly regulated, but by largely unknown mechanisms. Using murine T cells as a model system, we have addressed how specific signaling pathways influence mitochondrial biogenesis and mtDNA copy number. T cell receptor (TCR) activation results in a large increase in mitochondrial mass and membrane potential and a corresponding amplification of mtDNA, consistent with a vital role for mitochondrial function for growth and proliferation of these cells. Independent activation of protein kinase C (via PMA) or calcium-related pathways (via ionomycin) had differential and sub-maximal effects on these mitochondrial parameters, as did activation of naïve T cells with proliferative cytokines. Thus, the robust mitochondrial biogenesis response observed upon TCR activation requires synergy of multiple downstream signaling pathways. One such pathway involves AMP-activated protein kinase (AMPK), which we show has an unprecedented role in negatively regulating mitochondrial biogenesis that is mammalian target of rapamycin (mTOR)-dependent. That is, inhibition of AMPK after TCR signaling commences results in excessive, but uncoordinated mitochondrial proliferation. Thus mitochondrial biogenesis is not under control of a single master regulatory circuit, but rather requires the convergence of multiple signaling pathways with distinct downstream consequences on the organelle's structure, composition, and function.

  18. TATA-binding protein-like protein (TLP/TRF2/TLF) negatively regulates cell cycle progression and is required for the stress-mediated G(2) checkpoint.

    PubMed

    Shimada, Miho; Nakadai, Tomoyoshi; Tamura, Taka-Aki

    2003-06-01

    The TATA-binding protein (TBP) is a universal transcription factor required for all of the eukaryotic RNA polymerases. In addition to TBP, metazoans commonly express a distantly TBP-related protein referred to as TBP-like protein (TLP/TRF2/TLF). Although the function of TLP in transcriptional regulation is not clear, it is known that TLP is required for embryogenesis and spermiogenesis. In the present study, we investigated the cellular functions of TLP by using TLP knockout chicken DT40 cells. TLP was found to be dispensable for cell growth. Unexpectedly, TLP-null cells exhibited a 20% elevated cell cycle progression rate that was attributed to shortening of the G(2) phase. This indicates that TLP functions as a negative regulator of cell growth. Moreover, we found that TLP mainly existed in the cytoplasm and was translocated to the nucleus restrictedly at the G(2) phase. Ectopic expression of nuclear localization signal-carrying TLP resulted in an increase (1.5-fold) in the proportion of cells remaining in the G(2)/M phase and apoptotic state. Notably, TLP-null cells showed an insufficient G(2) checkpoint when the cells were exposed to stresses such as UV light and methyl methanesulfonate, and the population of apoptotic cells after stresses decreased to 40%. These phenomena in G(2) checkpoint regulation are suggested to be p53 independent because p53 does not function in DT40 cells. Moreover, TLP was transiently translocated to the nucleus shortly (15 min) after stress treatment. The expression of several stress response and cell cycle regulatory genes drifted in a both TLP- and stress-dependent manner. Nucleus-translocating TLP is therefore thought to work by checking cell integrity through its transcription regulatory ability. TLP is considered to be a signal-transducing transcription factor in cell cycle regulation and stress response.

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

    PubMed

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

    2014-06-01

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

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

    PubMed

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

    2014-06-01

    In this study, we report on the production of bulb scale-derived tissue cultures capable of efficient shoot and plant regeneration in three genotypes of snowdrop (Galanthus nivalis L., Amaryllidaceae), a protected ornamental plant. For culture line A, high auxin and low cytokinin concentration is required for callus production and plant regeneration. The type of auxin is of key importance: α-naphthaleneacetic acid (NAA) in combination with indole-3-acetic acid (IAA) at concentrations of 2 mg L-1 or 2-10 mg L-1 NAA with 1 mg L-1 N6-benzyladenine (BA), a cytokinin on full-strength media are required for regeneration. Cultures showing regeneration were embryogenic. When lines B and C were induced and maintained with 2 mg L-1 NAA and 1 mg L-1 BA, they produced mature bulblets with shoots, without roots. Line A produced immature bulblets with shoots under the above culture condition. Amplified Fragment Length Polymorphism (AFLP) analysis showed that (i) genetic differences between line A and its bulb explants were not significant, therefore these tissue cultures are suitable for germplasm preservation, and (ii) different morphogenetic responses of lines A, B and C originated from genetic differences. Culture line A is suitable for field-growing, cultivation and germplasm preservation of G. nivalis and for the production of Amaryllidaceae alkaloids. PMID:24873910

  1. Autism-Associated Chromatin Regulator Brg1/SmarcA4 Is Required for Synapse Development and Myocyte Enhancer Factor 2-Mediated Synapse Remodeling.

    PubMed

    Zhang, Zilai; Cao, Mou; Chang, Chia-Wei; Wang, Cindy; Shi, Xuanming; Zhan, Xiaoming; Birnbaum, Shari G; Bezprozvanny, Ilya; Huber, Kimberly M; Wu, Jiang I

    2016-01-01

    Synapse development requires normal neuronal activities and the precise expression of synapse-related genes. Dysregulation of synaptic genes results in neurological diseases such as autism spectrum disorders (ASD). Mutations in genes encoding chromatin-remodeling factor Brg1/SmarcA4 and its associated proteins are the genetic causes of several developmental diseases with neurological defects and autistic symptoms. Recent large-scale genomic studies predicted Brg1/SmarcA4 as one of the key nodes of the ASD gene network. We report that Brg1 deletion in early postnatal hippocampal neurons led to reduced dendritic spine density and maturation and impaired synapse activities. In developing mice, neuronal Brg1 deletion caused severe neurological defects. Gene expression analyses indicated that Brg1 regulates a significant number of genes known to be involved in synapse function and implicated in ASD. We found that Brg1 is required for dendritic spine/synapse elimination mediated by the ASD-associated transcription factor myocyte enhancer factor 2 (MEF2) and that Brg1 regulates the activity-induced expression of a specific subset of genes that overlap significantly with the targets of MEF2. Our analyses showed that Brg1 interacts with MEF2 and that MEF2 is required for Brg1 recruitment to target genes in response to neuron activation. Thus, Brg1 plays important roles in both synapse development/maturation and MEF2-mediated synapse remodeling. Our study reveals specific functions of the epigenetic regulator Brg1 in synapse development and provides insights into its role in neurological diseases such as ASD.

  2. Autism-Associated Chromatin Regulator Brg1/SmarcA4 Is Required for Synapse Development and Myocyte Enhancer Factor 2-Mediated Synapse Remodeling

    PubMed Central

    Zhang, Zilai; Cao, Mou; Chang, Chia-Wei; Wang, Cindy; Shi, Xuanming; Zhan, Xiaoming; Birnbaum, Shari G.; Bezprozvanny, Ilya; Huber, Kimberly M.

    2015-01-01

    Synapse development requires normal neuronal activities and the precise expression of synapse-related genes. Dysregulation of synaptic genes results in neurological diseases such as autism spectrum disorders (ASD). Mutations in genes encoding chromatin-remodeling factor Brg1/SmarcA4 and its associated proteins are the genetic causes of several developmental diseases with neurological defects and autistic symptoms. Recent large-scale genomic studies predicted Brg1/SmarcA4 as one of the key nodes of the ASD gene network. We report that Brg1 deletion in early postnatal hippocampal neurons led to reduced dendritic spine density and maturation and impaired synapse activities. In developing mice, neuronal Brg1 deletion caused severe neurological defects. Gene expression analyses indicated that Brg1 regulates a significant number of genes known to be involved in synapse function and implicated in ASD. We found that Brg1 is required for dendritic spine/synapse elimination mediated by the ASD-associated transcription factor myocyte enhancer factor 2 (MEF2) and that Brg1 regulates the activity-induced expression of a specific subset of genes that overlap significantly with the targets of MEF2. Our analyses showed that Brg1 interacts with MEF2 and that MEF2 is required for Brg1 recruitment to target genes in response to neuron activation. Thus, Brg1 plays important roles in both synapse development/maturation and MEF2-mediated synapse remodeling. Our study reveals specific functions of the epigenetic regulator Brg1 in synapse development and provides insights into its role in neurological diseases such as ASD. PMID:26459759

  3. Conjugative Transfer of p42a from Rhizobium etli CFN42, Which Is Required for Mobilization of the Symbiotic Plasmid, Is Regulated by Quorum Sensing

    PubMed Central

    Tun-Garrido, Cristina; Bustos, Patricia; González, Víctor; Brom, Susana

    2003-01-01

    Rhizobium etli CFN42 contains six plasmids. Only one of them, p42a, is self-conjugative at high frequency. This plasmid is strictly required for mobilization of the symbiotic plasmid (pSym). To study the transfer mechanism of p42a, a self-transmissible cosmid clone containing its transfer region was isolated. Its sequence showed that most of the tra genes are highly similar to genes of Agrobacterium tumefaciens pTiC58 and other related plasmids. Four putative regulatory genes were identified; three of these (traI, traR, and cinR) belong to the LuxR-LuxI family. Mutagenesis of these genes confirmed their requirement for p42a transfer. We found that the conjugative transfer of p42a is dependent on quorum sensing, and consequently pSym transfer also was found to be similarly regulated, establishing a complex link between environmental conditions and pSym transfer. Although R. etli has been shown to produce different N-acyl-homoserine lactones, only one of them, a 3-oxo-C8-homoserine lactone encoded by the traI gene described here, was involved in transfer. Mutagenesis of the fourth regulatory gene, traM, had no effect on transfer. Analysis of transcriptional fusions of the regulatory genes to a reporter gene suggests a complex regulation scheme for p42a conjugative transfer. Conjugal transfer gene expression was found to be directly upregulated by TraR and the 3-oxo-C8-homoserine lactone synthesized by TraI. The traI gene was autoregulated by these elements and positively regulated by CinR, while cinR expression required traI. Finally, we did not detect expression of traM, indicating that in p42a TraM may be expressed so weakly that it cannot inhibit conjugal transfer, leading to the unrepressed transfer of p42a. PMID:12591886

  4. Phosphothreonine 218 is required for the function of SR45.1 in regulating flower petal development in Arabidopsis

    PubMed Central

    Zhang, Xiao-Ning; Mo, Cecilia; Garrett, Wesley M; Cooper, Bret

    2014-01-01

    RNA splicing is crucial to the production of mature mRNAs (mRNA). In Arabidopsis thaliana, the protein Arginine/Serine-rich 45 (SR45) acts as an RNA splicing activator and initiates the spliceosome assembly. SR45 is alternatively spliced into 2 isoforms. Isoform 1 (SR45.1) plays an important role in the flower petal development whereas isoform 2 (SR45.2) is important for root growth. In this study, we used immunoprecipitation to isolate an SR45.1-GFP fusion protein from transgenic plants complementing a null mutant, sr45–1. Mass spectrometry suggested a single phosphorylation event in a peptide from the alternatively spliced region unique to SR45.1. Substituting alanine for threonine 218, a candidate site for phosphorylation, did not complement the sr45–1 mutant with narrow flower petals whereas substituting aspartic acid or glutamic acid for threonine 218 did complement the sr45–1 mutant. Mass spectrometry also revealed that other proteins involved in the spliceosome co-precipitated with SR45.1, and RT-qPCR revealed that phosphorylation of threonine 218 promotes the function of SR45.1 in promoting the constitutive splicing of SR30 mRNA. This is the first demonstration of a specific phosphorylation site that differentially regulates the function of a plant splicing activator in physiologically and morphologically distinct plant tissues. PMID:25763493

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

    PubMed

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

    2014-04-15

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

  6. Phosphothreonine 218 is required for the function of SR45.1 in regulating flower petal development in Arabidopsis.

    PubMed

    Zhang, Xiao-Ning; Mo, Cecilia; Garrett, Wesley M; Cooper, Bret

    2014-05-15

    RNA splicing is crucial to the production of mature mRNAs (mRNA). In Arabidopsis thaliana, the protein Arginine/Serine-rich 45 (SR45) acts as an RNA splicing activator and initiates the spliceosome assembly. SR45 is alternatively spliced into two isoforms. Isoform 1 (SR45.1) plays an important role in the flower petal development whereas isoform 2 (SR45.2) is important for root growth. In this study, we used immunoprecipitation to isolate an SR45.1-GFP fusion protein from transgenic plants complementing a null mutant, sr45-1. Mass spectrometry suggested a single phosphorylation event in a peptide from the alternatively spliced region unique to SR45.1. Substituting alanine for threonine 218, a candidate site for phosphorylation, did not complement the sr45-1 mutant with narrow flower petals whereas substituting aspartic acid or glutamic acid for threonine 218 did complement the sr45-1 mutant. Mass spectrometry also revealed that other proteins involved in the spliceosome co-precipitated with SR45.1, and RT-qPCR revealed that phosphorylation of threonine 218 promotes the function of SR45.1 in promoting the constitutive splicing of SR30 mRNA. This is the first demonstration of a specific phosphorylation site that differentially regulates the function of a plant splicing activator in physiologically and morphologically distinct plant tissues. PMID:24832081

  7. Phosphothreonine 218 is required for the function of SR45.1 in regulating flower petal development in Arabidopsis.

    PubMed

    Zhang, Xiao-Ning; Mo, Cecilia; Garrett, Wesley M; Cooper, Bret

    2014-01-01

    RNA splicing is crucial to the production of mature mRNAs (mRNA). In Arabidopsis thaliana, the protein Arginine/Serine-rich 45 (SR45) acts as an RNA splicing activator and initiates the spliceosome assembly. SR45 is alternatively spliced into 2 isoforms. Isoform 1 (SR45.1) plays an important role in the flower petal development whereas isoform 2 (SR45.2) is important for root growth. In this study, we used immunoprecipitation to isolate an SR45.1-GFP fusion protein from transgenic plants complementing a null mutant, sr45-1. Mass spectrometry suggested a single phosphorylation event in a peptide from the alternatively spliced region unique to SR45.1. Substituting alanine for threonine 218, a candidate site for phosphorylation, did not complement the sr45-1 mutant with narrow flower petals whereas substituting aspartic acid or glutamic acid for threonine 218 did complement the sr45-1 mutant. Mass spectrometry also revealed that other proteins involved in the spliceosome co-precipitated with SR45.1, and RT-qPCR revealed that phosphorylation of threonine 218 promotes the function of SR45.1 in promoting the constitutive splicing of SR30 mRNA. This is the first demonstration of a specific phosphorylation site that differentially regulates the function of a plant splicing activator in physiologically and morphologically distinct plant tissues. PMID:25763493

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

    PubMed

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

    2014-04-15

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

  9. Regulation of human epidermal stem cell proliferation and senescence requires polycomb- dependent and -independent functions of Cbx4.

    PubMed

    Luis, Nuno Miguel; Morey, Lluis; Mejetta, Stefania; Pascual, Gloria; Janich, Peggy; Kuebler, Bernd; Cozutto, Luca; Roma, Guglielmo; Nascimento, Elisabete; Frye, Michaela; Di Croce, Luciano; Benitah, Salvador Aznar

    2011-09-01

    Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex 1 (PRC1)-associated protein, maintains human epidermal stem cells as slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its antisenescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors such as Ezh2, Dnmt1, and Bmi1 to prevent the active stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation. PMID:21885019

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

    PubMed Central

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

    2014-01-01

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

  11. The WNT-controlled transcriptional regulator LBH is required for mammary stem cell expansion and maintenance of the basal lineage

    PubMed Central

    Lindley, Linsey E.; Curtis, Kevin M.; Sanchez-Mejias, Avencia; Rieger, Megan E.; Robbins, David J.; Briegel, Karoline J.

    2015-01-01

    The identification of multipotent mammary stem cells (MaSCs) has provided an explanation for the unique regenerative capacity of the mammary gland throughout adult life. However, it remains unclear what genes maintain MaSCs and control their specification into the two epithelial lineages: luminal and basal. LBH is a novel transcription co-factor in the WNT pathway with hitherto unknown physiological function. LBH is expressed during mammary gland development and aberrantly overexpressed in aggressive ‘basal’ subtype breast cancers. Here, we have explored the in vivo role of LBH in mammopoiesis. We show that in postnatal mammary epithelia, LBH is predominantly expressed in the Lin−CD29highCD24+ basal MaSC population. Upon conditional inactivation of LBH, mice exhibit pronounced delays in mammary tissue expansion during puberty and pregnancy, accompanied by increased luminal differentiation at the expense of basal lineage specification. These defects could be traced to a severe reduction in the frequency and self-renewal/differentiation potential of basal MaSCs. Mechanistically, LBH induces expression of key epithelial stem cell transcription factor ΔNp63 to promote a basal MaSC state and repress luminal differentiation genes, mainly that encoding estrogen receptor α (Esr1/ERα). Collectively, these studies identify LBH as an essential regulator of basal MaSC expansion/maintenance, raising important implications for its potential role in breast cancer pathogenesis. PMID:25655704

  12. Phosphorylation of Sox9 is required for neural crest delamination and is regulated downstream of BMP and canonical Wnt signaling.

    PubMed

    Liu, Jessica A J; Wu, Ming-Hoi; Yan, Carol H; Chau, Bolton K H; So, Henry; Ng, Alvis; Chan, Alan; Cheah, Kathryn S E; Briscoe, James; Cheung, Martin

    2013-02-19

    Coordination of neural crest cell (NCC) induction and delamination is orchestrated by several transcription factors. Among these, Sry-related HMG box-9 (Sox9) and Snail2 have been implicated in both the induction of NCC identity and, together with phoshorylation, NCC delamination. How phosphorylation effects this function has not been clear. Here we show, in the developing chick neural tube, that phosphorylation of Sox9 on S64 and S181 facilitates its SUMOylation, and the phosphorylated forms of Sox9 are essential for trunk neural crest delamination. Both phosphorylation and to a lesser extent SUMOylation, of Sox9 are required to cooperate with Snail2 to promote delamination. Moreover, bone morphogenetic protein and canonical Wnt signaling induce phosphorylation of Sox9, thereby connecting extracellular signals with the delamination of NCCs. Together the data suggest a model in which extracellular signals initiate phosphorylation of Sox9 and its cooperation with Snail2 to induce NCC delamination. PMID:23382206

  13. Phosphorylation of Sox9 is required for neural crest delamination and is regulated downstream of BMP and canonical Wnt signaling

    PubMed Central

    Liu, Jessica A. J.; Wu, Ming-Hoi; Yan, Carol H.; Chau, Bolton K. H.; So, Henry; Chan, Alan; Cheah, Kathryn S. E.; Briscoe, James; Cheung, Martin

    2013-01-01

    Coordination of neural crest cell (NCC) induction and delamination is orchestrated by several transcription factors. Among these, Sry-related HMG box-9 (Sox9) and Snail2 have been implicated in both the induction of NCC identity and, together with phoshorylation, NCC delamination. How phosphorylation effects this function has not been clear. Here we show, in the developing chick neural tube, that phosphorylation of Sox9 on S64 and S181 facilitates its SUMOylation, and the phosphorylated forms of Sox9 are essential for trunk neural crest delamination. Both phosphorylation and to a lesser extent SUMOylation, of Sox9 are required to cooperate with Snail2 to promote delamination. Moreover, bone morphogenetic protein and canonical Wnt signaling induce phosphorylation of Sox9, thereby connecting extracellular signals with the delamination of NCCs. Together the data suggest a model in which extracellular signals initiate phosphorylation of Sox9 and its cooperation with Snail2 to induce NCC delamination. PMID:23382206

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

    PubMed

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

    2008-02-01

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

  15. Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis.

    PubMed

    Sorkina, Tatiana; Caltagarone, John; Sorkin, Alexander

    2013-06-01

    Flotillins were proposed to mediate clathrin-independent endocytosis, and recently, flotillin-1 was implicated in the protein kinase C (PKC)-triggered endocytosis of the dopamine transporter (DAT). Since endocytosis of DAT was previously shown to be clathrin-mediated, we re-examined the role of clathrin coat proteins and flotillin in DAT endocytosis using DAT tagged with the hemagglutinin epitope (HA) in the extracellular loop and a quantitative HA antibody uptake assay. Depletion of flotillin-1, flotillin-2 or both flotillins together by small interfering RNAs (siRNAs) did not inhibit PKC-dependent internalization and degradation of HA-DAT. In contrast, siRNAs to clathrin heavy chain and μ2 subunit of clathrin adaptor complex AP-2 as well as a dynamin inhibitor Dyngo-4A significantly decreased PKC-dependent endocytosis of HA-DAT. Similarly, endocytosis and degradation of DAT that is not epitope-tagged were highly sensitive to the clathrin siRNAs and dynamin inhibition but were not affected by flotillin knockdown. Very little co-localization of DAT with flotillins was observed in cells ectopically expressing DAT and in cultured mouse dopaminergic neurons. Depletion of flotillins increased diffusion rates of HA-DAT in the plasma membrane, suggesting that flotillin-organized microdomains may regulate the lateral mobility of DAT. We propose that clathrin-mediated endocytosis is the major pathway of PKC-dependent internalization of DAT, and that flotillins may modulate functional association of DAT with plasma membrane rafts rather than mediate DAT endocytosis.

  16. Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa.

    PubMed Central

    Serino, L; Reimmann, C; Visca, P; Beyeler, M; Chiesa, V D; Haas, D

    1997-01-01

    The high-affinity siderophore salicylate is an intermediate in the biosynthetic pathway of pyochelin, another siderophore and chelator of transition metal ions, in Pseudomonas aeruginosa. The 2.5-kb region upstream of the salicylate biosynthetic genes pchBA was sequenced and found to contain two additional, contiguous genes, pchD and pchC, having the same orientation. The deduced amino acid sequence of the 60-kDa PchD protein was similar to those of the EntE protein (2,3-dihydroxybenzoate-AMP ligase) of Escherichia coli and other adenylate-forming enzymes, suggesting that salicylate might be adenylated at the carboxyl group by PchD. The 28-kDa PchC protein showed similarities to thioesterases of prokaryotic and eukaryotic origin and might participate in the release of the product(s) formed from activated salicylate. One potential product, dihydroaeruginoate (Dha), was identified in culture supernatants of iron-limited P. aeruginosa cells. The antifungal antibiotic Dha is thought to arise from the reaction of salicylate with cysteine, followed by cyclization of cysteine. Inactivation of the chromosomal pchD gene by insertion of the transcription and translation stop element omega Sm/Sp abolished the production of Dha and pyochelin, implying that PchD-mediated activation of salicylate may be a common first step in the synthesis of both metabolites. Furthermore, the pchD::omega Sm/Sp mutation had a strong polar effect on the expression of the pchBA genes, i.e., on salicylate synthesis, indicating that the pchDCBA genes constitute a transcriptional unit. A full-length pchDCBA transcript of ca. 4.4 kb could be detected in iron-deprived, growing cells of P. aeruginosa. Transcription of pchD started at tandemly arranged promoters, which overlapped with two Fur boxes (binding sites for the ferric uptake regulator) and the promoter of the divergently transcribed pchR gene encoding an activator of pyochelin biosynthesis. This promoter arrangement allows tight iron

  17. NK cell development requires Tsc1-dependent negative regulation of IL-15-triggered mTORC1 activation.

    PubMed

    Yang, Meixiang; Chen, Shasha; Du, Juan; He, Junming; Wang, Yuande; Li, Zehua; Liu, Guangao; Peng, Wanwen; Zeng, Xiaokang; Li, Dan; Xu, Panglian; Guo, Wei; Chang, Zai; Wang, Song; Tian, Zhigang; Dong, Zhongjun

    2016-01-01

    Activation of metabolic signalling by IL-15 is required for natural killer (NK) cell development. Here we show that Tsc1, a repressor of mTOR, is dispensable for the terminal maturation, survival and function of NK cells but is critical to restrict exhaustive proliferation of immature NK cells and activation downstream of IL-15 during NK cell development. Tsc1 is expressed in immature NK cells and is upregulated by IL-15. Haematopoietic-specific deletion of Tsc1 causes a marked decrease in the number of NK cells and compromises rejection of 'missing-self' haematopoietic tumours and allogeneic bone marrow. The residual Tsc1-null NK cells display activated, pro-apoptotic phenotype and elevated mTORC1 activity. Deletion of Raptor, a component of mTORC1, largely reverses these defects. Tsc1-deficient NK cells express increased levels of T-bet and downregulate Eomes and CD122, a subunit of IL-15 receptor. These results reveal a role for Tsc1-dependent inhibition of mTORC1 activation during immature NK cell development. PMID:27601261

  18. NK cell development requires Tsc1-dependent negative regulation of IL-15-triggered mTORC1 activation

    PubMed Central

    Yang, Meixiang; Chen, Shasha; Du, Juan; He, Junming; Wang, Yuande; Li, Zehua; Liu, Guangao; Peng, Wanwen; Zeng, Xiaokang; Li, Dan; Xu, Panglian; Guo, Wei; Chang, Zai; Wang, Song; Tian, Zhigang; Dong, Zhongjun

    2016-01-01

    Activation of metabolic signalling by IL-15 is required for natural killer (NK) cell development. Here we show that Tsc1, a repressor of mTOR, is dispensable for the terminal maturation, survival and function of NK cells but is critical to restrict exhaustive proliferation of immature NK cells and activation downstream of IL-15 during NK cell development. Tsc1 is expressed in immature NK cells and is upregulated by IL-15. Haematopoietic-specific deletion of Tsc1 causes a marked decrease in the number of NK cells and compromises rejection of ‘missing-self' haematopoietic tumours and allogeneic bone marrow. The residual Tsc1-null NK cells display activated, pro-apoptotic phenotype and elevated mTORC1 activity. Deletion of Raptor, a component of mTORC1, largely reverses these defects. Tsc1-deficient NK cells express increased levels of T-bet and downregulate Eomes and CD122, a subunit of IL-15 receptor. These results reveal a role for Tsc1-dependent inhibition of mTORC1 activation during immature NK cell development. PMID:27601261

  19. TNF-alpha/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species.

    PubMed

    Jin, Shi; Ray, Ramesh M; Johnson, Leonard R

    2008-04-01

    Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.

  20. Metabolically regulated endoplasmic reticulum-associated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase: evidence for requirement of a geranylgeranylated protein.

    PubMed

    Leichner, Gil S; Avner, Rachel; Harats, Dror; Roitelman, Joseph

    2011-09-16

    In mammalian cells, the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), which catalyzes the rate-limiting step in the mevalonate pathway, is ubiquitylated and degraded by the 26 S proteasome when mevalonate-derived metabolites accumulate, representing a case of metabolically regulated endoplasmic reticulum-associated degradation (ERAD). Here, we studied which mevalonate-derived metabolites signal for HMGR degradation and the ERAD step(s) in which these metabolites are required. In HMGR-deficient UT-2 cells that stably express HMGal, a chimeric protein between β-galactosidase and the membrane region of HMGR, which is necessary and sufficient for the regulated ERAD, we tested inhibitors specific to different steps in the mevalonate pathway. We found that metabolites downstream of farnesyl pyrophosphate but upstream to lanosterol were highly effective in initiating ubiquitylation, dislocation, and degradation of HMGal. Similar results were observed for endogenous HMGR in cells that express this protein. Ubiquitylation, dislocation, and proteasomal degradation of HMGal were severely hampered when production of geranylgeranyl pyrophosphate was inhibited. Importantly, inhibition of protein geranylgeranylation markedly attenuated ubiquitylation and dislocation, implicating for the first time a geranylgeranylated protein(s) in the metabolically regulated ERAD of HMGR.

  1. Regulation of Abiotic Stress Signalling by Arabidopsis C-Terminal Domain Phosphatase-Like 1 Requires Interaction with a K-Homology Domain-Containing Protein

    PubMed Central

    Jeong, In Sil; Fukudome, Akihito; Aksoy, Emre; Bang, Woo Young; Kim, Sewon; Guan, Qingmei; Bahk, Jeong Dong; May, Kimberly A.; Russell, William K.; Zhu, Jianhua; Koiwa, Hisashi

    2013-01-01

    Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD) PHOSPHATASE-LIKE 1 (CPL1) regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds) RNA binding motifs (dsRBMs) at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH) domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation. PMID:24303021

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    2016-08-31

    The Food and Drug Administration (FDA) is amending its regulations governing drug establishment registration and drug listing. These amendments reorganize, modify, and clarify current regulations concerning who must register establishments and list human drugs, human drugs that are also biological products, and animal drugs. The final rule requires electronic submission, unless waived in certain circumstances, of registration and listing information. This rulemaking pertains to finished drug products and to active pharmaceutical ingredients (APIs) alone or together with one or more other ingredients. The final rule describes how and when owners or operators of establishments at which drugs are manufactured or processed must register their establishments with FDA and list the drugs they manufacture or process. In addition, the rule makes certain changes to the National Drug Code (NDC) system. We are taking this action to improve management of drug establishment registration and drug listing requirements and make these processes more efficient and effective for industry and for us. This action also supports implementation of the electronic prescribing provisions of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (MMA) and the availability of current drug labeling information through DailyMed, a computerized repository of drug information maintained by the National Library of Medicine. PMID:27580511

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

    PubMed

    2016-08-31

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

  6. The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest.

    PubMed

    Gascoyne, Duncan M; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E; Croucher, Peter I; Banham, Alison H

    2015-01-01

    Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology.

  7. Actin Recruitment to the Chlamydia Inclusion Is Spatiotemporally Regulated by a Mechanism That Requires Host and Bacterial Factors

    PubMed Central

    Chin, Elizabeth; Kirker, Kelly; Zuck, Meghan; James, Garth; Hybiske, Kevin

    2012-01-01

    The ability to exit host cells at the end of their developmental growth is a critical step for the intracellular bacterium Chlamydia. One exit strategy, extrusion, is mediated by host signaling pathways involved with actin polymerization. Here, we show that actin is recruited to the chlamydial inclusion as a late event, occurring after 20 hours post-infection (hpi) and only within a subpopulation of cells. This event increases significantly in prevalence and extent from 20 to 68 hpi, and actin coats strongly correlated with extrusions. In contrast to what has been reported for other intracellular pathogens, actin nucleation on Chlamydia inclusions did not ‘flash’, but rather exhibited moderate depolymerization dynamics. By using small molecule agents to selectively disrupt host signaling pathways involved with actin nucleation, modulate actin polymerization dynamics and also to disable the synthesis and secretion of chlamydial proteins, we further show that host and bacterial proteins are required for actin coat formation. Transient disruption of either host or bacterial signaling pathways resulted in rapid loss of coats in all infected cells and a reduction in extrusion formation. Inhibition of Chlamydia type III secretion also resulted in rapid loss of actin association on inclusions, thus implicating chlamydial effector proteins(s) as being central factors for engaging with host actin nucleating factors, such as formins. In conclusion, our data illuminate the host and bacterial driven process by which a dense actin matrix is dynamically nucleated and maintained on the Chlamydia inclusion. This late stage event is not ubiquitous for all infected cells in a population, and escalates in prevalence and extent throughout the developmental cycle of Chlamydia, culminating with their exit from the host cell by extrusion. The initiation of actin recruitment by Chlamydia appears to be novel, and may serve as an upstream determinant of the extrusion mechanism. PMID

  8. GAS2 and GAS4, a Pair of Developmentally Regulated Genes Required for Spore Wall Assembly in Saccharomyces cerevisiae▿

    PubMed Central

    Ragni, Enrico; Coluccio, Alison; Rolli, Eleonora; Rodriguez-Peña, José Manuel; Colasante, Gaia; Arroyo, Javier; Neiman, Aaron M.; Popolo, Laura

    2007-01-01

    The GAS multigene family of Saccharomyces cerevisiae is composed of five paralogs (GAS1 to GAS5). GAS1 is the only one of these genes that has been characterized to date. It encodes a glycosylphosphatidylinositol-anchored protein functioning as a β(1,3)-glucan elongase and required for proper cell wall assembly during vegetative growth. In this study, we characterize the roles of the GAS2 and GAS4 genes. These genes are expressed exclusively during sporulation. Their mRNA levels showed a peak at 7 h from induction of sporulation and then decreased. Gas2 and Gas4 proteins were detected and reached maximum levels between 8 and 10 h from induction of sporulation, a time roughly coincident with spore wall assembly. The double null gas2 gas4 diploid mutant showed a severe reduction in the efficiency of sporulation, an increased permeability of the spores to exogenous substances, and production of inviable spores, whereas the single gas2 and gas4 null diploids were similar to the parental strain. An analysis of spore ultrastructure indicated that the loss of Gas2 and Gas4 proteins affected the proper attachment of the glucan to the chitosan layer, probably as a consequence of the lack of coherence of the glucan layer. The ectopic expression of GAS2 and GAS4 genes in a gas1 null mutant revealed that these proteins are redundant versions of Gas1p specialized to function in a compartment at a pH value close to neutral. PMID:17189486

  9. Full activation of mouse platelets requires ADP secretion regulated by SERCA3 ATPase-dependent calcium stores.

    PubMed

    Elaïb, Ziane; Adam, Frédéric; Berrou, Eliane; Bordet, Jean-Claude; Prévost, Nicolas; Bobe, Régis; Bryckaert, Marijke; Rosa, Jean-Philippe

    2016-08-25

    The role of the sarco-endoplasmic reticulum calcium (Ca(2+)) adenosine triphosphatase (ATPase) 3 (SERCA3) in platelet physiology remains poorly understood. Here, we show that SERCA3 knockout (SERCA3(-/-)) mice exhibit prolonged tail bleeding time and rebleeding. Thrombus formation was delayed both in arteries and venules in an in vivo ferric chloride-induced thrombosis model. Defective platelet adhesion and thrombus growth over collagen was confirmed in vitro. Adenosine 5'-diphosphate (ADP) removal by apyrase diminished adhesion and thrombus growth of control platelets to the level of SERCA3(-/-) platelets. Aggregation, dense granule secretion, and Ca(2+) mobilization of SERCA3(-/-) platelets induced by low collagen or low thrombin concentration were weaker than controls. Accordingly, SERCA3(-/-) platelets exhibited a partial defect in total stored Ca(2+) and in Ca(2+) store reuptake following thrombin stimulation. Importantly ADP, but not serotonin, rescued aggregation, secretion, and Ca(2+) mobilization in SERCA3(-/-) platelets, suggesting specificity. Dense granules appeared normal upon electron microscopy, mepacrine staining, and total serotonin content, ruling out a dense granule defect. ADP induced normal platelet aggregation, excluding a defect in ADP activation pathways. The SERCA3-specific inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone diminished both Ca(2+) mobilization and secretion of control platelets, as opposed to the SERCA2b inhibitor thapsigargin. This confirmed the specific role of catalytically active SERCA3 in ADP secretion. Accordingly, SERCA3-dependent Ca(2+) stores appeared depleted in SERCA3(-/-) platelets. Finally, αIIbβ3 integrin blockade did not affect SERCA3-dependent secretion, therefore proving independent of αIIbβ3 engagement. Altogether, these results show that SERCA3-dependent Ca(2+) stores control a specific ADP secretion pathway required for full platelet secretion induced by agonists at low concentration and independent

  10. Federal environmental and occupational toxicology regulations and reporting requirements: a practical approach to what the medical toxicologist needs to know, part 1.

    PubMed

    Schwartz, Michael D; Dell'Aglio, Damon M; Nickle, Richard; Hornsby-Myers, Jennifer

    2014-09-01

    Toxicologists are often called upon to assist in environmental, industrial, occupational and public health assessments. Accordingly, medical toxicologists may find it prudent to be aware of applicable federal toxicological regulations and reporting requirements and of the roles of relevant federal agencies. These regulations are numerous, complex, and have evolved and expanded over time, making it difficult for toxicologists to sustain a current knowledge base. This article reviews the pertinent federal toxicological reporting requirements with regard to the Toxic Substances Control Act (TSCA), the Atomic Energy Act (AEA), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the Resource Conservation and Recovery Act (RCRA), the Clean Air Act, the Clean Water Act, the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), the Emergency Planning and Community Right to Know Act (EPCRA), the Occupational Safety and Health Act, the Department of Transportation, and information about the National Response Center. We reference internet-based government resources and offer direct links to applicable websites in an attempt to offer rapid and current sources of practical information. The format of the article is a series of hypothetical scenarios followed by commentary. Discussions of the Safe Drinking Water Act, the Food, Drug, and Cosmetic Act, and the Dietary Supplement Health and Education Act are beyond the scope of this paper. For those desiring a more in-depth discussion of the relevant federal environmental laws and statutes and applicable case law, the reader is directed to resources such as the Environmental Law Handbook, the websites of individual laws found at www.epa.gov and the decisions of individual courts of appeal. It is our hope that this article provides not only useful practical information for the practicing toxicologist but also serves as a key reference for medical toxicology core content on environmental laws and

  11. Federal environmental and occupational toxicology regulations and reporting requirements: a practical approach to what the medical toxicologist needs to know, part 2.

    PubMed

    Schwartz, Michael D; Dell'Aglio, Damon M; Nickle, Richard; Hornsby-Myers, Jennifer

    2014-12-01

    Toxicologists are often called upon to assist in environmental, industrial, occupational and public health assessments. Accordingly, medical toxicologists may find it prudent to be aware of applicable federal toxicological regulations and reporting requirements and of the roles of relevant federal agencies. These regulations are numerous, complex, and have evolved and expanded over time, making it difficult for toxicologists to sustain a current knowledge base. This article reviews the pertinent federal toxicological reporting requirements with regards to the Toxic Substances Control Act (TSCA), the Atomic Energy Act (AEA), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the Resource Conservation and Recovery Act (RCRA), the Clean Air Act, the Clean Water Act, the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), the Emergency Planning and Community Right to Know Act (EPCRA), the Occupational Safety and Health Act, the Department of Transportation, and information about the National Response Center. We reference internet-based government resources and offer direct links to applicable websites in an attempt to offer rapid and current sources of practical information. The format of the article is a series of hypothetical scenarios followed by commentary. Discussions of the Safe Drinking Water Act and the Food, Drug, and Cosmetic Act and the Dietary Supplement Health and Education Act are beyond the scope of this paper. For those desiring a more in depth discussion of the relevant federal environmental laws and statutes, and applicable case law, the reader is directed to resources such as the Environmental Law Handbook, the websites of individual laws found at www.epa.gov and the decisions of individual courts of appeal. It is our hope that this article provides not only useful practical information for the practicing toxicologist, but also serves as a key reference for Medical Toxicology core content on environmental

  12. Federal environmental and occupational toxicology regulations and reporting requirements: a practical approach to what the medical toxicologist needs to know, part 2.

    PubMed

    Schwartz, Michael D; Dell'Aglio, Damon M; Nickle, Richard; Hornsby-Myers, Jennifer

    2014-12-01

    Toxicologists are often called upon to assist in environmental, industrial, occupational and public health assessments. Accordingly, medical toxicologists may find it prudent to be aware of applicable federal toxicological regulations and reporting requirements and of the roles of relevant federal agencies. These regulations are numerous, complex, and have evolved and expanded over time, making it difficult for toxicologists to sustain a current knowledge base. This article reviews the pertinent federal toxicological reporting requirements with regards to the Toxic Substances Control Act (TSCA), the Atomic Energy Act (AEA), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the Resource Conservation and Recovery Act (RCRA), the Clean Air Act, the Clean Water Act, the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), the Emergency Planning and Community Right to Know Act (EPCRA), the Occupational Safety and Health Act, the Department of Transportation, and information about the National Response Center. We reference internet-based government resources and offer direct links to applicable websites in an attempt to offer rapid and current sources of practical information. The format of the article is a series of hypothetical scenarios followed by commentary. Discussions of the Safe Drinking Water Act and the Food, Drug, and Cosmetic Act and the Dietary Supplement Health and Education Act are beyond the scope of this paper. For those desiring a more in depth discussion of the relevant federal environmental laws and statutes, and applicable case law, the reader is directed to resources such as the Environmental Law Handbook, the websites of individual laws found at www.epa.gov and the decisions of individual courts of appeal. It is our hope that this article provides not only useful practical information for the practicing toxicologist, but also serves as a key reference for Medical Toxicology core content on environmental

  13. Federal environmental and occupational toxicology regulations and reporting requirements: a practical approach to what the medical toxicologist needs to know, part 1.

    PubMed

    Schwartz, Michael D; Dell'Aglio, Damon M; Nickle, Richard; Hornsby-Myers, Jennifer

    2014-09-01

    Toxicologists are often called upon to assist in environmental, industrial, occupational and public health assessments. Accordingly, medical toxicologists may find it prudent to be aware of applicable federal toxicological regulations and reporting requirements and of the roles of relevant federal agencies. These regulations are numerous, complex, and have evolved and expanded over time, making it difficult for toxicologists to sustain a current knowledge base. This article reviews the pertinent federal toxicological reporting requirements with regard to the Toxic Substances Control Act (TSCA), the Atomic Energy Act (AEA), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the Resource Conservation and Recovery Act (RCRA), the Clean Air Act, the Clean Water Act, the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), the Emergency Planning and Community Right to Know Act (EPCRA), the Occupational Safety and Health Act, the Department of Transportation, and information about the National Response Center. We reference internet-based government resources and offer direct links to applicable websites in an attempt to offer rapid and current sources of practical information. The format of the article is a series of hypothetical scenarios followed by commentary. Discussions of the Safe Drinking Water Act, the Food, Drug, and Cosmetic Act, and the Dietary Supplement Health and Education Act are beyond the scope of this paper. For those desiring a more in-depth discussion of the relevant federal environmental laws and statutes and applicable case law, the reader is directed to resources such as the Environmental Law Handbook, the websites of individual laws found at www.epa.gov and the decisions of individual courts of appeal. It is our hope that this article provides not only useful practical information for the practicing toxicologist but also serves as a key reference for medical toxicology core content on environmental laws and

  14. A Non-Classical LysR-Type Transcriptional Regulator PA2206 Is Required for an Effective Oxidative Stress Response in Pseudomonas aeruginosa

    PubMed Central

    Mooij, Marlies J.; O'Gara, Fergal

    2013-01-01

    LysR-type transcriptional regulators (LTTRs) are emerging as key circuit components in regulating microbial stress responses and are implicated in modulating oxidative stress in the human opportunistic pathogen Pseudomonas aeruginosa. The oxidative stress response encapsulates several strategies to overcome the deleterious effects of reactive oxygen species. However, many of the regulatory components and associated molecular mechanisms underpinning this key adaptive response remain to be characterised. Comparative analysis of publically available transcriptomic datasets led to the identification of a novel LTTR, PA2206, whose expression was altered in response to a range of host signals in addition to oxidative stress. PA2206 was found to be required for tolerance to H2O2 in vitro and lethality in vivo in the Zebrafish embryo model of infection. Transcriptomic analysis in the presence of H2O2 showed that PA2206 altered the expression of 58 genes, including a large repertoire of oxidative stress and iron responsive genes, independent of the master regulator of oxidative stress, OxyR. Contrary to the classic mechanism of LysR regulation, PA2206 did not autoregulate its own expression and did not influence expression of adjacent or divergently transcribed genes. The PA2214-15 operon was identified as a direct target of PA2206 with truncated promoter fragments revealing binding to the 5′-ATTGCCTGGGGTTAT-3′ LysR box adjacent to the predicted −35 region. PA2206 also interacted with the pvdS promoter suggesting a global dimension to the PA2206 regulon, and suggests PA2206 is an important regulatory component of P. aeruginosa adaptation during oxidative stress. PMID:23382903

  15. Convergence of multiple signaling pathways is required to coordinately up-regulate mtDNA and mitochondrial biogenesis during T cell activation

    PubMed Central

    D’Souza, Anthony D.; Parikh, Neal; Kaech, Susan M.; Shadel, Gerald S.

    2009-01-01

    The quantity and activity of mitochondria vary dramatically in tissues and are modulated in response to changing cellular energy demands and environmental factors. The amount of mitochondrial DNA (mtDNA), which encodes essential subunits of the oxidative phosphorylation complexes required for cellular ATP production, is also tightly regulated, but by largely unknown mechanisms. Using murine T cells as a model system, we have addressed how specific signaling pathways influence mitochondrial biogenesis and mtDNA levels. T cell receptor (TCR) activation results in a large increase in mitochondrial mass and membrane potential and a corresponding increase of mtDNA copy number, indicating the vital role for mitochondrial function for the growth and proliferation of these cells. Independent activation of protein kinase C (via PMA) or calcium-related pathways (via ionomycin) had differential and sub-maximal effects on these mitochondrial parameters, as did activation of naïve T cells with proliferative cytokines. Thus, the robust mitochondrial biogenesis response observed upon TCR activation requires synergy of multiple downstream signaling pathways. One such pathway involves AMP-activated protein kinase (AMPK), which we show has an unprecedented role in negatively regulating mitochondrial biogenesis that is mammalian target of rapamycin (mTOR)-dependent. That is, inhibition of AMPK after TCR signaling commences results in excessive, but uncoordinated mitochondrial proliferation. We propose that mitochondrial biogenesis is not under control of a master regulatory circuit, but rather requires the convergence of multiple signaling pathways with distinct downstream consequences on the organelle’s structure, composition, and function. PMID:17890163

  16. Splicing of Receptor-Like Kinase-Encoding SNC4 and CERK1 is Regulated by Two Conserved Splicing Factors that Are Required for Plant Immunity

    PubMed Central

    Zhang, Zhibin; Liu, Yanan; Ding, Pingtao; Li, Yan; Kong, Qing; Zhang, Yuelin

    2014-01-01

    Plant immune receptors belonging to the receptor-like kinase (RLK) family play important roles in the recognition of microbial pathogens and activation of downstream defense responses. The Arabidopsis mutant snc4-1D contains a gain-of-function mutation in the RLK SNC4 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE4), which leads to constitutive activation of defense responses. Analysis of suppressor mutants of snc4-1D identified two conserved splicing factors, SUA (SUPPRESSOR OF ABI3-5) and RSN2 (REQUIRED FOR SNC4-1D 2), that are required for the constitutive defense responses in snc4-1D. In sua and rsn2 mutants, SNC4 splicing is altered and the amount of SNC4 transcripts is reduced. Further analysis showed that SUA and RSN2 are also required for the proper splicing of CERK1 (CHITIN ELICITOR RECEPTOR KINASE1), which encodes another RLK that functions as a receptor for chitin. In sua and rsn2 mutants, induction of reactive oxygen species by chitin is reduced and the non-pathogenic bacteria Pseudomonas syringae pv. tomato DC3000hrcC grows to higher titers than in wild-type plants. Our study suggests that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1. PMID:25267732

  17. Splicing of receptor-like kinase-encoding SNC4 and CERK1 is regulated by two conserved splicing factors that are required for plant immunity.

    PubMed

    Zhang, Zhibin; Liu, Yanan; Ding, Pingtao; Li, Yan; Kong, Qing; Zhang, Yuelin

    2014-12-01

    Plant immune receptors belonging to the receptor-like kinase (RLK) family play important roles in the recognition of microbial pathogens and activation of downstream defense responses. The Arabidopsis mutant snc4-1D contains a gain-of-function mutation in the RLK SNC4 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE4), which leads to constitutive activation of defense responses. Analysis of suppressor mutants of snc4-1D identified two conserved splicing factors, SUA (SUPPRESSOR OF ABI3-5) and RSN2 (REQUIRED FOR SNC4-1D 2), that are required for the constitutive defense responses in snc4-1D. In sua and rsn2 mutants, SNC4 splicing is altered and the amount of SNC4 transcripts is reduced. Further analysis showed that SUA and RSN2 are also required for the proper splicing of CERK1 (CHITIN ELICITOR RECEPTOR KINASE1), which encodes another RLK that functions as a receptor for chitin. In sua and rsn2 mutants, induction of reactive oxygen species by chitin is reduced and the non-pathogenic bacteria Pseudomonas syringae pv. tomato DC3000hrcC grows to higher titers than in wild-type plants. Our study suggests that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1. PMID:25267732

  18. Proper regulation of Cdc42 activity is required for tight actin concentration at the equator during cytokinesis in adherent mammalian cells.

    PubMed

    Zhu, Xiaodong; Wang, Junxia; Moriguchi, Kazuki; Liow, Lu Ting; Ahmed, Sohail; Kaverina, Irina; Murata-Hori, Maki

    2011-10-01

    Cytokinesis in mammalian cells requires actin assembly at the equatorial region. Although functions of RhoA in this process have been well established, additional mechanisms are likely involved. We have examined if Cdc42 is involved in actin assembly during cytokinesis. Depletion of Cdc42 had no apparent effects on the duration of cytokinesis, while overexpression of constitutively active Cdc42 (CACdc42) caused cytokinesis failure in normal rat kidney epithelial cells. Cells depleted of Cdc42 displayed abnormal cell morphology and caused a failure of tight accumulation of actin and RhoA at the equator. In contrast, in cells overexpressing CACdc42, actin formed abnormal bundles and RhoA was largely eliminated from the equator. Our results suggest that accurate regulation of Cdc42 activity is crucial for proper equatorial actin assembly and RhoA localization during cytokinesis. Notably, our observations also suggest that tight actin concentration is not essential for cytokinesis in adherent mammalian cells.

  19. [Protection against electromagnetic fields 0-300 GHz in Poland. New regulations and perspectives if their harmonization wit the European Union requirements].

    PubMed

    Trzaska, Hubert

    2003-01-01

    A critical review of protection regulations (or their proposals) on work safety regarding electromagnetic fields as well as the general public protection against unwanted exposure to the fields are presented in this paper. The standards of the Western World are illogical in both aspects, biomedical knowledge and technical. The results (permissible exposures) are defined more precisely than the input data. In Poland, two independent acts are in force: one on occupational exposures and the other on non-occupational exposures. The former is "a world-better", but requires to be immediately changed to a standard that is applicable and friendly to its users. The latter can be accepted after introducing several amendments. The enlargement of the environmental standard to include occupational issues may create a good Polish proposal also useful at the international level.

  20. The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence

    PubMed Central

    Gottig, Natalia; Ottado, Jorgelina

    2015-01-01

    Xanthomonas citri subsp. citri colonizes its hosts through the trafficking of effector proteins to the plant cell by the type III protein secretion system. In X. citri subsp. citri, as in other plant pathogens, the hrp cluster encodes the type III protein secretion system and is regulated by the transcription factors HrpG and HrpX. HrpG belongs to the OmpR family’s response regulator of EnvZ/OmpR two-component signal transduction system. Here, we show that the arginine 210 residue is crucial for the transcriptional activity of HrpG revealed by the absence of disease in host plants and hypersensitive response in non-host plants when a strain carrying this point mutation is used in plant infiltration assays. Also, this strain showed decreased expression levels of hrp genes in bacteria grown in culture or when they were recovered from citrus leaves. Moreover, we show for the first time that HrpG binds to both hrpX and its own promoter, and the change of the arginine 210 by a cysteine does not prevent the binding to both promoters. Nevertheless, in vitro hrpX transcription was observed only with HrpG whereas no transcription was detected with the R210C mutant. HrpG was able to interact with itself as well as with the mutant R210C suggesting that it functions as a dimer. The mutant protein R210C showed altered protease sensitivity, suggesting that Arg210 is essential for protein active conformation and thus for transcriptional activity. Our results indicate that arginine 210 in HrpG, as it may occur with this conserved residue in other members of this family of response regulators, is not required for DNA binding whereas is essential for hrp genes transcription and therefore for pathogenicity and HR induction. PMID:25961560

  1. The J-protein AtDjB1 is required for mitochondrial complex I activity and regulates growth and development through ROS-mediated auxin signalling.

    PubMed

    Jia, Ning; Lv, Ting-Ting; Li, Mi-Xin; Wei, Shan-Shan; Li, Yan-Yi; Zhao, Chun-Lan; Li, Bing

    2016-05-01

    AtDjB1 is a mitochondria-located J-protein in Arabidopsis thaliana It is involved in the regulation of plant growth and development; however, the exact mechanisms remain to be determined. We performed comparison analyses of phenotypes, auxin signalling, redox status, mitochondrial structure and function using wild-type plants, AtDjB1 mutants, rescued AtDjB1 mutants by AtDjB1 or YUCCA2 (an auxin synthesis gene), and AtDjB1 overexpression plants. AtDjB1 mutants (atj1-1 or atj1-4) exhibited inhibition of growth and development and reductions in the level of IAA and the expression of YUCCA genes compared to wild-type plants. The introduction of AtDjB1 or YUCCA2 into atj1-1 largely rescued phenotypic defects and the IAA level, indicating that AtDjB1 probably regulates growth and development via auxin. Furthermore, atj1-1 plants displayed a significant reduction in amount/activity of mitochondrial complex I compared to wild-type plants; this resulted in the accumulation of reactive oxygen species (ROS). Moreover, exogenous H2O2 markedly inhibited the expression of YUCCA genes in wild-type plants. In contrast, the reducing agent ascorbate increased the expression of YUCCA genes and IAA level in atj1-1 plants, indicating that the low auxin level observed in atj1-1 was probably due to the high oxidation status. Overall, the data presented here suggest that AtDjB1 is required for mitochondrial complex I activity and regulates growth and development through ROS-mediated auxin signalling in Arabidopsis.

  2. The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence.

    PubMed

    Ficarra, Florencia A; Garofalo, Cecilia G; Gottig, Natalia; Ottado, Jorgelina

    2015-01-01

    Xanthomonas citri subsp. citri colonizes its hosts through the trafficking of effector proteins to the plant cell by the type III protein secretion system. In X. citri subsp. citri, as in other plant pathogens, the hrp cluster encodes the type III protein secretion system and is regulated by the transcription factors HrpG and HrpX. HrpG belongs to the OmpR family's response regulator of EnvZ/OmpR two-component signal transduction system. Here, we show that the arginine 210 residue is crucial for the transcriptional activity of HrpG revealed by the absence of disease in host plants and hypersensitive response in non-host plants when a strain carrying this point mutation is used in plant infiltration assays. Also, this strain showed decreased expression levels of hrp genes in bacteria grown in culture or when they were recovered from citrus leaves. Moreover, we show for the first time that HrpG binds to both hrpX and its own promoter, and the change of the arginine 210 by a cysteine does not prevent the binding to both promoters. Nevertheless, in vitro hrpX transcription was observed only with HrpG whereas no transcription was detected with the R210C mutant. HrpG was able to interact with itself as well as with the mutant R210C suggesting that it functions as a dimer. The mutant protein R210C showed altered protease sensitivity, suggesting that Arg210 is essential for protein active conformation and thus for transcriptional activity. Our results indicate that arginine 210 in HrpG, as it may occur with this conserved residue in other members of this family of response regulators, is not required for DNA binding whereas is essential for hrp genes transcription and therefore for pathogenicity and HR induction.

  3. The J-protein AtDjB1 is required for mitochondrial complex I activity and regulates growth and development through ROS-mediated auxin signalling.

    PubMed

    Jia, Ning; Lv, Ting-Ting; Li, Mi-Xin; Wei, Shan-Shan; Li, Yan-Yi; Zhao, Chun-Lan; Li, Bing

    2016-05-01

    AtDjB1 is a mitochondria-located J-protein in Arabidopsis thaliana It is involved in the regulation of plant growth and development; however, the exact mechanisms remain to be determined. We performed comparison analyses of phenotypes, auxin signalling, redox status, mitochondrial structure and function using wild-type plants, AtDjB1 mutants, rescued AtDjB1 mutants by AtDjB1 or YUCCA2 (an auxin synthesis gene), and AtDjB1 overexpression plants. AtDjB1 mutants (atj1-1 or atj1-4) exhibited inhibition of growth and development and reductions in the level of IAA and the expression of YUCCA genes compared to wild-type plants. The introduction of AtDjB1 or YUCCA2 into atj1-1 largely rescued phenotypic defects and the IAA level, indicating that AtDjB1 probably regulates growth and development via auxin. Furthermore, atj1-1 plants displayed a significant reduction in amount/activity of mitochondrial complex I compared to wild-type plants; this resulted in the accumulation of reactive oxygen species (ROS). Moreover, exogenous H2O2 markedly inhibited the expression of YUCCA genes in wild-type plants. In contrast, the reducing agent ascorbate increased the expression of YUCCA genes and IAA level in atj1-1 plants, indicating that the low auxin level observed in atj1-1 was probably due to the high oxidation status. Overall, the data presented here suggest that AtDjB1 is required for mitochondrial complex I activity and regulates growth and development through ROS-mediated auxin signalling in Arabidopsis. PMID:27117341

  4. Proper Actin Ring Formation and Septum Constriction Requires Coordinated Regulation of SIN and MOR Pathways through the Germinal Centre Kinase MST-1

    PubMed Central

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

    2014-01-01

    Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in Δmst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1-dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes. PMID:24762679

  5. The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence.

    PubMed

    Ficarra, Florencia A; Garofalo, Cecilia G; Gottig, Natalia; Ottado, Jorgelina

    2015-01-01

    Xanthomonas citri subsp. citri colonizes its hosts through the trafficking of effector proteins to the plant cell by the type III protein secretion system. In X. citri subsp. citri, as in other plant pathogens, the hrp cluster encodes the type III protein secretion system and is regulated by the transcription factors HrpG and HrpX. HrpG belongs to the OmpR family's response regulator of EnvZ/OmpR two-component signal transduction system. Here, we show that the arginine 210 residue is crucial for the transcriptional activity of HrpG revealed by the absence of disease in host plants and hypersensitive response in non-host plants when a strain carrying this point mutation is used in plant infiltration assays. Also, this strain showed decreased expression levels of hrp genes in bacteria grown in culture or when they were recovered from citrus leaves. Moreover, we show for the first time that HrpG binds to both hrpX and its own promoter, and the change of the arginine 210 by a cysteine does not prevent the binding to both promoters. Nevertheless, in vitro hrpX transcription was observed only with HrpG whereas no transcription was detected with the R210C mutant. HrpG was able to interact with itself as well as with the mutant R210C suggesting that it functions as a dimer. The mutant protein R210C showed altered protease sensitivity, suggesting that Arg210 is essential for protein active conformation and thus for transcriptional activity. Our results indicate that arginine 210 in HrpG, as it may occur with this conserved residue in other members of this family of response regulators, is not required for DNA binding whereas is essential for hrp genes transcription and therefore for pathogenicity and HR induction. PMID:25961560

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

    PubMed

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

    2016-04-01

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

  7. Inside-out Regulation of Ectodomain Cleavage of Cluster-of-Differentiation-44 (CD44) and of Neuregulin-1 Requires Substrate Dimerization*♦

    PubMed Central

    Hartmann, Monika; Parra, Liseth M.; Ruschel, Anne; Lindner, Christina; Morrison, Helen; Herrlich, Andreas; Herrlich, Peter

    2015-01-01

    Ectodomain shedding of transmembrane precursor proteins generates numerous life-essential molecules, such as epidermal growth factor receptor ligands. This cleavage not only releases the regulatory growth factor, but it is also the required first step for the subsequent processing by γ-secretase and the release of gene regulatory intracellular fragments. Signaling within the cell modifies the cytoplasmic tails of substrates, a step important in starting the specific and regulated cleavage of a large number of studied substrates. Ectodomain cleavage occurs, however, on the outside of the plasma membrane and is carried out by membrane-bound metalloproteases. How the intracellular domain modification communicates with the ectodomain of the substrate to allow for cleavage to occur is unknown. Here, we show that homodimerization of a cluster-of-differentiation-44 or of pro-neuregulin-1 monomers represents an essential pre-condition for their regulated ectodomain cleavage. Both substrates are associated with their respective metalloproteases under both basal or cleavage-stimulated conditions. These interactions only turn productive by specific intracellular signal-induced intracellular domain modifications of the substrates, which in turn regulate metalloprotease access to the substrates' ectodomain and cleavage. We propose that substrate intracellular domain modification induces a relative rotation or other positional change of the dimerization partners that allow metalloprotease cleavage in the extracellular space. Our findings fill an important gap in understanding substrate-specific inside-out signal transfer along cleaved transmembrane proteins and suggest that substrate dimerization (homo- or possibly heterodimerization) might represent a general principle in ectodomain shedding. PMID:25925953

  8. Federal Register Volume 61 No. 94, Tuesday, May 14, 1996 rules and regulations. Part 2. 40 CFR part 141. National primary drinking water regulations: Monitoring requirements for public drinking water supplies; final rule

    SciTech Connect

    1996-05-14

    Today the Environmental Protection Agency (EPA) is promulgating an information collection rule (ICR) which establishes monitoring and data reporting requirements for large public water systems (PWSs). This rule is intended to provide EPA with information on the occurrence in drinking water of (1) chemical byproducts that form when disinfectants used for microbial control react with chemicals already present in source water (disinfection byproducts (DBPs)) and (2) disease-causing microorganisms (pathogens), including Cryptosporidium. Also, EPA will collect engineering data on how PWSs currently control such contaminants. EPA will use information generated by this rule, along with concurrent research, to determine whether revisions need to be made to EPA`s current drinking water filtration and disinfection rule and to determine the need for new regulations for disinfectants and DBPs.

  9. Glioma invasion mediated by the p75 neurotrophin receptor (p75NTR/CD271) requires regulated interaction with PDLIM1

    PubMed Central

    Ahn, B Y; Saldanha-Gama, R F G; Rahn, J J; Hao, X; Zhang, J; Dang, N-H; Alshehri, M; Robbins, S M; Senger, D L

    2016-01-01

    The invasive nature of glioblastoma renders them incurable by current therapeutic interventions. Using a novel invasive human glioma model, we previously identified the neurotrophin receptor p75NTR (aka CD271) as a mediator of glioma invasion. Herein, we provide evidence that preventing phosphorylation of p75NTR on S303 by pharmacological inhibition of PKA, or by a mutational strategy (S303G), cripples p75NTR-mediated glioma invasion resulting in serine phosphorylation within the C-terminal PDZ-binding motif (SPV) of p75NTR. Consistent with this, deletion (ΔSPV) or mutation (SPM) of the PDZ motif results in abrogation of p75NTR-mediated invasion. Using a peptide-based strategy, we identified PDLIM1 as a novel signaling adaptor for p75NTR and provide the first evidence for a regulated interaction via S425 phosphorylation. Importantly, PDLIM1 was shown to interact with p75NTR in highly invasive patient-derived glioma stem cells/tumor-initiating cells and shRNA knockdown of PDLIM1 in vitro and in vivo results in complete ablation of p75NTR-mediated invasion. Collectively, these data demonstrate a requirement for a regulated interaction of p75NTR with PDLIM1 and suggest that targeting either the PDZ domain interactions and/or the phosphorylation of p75NTR by PKA could provide therapeutic strategies for patients with glioblastoma. PMID:26119933

  10. Post-prandial regulation of hepatic glucokinase and lipogenesis requires the activation of TORC1 signalling in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Dai, Weiwei; Panserat, Stéphane; Mennigen, Jan A; Terrier, Frédéric; Dias, Karine; Seiliez, Iban; Skiba-Cassy, Sandrine

    2013-12-01

    To assess the potential involvement of TORC1 (target of rapamycin complex 1) signalling in the regulation of post-prandial hepatic lipid and glucose metabolism-related gene expression in trout, we employed intraperitoneal administration of rapamycin to achieve an acute inhibition of the TOR pathway. Our results reveal that rapamycin inhibits the phosphorylation of TORC1 and its downstream effectors (S6K1, S6 and 4E-BP1), without affecting Akt and the Akt substrates Forkhead-box Class O1 (FoxO1) and glycogen synthase kinase 3α/β (GSK 3α/β). These results indicate that acute administration of rapamycin in trout leads to the inhibition of TORC1 activation. No effect is observed on the expression of genes involved in gluconeogenesis, glycolysis and fatty acid oxidation, but hepatic TORC1 inhibition results in decreased sterol regulatory element binding protein 1c (SREBP1c) gene expression and suppressed fatty acid synthase (FAS) and glucokinase (GK) at gene expression and activity levels, indicating that FAS and GK activity is controlled at a transcriptional level in a TORC1-dependent manner. This study demonstrates for the first time in fish that post-prandial regulation of hepatic lipogenesis and glucokinase in rainbow trout requires the activation of TORC1 signalling. PMID:24031053

  11. Requirement of miR-9-dependent regulation of Myocd in PASMCs phenotypic modulation and proliferation induced by hepatopulmonary syndrome rat serum

    PubMed Central

    Xu, Duo; Gu, Jian-teng; Yi, Bin; Chen, Lin; Wang, Guan-song; Qian, Gui-sheng; Lu, Kai-zhi

    2015-01-01

    Hepatopulmonary syndrome (HPS) is characterized by a triad of severe liver disease, intrapulmonary vascular dilation and hypoxaemia. Pulmonary vascular remodelling (PVR) is a key feature of HPS pathology. Our previous studies have established the role of the pulmonary artery smooth muscle cell (PASMC) phenotypic modulation and proliferation in HPS-associated PVR. Myocardin, a robust transcriptional coactivator of serum response factor, plays a critical role in the vascular smooth muscle cell phenotypic switch. However, the mechanism regulating myocardin upstream signalling remains unclear. In this study, treatment of rat PASMCs with serum drawn from common bile duct ligation rats, which model symptoms of HPS, resulted in a significant increase in miR-9 expression correlated with a decrease in expression of myocardin and the phenotypic markers SM-α-actin and smooth muscle-specific myosin heavy chain (SM-MHC). Furthermore, miRNA functional analysis and luciferase reporter assay demonstrated that miR-9 effectively regulated myocardin expression by directly binding to its 3′-untranslated region. Both the knockdown of miR-9 and overexpression of myocardin effectively attenuated the HPS rat serum-induced phenotype switch and proliferation of PASMCs. Taken together, the findings of our present study demonstrate that miR-9 is required in HPS rat serum-induced phenotypic modulation and proliferation of PASMCs for targeting of myocardin and that miR-9 may serve as a potential therapeutic target in HPS. PMID:26147104

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

    SciTech Connect

    Branda, Steven S.; Losick, Richard; Kolter, Roberto; Kearns, Daniel B.; Chu, Frances

    2005-08-01

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

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

    PubMed

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

    2016-06-21

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

  14. crooked legs encodes a family of zinc finger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis.

    PubMed

    D'Avino, P P; Thummel, C S

    1998-05-01

    Drosophila imaginal discs undergo extensive pattern formation during larval development, resulting in each cell acquiring a specific adult fate. The final manifestation of this pattern into adult structures is dependent on pulses of the steroid hormone ecdysone during metamorphosis, which trigger disc eversion, elongation and differentiation. We have defined genetic criteria that allow us to screen for ecdysone-inducible regulatory genes that are required for this transformation from patterned disc to adult structure. We describe here the first genetic locus isolated using these criteria: crooked legs (crol). crol mutants die during pupal development with defects in adult head eversion and leg morphogenesis. The crol gene is induced by ecdysone during the onset of metamorphosis and encodes at least three protein isoforms that contain 12-18 C2H2 zinc fingers. Consistent with this sequence motif, crol mutations have stage-specific effects on ecdysone-regulated gene expression. The EcR ecdysone receptor, and the BR-C, E74 and E75 early regulatory genes, are submaximally induced in crol mutants in response to the prepupal ecdysone pulse. These changes in gene activity are consistent with the crol lethal phenotypes and provide a basis for understanding the molecular mechanisms of crol action. The genetic criteria described here provide a new direction for identifying regulators of adult tissue development during insect metamorphosis. PMID:9521911

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

    PubMed

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

    2016-01-01

    Cyclins play a central role in cell-cycle regulation; in mammals, the D family of cyclins consists of cyclin D1, D2, and D3. In Xenopus, only homologs of cyclins D1 and D2 have been reported, while a novel cyclin, cyclin Dx (ccndx), was found to be required for the maintenance of motor neuron progenitors during embryogenesis. It remains unknown whether zebrafish possess cyclin D3 or cyclin Dx. In this study, we identified a zebrafish ccndx gene encoding a protein which can form a complex with Cdk4. Through whole-mount in situ hybridization, we observed that zccndx mRNA is expressed in the motor neurons of hindbrain and spinal cord during development. Analysis of a 4-kb promoter sequence of the zccndx gene revealed the presence of HRE sites, which can be regulated by HIF2α. Morpholino knockdown of zebrafish Hif2α and cyclin Dx resulted in the abolishment of isl1 and oligo2 expression in the precursors of motor neurons, and also disrupted axon growth. Overexpression of cyclin Dx mRNA in Hif2α morphants partially rescued zccndx expression. Taken together, our data indicate that zebrafish cyclin Dx plays a role in maintaining the precursors of motor neurons. PMID:27323909

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

    PubMed

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

    2006-03-01

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

  17. Differential regulation of transcription: repression by unactivated mitogen-activated protein kinase Kss1 requires the Dig1 and Dig2 proteins.

    PubMed

    Bardwell, L; Cook, J G; Zhu-Shimoni, J X; Voora, D; Thorner, J

    1998-12-22

    Kss1, a yeast mitogen-activated protein kinase (MAPK), in its unphosphorylated (unactivated) state binds directly to and represses Ste12, a transcription factor necessary for expression of genes whose promoters contain filamentous response elements (FREs) and genes whose promoters contain pheromone response elements (PREs). Herein we show that two nuclear proteins, Dig1 and Dig2, are required cofactors in Kss1-imposed repression. Dig1 and Dig2 cooperate with Kss1 to repress Ste12 action at FREs and regulate invasive growth in a naturally invasive strain. Kss1-imposed Dig-dependent repression of Ste12 also occurs at PREs. However, maintenance of repression at PREs is more dependent on Dig1 and/or Dig2 and less dependent on Kss1 than repression at FREs. In addition, derepression at PREs is more dependent on MAPK-mediated phosphorylation than is derepression at FREs. Differential utilization of two types of MAPK-mediated regulation (binding-imposed repression and phosphorylation-dependent activation), in combination with distinct Ste12-containing complexes, contributes to the mechanisms by which separate extracellular stimuli that use the same MAPK cascade can elicit two different transcriptional responses. PMID:9860980

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

    PubMed Central

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

    2016-01-01

    The RecQ helicase Sgs1 plays critical roles during DNA repair by homologous recombination, from end resection to Holliday junction (HJ) dissolution. Sgs1 has both pro- and anti-recombinogenic roles, and therefore its activity must be tightly regulated. However, the controls involved in recruitment and activation of Sgs1 at damaged sites are unknown. Here we show a two-step role for Smc5/6 in recruiting and activating Sgs1 through SUMOylation. First, auto-SUMOylation of Smc5/6 subunits leads to recruitment of Sgs1 as part of the STR (Sgs1–Top3–Rmi1) complex, mediated by two SUMO-interacting motifs (SIMs) on Sgs1 that specifically recognize SUMOylated Smc5/6. Second, Smc5/6-dependent SUMOylation of Sgs1 and Top3 is required for the efficient function of STR. Sgs1 mutants impaired in recognition of SUMOylated Smc5/6 (sgs1-SIMΔ) or SUMO-dead alleles (sgs1-KR) exhibit unprocessed HJs at damaged replication forks, increased crossover frequencies during double-strand break repair, and severe impairment in DNA end resection. Smc5/6 is a key regulator of Sgs1's recombination functions. PMID:27298337

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

    PubMed Central

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

    2016-01-01

    Cyclins play a central role in cell-cycle regulation; in mammals, the D family of cyclins consists of cyclin D1, D2, and D3. In Xenopus, only homologs of cyclins D1 and D2 have been reported, while a novel cyclin, cyclin Dx (ccndx), was found to be required for the maintenance of motor neuron progenitors during embryogenesis. It remains unknown whether zebrafish possess cyclin D3 or cyclin Dx. In this study, we identified a zebrafish ccndx gene encoding a protein which can form a complex with Cdk4. Through whole-mount in situ hybridization, we observed that zccndx mRNA is expressed in the motor neurons of hindbrain and spinal cord during development. Analysis of a 4-kb promoter sequence of the zccndx gene revealed the presence of HRE sites, which can be regulated by HIF2α. Morpholino knockdown of zebrafish Hif2α and cyclin Dx resulted in the abolishment of isl1 and oligo2 expression in the precursors of motor neurons, and also disrupted axon growth. Overexpression of cyclin Dx mRNA in Hif2α morphants partially rescued zccndx expression. Taken together, our data indicate that zebrafish cyclin Dx plays a role in maintaining the precursors of motor neurons. PMID:27323909

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

    PubMed Central

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

    2016-01-01

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

  1. Regulation of the Osem gene by abscisic acid and the transcriptional activator VP1: analysis of cis-acting promoter elements required for regulation by abscisic acid and VP1.

    PubMed

    Hattori, T; Terada, T; Hamasuna, S

    1995-06-01

    Osem, a rice gene homologous to the wheat Em gene, which encodes one of the late-embryogenesis abundant proteins was isolated. The gene was characterized with respect to control of transcription by abscisic acid (ABA) and the transcriptional activator VP1, which is involved in the ABA-regulated gene expression during late embryo-genesis. A fusion gene (Osem-GUS) consisting of the Osem promoter and the bacterial beta-glucuronidase (GUS) gene was constructed and tested in a transient expression system, using protoplasts derived from a suspension-cultured line of rice cells, for activation by ABA and by co-transfection with an expression vector (35S-Osvp1) for the rice VP1 (OSVP1) cDNA. The expression of Osem-GUS was strongly (40- to 150-fold) activated by externally applied ABA and by over-expression of (OS)VP1. The Osem promoter has three ACGTG-containing sequences, motif A, motif B and motif A', which resemble the abscisic acid-responsive element (ABRE) that was previously identified in the wheat Em and the rice Rab16. There is also a CATGCATG sequence, which is known as the Sph box and is shown to be essential for the regulation by VP1 of the maize anthocyanin regulatory gene C1. Focusing on these sequence elements, various mutant derivatives of the Osem promoter in the transient expression system were assayed. The analysis revealed that motif A functions not only as an ABRE but also as a sequence element required for the regulation by (OS)VP1.

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

    PubMed

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

    2004-09-01

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

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

    PubMed

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

    2011-06-01

    Thiamine is a key primary metabolite which is necessary for the viability of all organisms. It is a dietary requirement for mammals because only prokaryotes, fungi and plants are thiamine prototrophs. In contrast to the well documented biosynthetic mechanism in bacteria, much remains to be deciphered in plants. In this work, a tomato thiamine-auxotrophic (thiamineless, tl) mutant was characterized. The tl mutant occurs due to inactivation of LeTHIC transcription as a result of insertion of a large unknown DNA fragment in its 5'-untranslated region. Expression of wild-type LeTHIC in tl plants was able to complement the mutant to wild type. LeTHIC possessed the same function as E.cTHIC [an Escherichia coli 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase involved in synthesis of the pyrimidine moiety of thiamine] because expression of LeTHIC rescued THIC-deficient strains of E. coli under culture conditions without thiamine supplementation, suggesting that plants employ a bacteria-like route of pyrimidine moiety synthesis. LeTHIC is an Fe-S cluster protein localized in chloroplasts, and Fe is required for maintenance of its enzyme activity because Fe deficiency resulted in a significant reduction of thiamine content in tomato leaves. Further, we also showed that the expression of LeTHIC is tightly regulated at the transcriptional and post-transcriptional level by multiple factors, such as light, Fe status and thiamine pyrophosphate (TPP)-riboswitch. The results clearly demonstrated that a feedback regulation mechanism is involved in synthesis of the pyrimidine moiety for controlling thiamine synthesis in tomato. Our results provide a new insight into understanding the molecular mechanism of thiamine biosynthesis in plants. PMID:21511719

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

    PubMed

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

    2013-08-28

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

  5. Vehicle Efficiency and Tractive Work: Rate of Change for the Past Decade and Accelerated Progress Required for U.S. Fuel Economy and CO2 Regulations

    DOE PAGESBeta

    Thomas, John

    2016-01-01

    A major driving force for change in light-duty vehicle design and technology is the National Highway Traffic Safety Administration (NHTSA) and the U.S. Environmental Protection Agency (EPA) joint final rules concerning Corporate Average Fuel Economy (CAFE) and greenhouse gas (GHG) emissions for model years (MY) 2016 through 2025 passenger cars and light trucks. The chief goal of this current study is to compare the already rapid pace of fuel economy improvement and technological change over the previous decade to the needed rate of change to meet regulations over the next decade. EPA and NHTSA comparisons of the MY 2004 USmore » light-duty vehicle fleet to the MY 2014 fleet shows improved fuel economy (FE) of approximately 28% using the same FE estimating method mandated for CAFE regulations. Future predictions by EPA and NHTSA concerning ensemble fleet fuel economy are examined as an indicator of needed vehicle rate-of-change. A set of 40 same-model vehicle pairs for MY 2005 and MY 2015 is compared to examine changes in energy use and related technological change over the 10 year period. Powertrain improvements measured as increased vehicle efficiency, and vehicle mass-glider improvements measured as decreased tractive work requirements are quantified. The focus is first on conventional gasoline powertrain vehicles which currently dominate the market, with hybrids also examined due to their high potential importance for CAFE compliance. Most hybrid vehicles with significant sales in 2014 were represented in the study. Results show 10 years of progress for the studied vehicle set includes lowered tractive effort of about 5.6% and improved powertrain efficiency of about 16.5%. Further analysis shows that this high rate of past progress must increase by about 50% in order to meet the 2025 CAFE standards. Examination of where certain MY 2015 vehicle compare to CAFE regulations is offered as well as some simple conjecture on what is needed to meet regulations under

  6. Calcium responses mediated by type 2 IP3-receptors are required for osmotic volume regulation of retinal glial cells in mice.

    PubMed

    Lipp, Stephan; Wurm, Antje; Pannicke, Thomas; Wiedemann, Peter; Reichenbach, Andreas; Chen, Ju; Bringmann, Andreas

    2009-06-26

    Prevention of osmotic swelling of retinal glial (Müller) cells is required to avoid detrimental decreases in the extracellular space volume during intense neuronal activity. Here, we show that glial cells in slices of the wildtype mouse retina maintain the volume of their somata constant up to approximately 4 min of perfusion with a hypoosmolar solution. However, calcium chelation with BAPTA/AM induced a rapid swelling of glial cell bodies. In glial cells of retinas from inositol-1,4,5-trisphosphate-receptor type 2-deficient (IP(3)R2(-/-)) mice, hypotonic conditions caused swelling of the cell bodies without delay. Exogenous ATP (acting at P2Y(1) receptors) prevented the swelling of glial cells in retinal slices from wildtype but not from IP(3)R2(-/-) mice. Müller cells from IP(3)R2(-/-) mice displayed a strongly reduced amplitude of the ATP-evoked calcium responses as compared to cells from wildtype mice. It is concluded that endogenous calcium signaling mediated by IP(3)R2 is required for the osmotic volume regulation of retinal glial cells. PMID:19429168

  7. The vaccinia virus O1 protein is required for sustained activation of extracellular signal-regulated kinase 1/2 and promotes viral virulence.

    PubMed

    Schweneker, Marc; Lukassen, Susanne; Späth, Michaela; Wolferstätter, Michael; Babel, Eveline; Brinkmann, Kay; Wielert, Ursula; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

    2012-02-01

    Sustained activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway in infected cells has been shown to be crucial for full replication efficiency of orthopoxviruses in cell culture. In infected cells, this pathway is mainly activated by the vaccinia virus growth factor (VGF), an epidermal growth factor (EGF)-like protein. We show here that chorioallantois vaccinia virus Ankara (CVA), but not modified vaccinia virus Ankara (MVA), induced sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in infected human 293 cells, although both viruses direct secretion of functional VGF. A CVA mutant lacking the O1L gene (CVA-ΔO1L) demonstrated that the O1 protein was required for sustained upregulation of the ERK1/2 pathway in 293 cells as well as in other mammalian cell lines. The highly conserved orthopoxvirus O1L gene encodes a predicted 78-kDa protein with a hitherto-unknown function. CVA-ΔO1L showed reduced plaque size and an attenuated cytopathic effect (CPE) in infected cell cultures and reduced virulence and spread from lungs to ovaries in intranasally infected BALB/c mice. Reinsertion of an intact O1L gene into MVA, which in its original form harbors a fragmented O1L open reading frame (ORF), restored ERK1/2 activation in 293 cells but did not increase replication and spread of MVA in human or other mammalian cell lines. Thus, the O1 protein was crucial for sustained ERK1/2 activation in CVA- and MVA-infected human cells, complementing the autocrine function of VGF, and enhanced virulence in vivo.

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

    PubMed Central

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

    1995-01-01

    Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs. PMID:7565670

  9. Vimentin, a Novel NF-κB Regulator, Is Required for Meningitic Escherichia coli K1-Induced Pathogen Invasion and PMN Transmigration across the Blood-Brain Barrier

    PubMed Central

    Zhang, Bao; Liu, Li-Qun; Wu, Xuedong; Mor-Vaknin, Nirit; Markovitz, David M.; Cao, Hong; Zhou, Yan-Hong

    2016-01-01

    Background NF-κB activation, pathogen invasion, polymorphonuclear leukocytes (PMN) transmigration (PMNT) across the blood-brain barrier (BBB) are the pathogenic triad hallmark features of bacterial meningitis, but the mechanisms underlying these events remain largely unknown. Vimentin, which is a novel NF-κB regulator, is the primary receptor for the major Escherichia coli K1 virulence factor IbeA that contributes to the pathogenesis of neonatal bacterial sepsis and meningitis (NSM). We have previously shown that IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PTB-associated splicing factor (PSF) is required for pathogen penetration and leukocyte transmigration across the BBB. This is the first in vivo study to demonstrate how vimentin and related factors contributed to the pathogenic triad of bacterial meningitis. Methodology/Principal Findings The role of vimentin in IbeA+ E. coli K1-induced NF-κB activation, pathogen invasion, leukocyte transmigration across the BBB has now been demonstrated by using vimentin knockout (KO) mice. In the in vivo studies presented here, IbeA-induced NF-κB activation, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the BBB were significantly reduced in Vim-/- mice. Decreased neuronal injury in the hippocampal dentate gyrus was observed in Vim-/- mice with meningitis. The major inflammatory regulator α7 nAChR and several signaling molecules contributing to NF-κB activation (p65 and p-CamKII) were significantly reduced in the brain tissues of the Vim-/- mice with E. coli meningitis. Furthermore, Vim KO resulted in significant reduction in neuronal injury and in α7 nAChR-mediated calcium signaling. Conclusion/Significance Vimentin, a novel NF-κB regulator, plays a detrimental role in the host defense against meningitic infection by modulating the NF-κB signaling pathway to increase pathogen invasion, PMN recruitment, BBB permeability and neuronal

  10. The Lectin Receptor Kinase-VI.2 Is Required for Priming and Positively Regulates Arabidopsis Pattern-Triggered Immunity[C][W

    PubMed Central

    Singh, Prashant; Kuo, Yi-Chun; Mishra, Swati; Tsai, Chia-Hong; Chien, Chih-Cheng; Chen, Ching-Wei; Desclos-Theveniau, Marie; Chu, Po-Wei; Schulze, Birgit; Chinchilla, Delphine; Boller, Thomas; Zimmerli, Laurent

    2012-01-01

    Plant cells can be sensitized toward a subsequent pathogen attack by avirulent pathogens or by chemicals such as β-aminobutyric acid (BABA). This process is called priming. Using a reverse genetic approach in Arabidopsis thaliana, we demonstrate that the BABA-responsive L-type lectin receptor kinase-VI.2 (LecRK-VI.2) contributes to disease resistance against the hemibiotrophic Pseudomonas syringae and the necrotrophic Pectobacterium carotovorum bacteria. Accordingly, LecRK-VI.2 mRNA levels increased after bacterial inoculation or treatments with microbe-associated molecular patterns (MAMPs). We also show that LecRK-VI.2 is required for full activation of pattern-triggered immunity (PTI); notably, lecrk-VI.2-1 mutants show reduced upregulation of PTI marker genes, impaired callose deposition, and defective stomatal closure. Overexpression studies combined with genome-wide microarray analyses indicate that LecRK-VI.2 positively regulates the PTI response. LecRK-VI.2 is demonstrated to act upstream of mitogen-activated protein kinase signaling, but independently of reactive oxygen production and BOTRYTIS-INDUCED KINASE1 phosphorylation. In addition, complex formation between the MAMP receptor FLAGELLIN SENSING2 and its signaling partner BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 is observed in flg22-treated lecrk-VI.2-1 mutants. LecRK-VI.2 is also required for full BABA-induced resistance and priming of PTI. Our work identifies LecRK-VI.2 as a novel mediator of the Arabidopsis PTI response and provides insight into molecular mechanisms governing priming. PMID:22427336

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. Extracellular Signal-regulated Kinase Activation Is Required for Serine 727 Phosphorylation of STAT3 in Schwann Cells in vitro and in vivo

    PubMed Central

    Lee, Hyun Kyoung; Jung, Junyang; Lee, Sang Hwa; Seo, Su-Yeong; Suh, Duk Joon

    2009-01-01

    In the peripheral nerves, injury-induced cytokines and growth factors perform critical functions in the activation of both the MEK/ERK and JAK/STAT3 pathways. In this study, we determined that nerve injury-induced ERK activation was temporally correlated with STAT3 phosphorylation at the serine 727 residue. In cultured Schwann cells, we noted that ERK activation is required for the serine phosphorylation of STAT3 by neuropoietic cytokine interleukin-6 (IL-6). Serine phosphorylated STAT3 by IL-6 was transported into Schwann cell nuclei, thereby indicating that ERK may regulate the transcriptional activity of STAT3 via the induction of serine phosphorylation of STAT3. Neuregulin-1 (NRG) also induced the serine phosphorylation of STAT3 in an ERK-dependent fashion. In contrast with the IL-6 response, serine phosphorylated STAT3 induced by NRG was not detected in the nucleus, thus indicating the non-nuclear function of serine phosphorylated STAT3 in response to NRG. Finally, we determined that the inhibition of ERK prevented injury-induced serine phosphorylation of STAT3 in an ex-vivo explants culture of the sciatic nerves. Collectively, the results of this study show that ERK may be an upstream kinase for the serine phosphorylation of STAT3 induced by multiple stimuli in Schwann cells after peripheral nerve injury. PMID:19885032

  13. Cooperation between SAGA and SWI/SNF complexes is required for efficient transcriptional responses regulated by the yeast MAPK Slt2

    PubMed Central

    Sanz, Ana Belén; García, Raúl; Rodríguez-Peña, José Manuel; Nombela, César; Arroyo, Javier

    2016-01-01

    The transcriptional response of Saccharomyces cerevisiae to cell wall stress is mainly mediated by the cell wall integrity (CWI) pathway through the MAPK Slt2 and the transcription factor Rlm1. Once activated, Rlm1 interacts with the chromatin remodeling SWI/SNF complex which locally alters nucleosome positioning at the target promoters. Here we show that the SAGA complex plays along with the SWI/SNF complex an important role for eliciting both early induction and sustained gene expression upon stress. Gcn5 co-regulates together with Swi3 the majority of the CWI transcriptional program, except for a group of genes which are only dependent on the SWI/SNF complex. SAGA subunits are recruited to the promoter of CWI-responsive genes in a Slt2, Rlm1 and SWI/SNF-dependent manner. However, Gcn5 mediates acetylation and nucleosome eviction only at the promoters of the SAGA-dependent genes. This process is not essential for pre-initiation transcriptional complex assembly but rather increase the extent of the remodeling mediated by SWI/SNF. As a consequence, H3 eviction and Rlm1 recruitment is completely blocked in a swi3Δ gcn5Δ double mutant. Therefore, SAGA complex, through its histone acetylase activity, cooperates with the SWI/SNF complex for the mandatory nucleosome displacement required for full gene expression through the CWI pathway. PMID:27112564

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

    PubMed Central

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

    2012-01-01

    The plant growth-promoting rhizobacterium Pseudomonas fluorescens WCS374r produces several iron-regulated metabolites, including the fluorescent siderophore pseudobactin (Psb374), salicylic acid (SA), and pseudomonine (Psm), a siderophore that contains a SA moiety. After purification of Psb374 from culture supernatant of WCS374r, its structure was determined following isoelectrofocusing and tandem mass spectrometry, and found to be identical to the fluorescent siderophore produced by P. fluorescens ATCC 13525. To study the role of SA and Psm production in colonization of Arabidopsis thaliana roots and in induced systemic resistance (ISR) against Pseudomonas syringae pv. tomato (Pst) by strain WCS374r, mutants disrupted in the production of these metabolites were obtained by homologous recombination. These mutants were further subjected to transposon Tn5 mutagenesis to generate mutants also deficient in Psb374 production. The mutants behaved similar to the wild type in both their Arabidopsis rhizosphere-colonizing capacity and their ability to elicit ISR against Pst. We conclude that Psb374, SA, and Psm production by P. fluorescens WCS374r are not required for eliciting ISR in Arabidopsis. PMID:23170230

  15. Cooperation between SAGA and SWI/SNF complexes is required for efficient transcriptional responses regulated by the yeast MAPK Slt2.

    PubMed

    Sanz, Ana Belén; García, Raúl; Rodríguez-Peña, José Manuel; Nombela, César; Arroyo, Javier

    2016-09-01

    The transcriptional response of Saccharomyces cerevisiae to cell wall stress is mainly mediated by the cell wall integrity (CWI) pathway through the MAPK Slt2 and the transcription factor Rlm1. Once activated, Rlm1 interacts with the chromatin remodeling SWI/SNF complex which locally alters nucleosome positioning at the target promoters. Here we show that the SAGA complex plays along with the SWI/SNF complex an important role for eliciting both early induction and sustained gene expression upon stress. Gcn5 co-regulates together with Swi3 the majority of the CWI transcriptional program, except for a group of genes which are only dependent on the SWI/SNF complex. SAGA subunits are recruited to the promoter of CWI-responsive genes in a Slt2, Rlm1 and SWI/SNF-dependent manner. However, Gcn5 mediates acetylation and nucleosome eviction only at the promoters of the SAGA-dependent genes. This process is not essential for pre-initiation transcriptional complex assembly but rather increase the extent of the remodeling mediated by SWI/SNF. As a consequence, H3 eviction and Rlm1 recruitment is completely blocked in a swi3Δ gcn5Δ double mutant. Therefore, SAGA complex, through its histone acetylase activity, cooperates with the SWI/SNF complex for the mandatory nucleosome displacement required for full gene expression through the CWI pathway. PMID:27112564

  16. The lemA gene required for pathogenicity of Pseudomonas syringae pv. syringae on bean is a member of a family of two-component regulators.

    PubMed Central

    Hrabak, E M; Willis, D K

    1992-01-01

    The lemA gene of the plant pathogen Pseudomonas syringae pv. syringae is required for disease lesion formation on bean plants. Cosmid clones that complemented a lemA mutant in trans were isolated previously. The lemA gene was localized by subcloning and transposon mutagenesis. The lemA region and flanking DNA were sequenced, and an open reading frame of 2.7 kb was identified. The nucleotide and predicted amino acid sequences of the lemA gene showed sequence similarity to a family of prokaryotic two-component regulatory proteins. Unlike most of the previously described two-component systems, the lemA gene product contained homology to both components in one protein. Mutations introduced upstream and downstream of the lemA gene failed to locate a gene for a second protein component but identified the putative cysM gene of P. syringae pv. syringae. The cysM gene was located upstream of the lemA gene and was divergently transcribed. The lemA gene product was expressed at low levels in P. syringae pv. syringae and appeared to be positively auto-regulated. PMID:1314807

  17. Activation of ADF/cofilin by phosphorylation-regulated Slingshot phosphatase is required for the meiotic spindle assembly in Xenopus laevis oocytes

    PubMed Central

    Iwase, Shohei; Sato, Ryuhei; De Bock, Pieter-Jan; Gevaert, Kris; Fujiki, Saburo; Tawada, Toshinobu; Kuchitsu, Miyako; Yamagishi, Yuka; Ono, Shoichiro; Abe, Hiroshi

    2013-01-01

    We identify Xenopus ADF/cofilin (XAC) and its activator, Slingshot phosphatase (XSSH), as key regulators of actin dynamics essential for spindle microtubule assembly during Xenopus oocyte maturation. Phosphorylation of XSSH at multiple sites within the tail domain occurs just after germinal vesicle breakdown (GVBD) and is accompanied by dephosphorylation of XAC, which was mostly phosphorylated in immature oocytes. This XAC dephosphorylation after GVBD is completely suppressed by latrunculin B, an actin monomer–sequestering drug. On the other hand, jasplakinolide, an F-actin–stabilizing drug, induces dephosphorylation of XAC. Effects of latrunculin B and jasplakinolide are reconstituted in cytostatic factor–arrested extracts (CSF extracts), and XAC dephosphorylation is abolished by depletion of XSSH from CSF extracts, suggesting that XSSH functions as an actin filament sensor to facilitate actin filament dynamics via XAC activation. Injection of anti-XSSH antibody, which blocks full phosphorylation of XSSH after GVBD, inhibits both meiotic spindle formation and XAC dephosphorylation. Coinjection of constitutively active XAC with the antibody suppresses this phenotype. Treatment of oocytes with jasplakinolide also impairs spindle formation. These results strongly suggest that elevation of actin dynamics by XAC activation through XSSH phosphorylation is required for meiotic spindle assembly in Xenopus laevis. PMID:23615437

  18. Regulation of Tlx3 by Pax6 is required for the restricted expression of Chrnα3 in Cerebellar Granule Neuron progenitors during development

    PubMed Central

    Divya, Thulasi Sheela; Lalitha, Soundararajan; Parvathy, Surendran; Subashini, Chandramohan; Sanalkumar, Rajendran; Dhanesh, Sivadasan Bindu; Rasheed, Vazhanthodi Abdul; Divya, Mundackal Sivaraman; Tole, Shubha; James, Jackson

    2016-01-01

    Homeobox gene Tlx3 is known to promote glutamatergic differentiation and is expressed in post-mitotic neurons of CNS. Contrary to this here, we discovered that Tlx3 is expressed in the proliferating progenitors of the external granule layer in the cerebellum, and examined factors that regulate this expression. Using Pax6−/−Sey mouse model and molecular interaction studies we demonstrate Pax6 is a key activator of Tlx3 specifically in cerebellum, and induces its expression starting at embryonic day (E)15. By Postnatal day (PN)7, Tlx3 is expressed in a highly restricted manner in the cerebellar granule neurons of the posterior cerebellar lobes, where it is required for the restricted expression of nicotinic cholinergic receptor-α3 subunit (Chrnα3) and other genes involved in formation of synaptic connections and neuronal migration. These results demonstrate a novel role for Tlx3 and indicate that Pax6-Tlx3 expression and interaction is part of a region specific regulatory network in cerebellum and its deregulation during development could possibly lead to Autistic spectral disorders (ASD). PMID:27452274

  19. Cooperation between SAGA and SWI/SNF complexes is required for efficient transcriptional responses regulated by the yeast MAPK Slt2.

    PubMed

    Sanz, Ana Belén; García, Raúl; Rodríguez-Peña, José Manuel; Nombela, César; Arroyo, Javier

    2016-09-01

    The transcriptional response of Saccharomyces cerevisiae to cell wall stress is mainly mediated by the cell wall integrity (CWI) pathway through the MAPK Slt2 and the transcription factor Rlm1. Once activated, Rlm1 interacts with the chromatin remodeling SWI/SNF complex which locally alters nucleosome positioning at the target promoters. Here we show that the SAGA complex plays along with the SWI/SNF complex an important role for eliciting both early induction and sustained gene expression upon stress. Gcn5 co-regulates together with Swi3 the majority of the CWI transcriptional program, except for a group of genes which are only dependent on the SWI/SNF complex. SAGA subunits are recruited to the promoter of CWI-responsive genes in a Slt2, Rlm1 and SWI/SNF-dependent manner. However, Gcn5 mediates acetylation and nucleosome eviction only at the promoters of the SAGA-dependent genes. This process is not essential for pre-initiation transcriptional complex assembly but rather increase the extent of the remodeling mediated by SWI/SNF. As a consequence, H3 eviction and Rlm1 recruitment is completely blocked in a swi3Δ gcn5Δ double mutant. Therefore, SAGA complex, through its histone acetylase activity, cooperates with the SWI/SNF complex for the mandatory nucleosome displacement required for full gene expression through the CWI pathway.

  20. Muscle-Derived Extracellular Signal-Regulated Kinases 1 and 2 Are Required for the Maintenance of Adult Myofibers and Their Neuromuscular Junctions

    PubMed Central

    Seaberg, Bonnie; Henslee, Gabrielle; Wang, Shuo; Paez-Colasante, Ximena; Landreth, Gary E.

    2015-01-01

    The Ras–extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway appears to be important for the development, maintenance, aging, and pathology of mammalian skeletal muscle. Yet no gene targeting of Erk1/2 in muscle fibers in vivo has been reported to date. We combined a germ line Erk1 mutation with Cre-loxP Erk2 inactivation in skeletal muscle to produce, for the first time, mice lacking ERK1/2 selectively in skeletal myofibers. Animals lacking muscle ERK1/2 displayed stunted postnatal growth, muscle weakness, and a shorter life span. Their muscles examined in this study, sternomastoid and tibialis anterior, displayed fragmented neuromuscular synapses and a mixture of modest fiber atrophy and loss but failed to show major changes in fiber type composition or absence of cell surface dystrophin. Whereas the lack of only ERK1 had no effects on the phenotypes studied, the lack of myofiber ERK2 explained synaptic fragmentation in the sternomastoid but not the tibialis anterior and a decrease in the expression of the acetylcholine receptor (AChR) epsilon subunit gene mRNA in both muscles. A reduction in AChR protein was documented in line with the above mRNA results. Evidence of partial denervation was found in the sternomastoid but not the tibialis anterior. Thus, myofiber ERK1/2 are differentially required for the maintenance of myofibers and neuromuscular synapses in adult mice. PMID:25605336

  1. The dicer-like1 homolog fuzzy tassel is required for the regulation of meristem determinacy in the inflorescence and vegetative growth in maize.

    PubMed

    Thompson, Beth E; Basham, Christine; Hammond, Reza; Ding, Queying; Kakrana, Atul; Lee, Tzuu-Fen; Simon, Stacey A; Meeley, Robert; Meyers, Blake C; Hake, Sarah

    2014-12-01

    Plant architecture is determined by meristems that initiate leaves during vegetative development and flowers during reproductive development. Maize (Zea mays) inflorescences are patterned by a series of branching events, culminating in floral meristems that produce sexual organs. The maize fuzzy tassel (fzt) mutant has striking inflorescence defects with indeterminate meristems, fasciation, and alterations in sex determination. fzt plants have dramatically reduced plant height and shorter, narrower leaves with leaf polarity and phase change defects. We positionally cloned fzt and discovered that it contains a mutation in a dicer-like1 homolog, a key enzyme required for microRNA (miRNA) biogenesis. miRNAs are small noncoding RNAs that reduce target mRNA levels and are key regulators of plant development and physiology. Small RNA sequencing analysis showed that most miRNAs are moderately reduced in fzt plants and a few miRNAs are dramatically reduced. Some aspects of the fzt phenotype can be explained by reduced levels of known miRNAs, including miRNAs that influence meristem determinacy, phase change, and leaf polarity. miRNAs responsible for other aspects of the fzt phenotype are unknown and likely to be those miRNAs most severely reduced in fzt mutants. The fzt mutation provides a tool to link specific miRNAs and targets to discrete phenotypes and developmental roles. PMID:25465405

  2. Vernalization Requirement and the Chromosomal VRN1-Region can Affect Freezing Tolerance and Expression of Cold-Regulated Genes in Festuca pratensis

    PubMed Central

    Ergon, Åshild; Melby, Tone I.; Höglind, Mats; Rognli, Odd A.

    2016-01-01

    Plants adapted to cold winters go through annual cycles of gain followed by loss of freezing tolerance (cold acclimation and deacclimation). Warm spells during winter and early spring can cause deacclimation, and if temperatures drop, freezing damage may occur. Many plants are vernalized during winter, a process making them competent to flower in the following summer. In winter cereals, a coincidence in the timing of vernalization saturation, deacclimation, downregulation of cold-induced genes, and reduced ability to reacclimate, occurs under long photoperiods and is under control of the main regulator of vernalization requirement in cereals, VRN1, and/or closely linked gene(s). Thus, the probability of freezing damage after a warm spell may depend on both vernalization saturation and photoperiod. We investigated the role of vernalization and the VRN1-region on freezing tolerance of meadow fescue (Festuca pratensis Huds.), a perennial grass species. Two F2 populations, divergently selected for high and low vernalization requirement, were studied. Each genotype was characterized for the copy number of one of the four parental haplotypes of the VRN1-region. Clonal plants were cold acclimated for 2 weeks or vernalized/cold acclimated for a total of 9 weeks, after which the F2 populations reached different levels of vernalization saturation. Vernalized and cold acclimated plants were deacclimated for 1 week and then reacclimated for 2 weeks. All treatments were given at 8 h photoperiod. Flowering response, freezing tolerance and expression of the cold-induced genes VRN1, MADS3, CBF6, COR14B, CR7 (BLT14), LOS2, and IRI1 was measured. We found that some genotypes can lose some freezing tolerance after vernalization and a deacclimation–reacclimation cycle. The relationship between vernalization and freezing tolerance was complex. We found effects of the VRN1-region on freezing tolerance in plants cold acclimated for 2 weeks, timing of heading after 9 weeks of

  3. Cotton plasma membrane intrinsic protein 2s (PIP2s) selectively interact to regulate their water channel activities and are required for fibre development.

    PubMed

    Li, Deng-Di; Ruan, Xiang-Mei; Zhang, Jie; Wu, Ya-Jie; Wang, Xiu-Lan; Li, Xue-Bao

    2013-08-01

    Aquaporins are thought to be associated with water transport and play important roles in cotton (Gossypium hirsutum) fibre elongation. Among aquaporins, plasma membrane intrinsic proteins (PIPs) constitute a plasma-membrane-specific subfamily and are further subdivided into PIP1 and PIP2 groups. In this study, four fibre-preferential GhPIP2 genes were functionally characterized. The selective interactions among GhPIP2s and their interaction proteins were studied in detail to elucidate the molecular mechanism of cotton fibre development. GhPIP2;3 interacted with GhPIP2;4 and GhPIP2;6, but GhPIP2;6 did not interact with GhPIP2;4. Coexpression of GhPIP2;3/2;4 or GhPIP2;3/2;6 resulted in a positive cooperative effect which increased the permeability coefficient of oocytes, while GhPIP2;4/2;6 did not. GhBCP2 (a blue copper-binding protein) inhibited GhPIP2;6 water channel activity through their interaction. Overexpression of GhPIP2 genes in yeast induced longitudinal growth of the host cells. By contrast, knockdown of expression of GhPIP2 genes in cotton by RNA interference markedly hindered fibre elongation. In conclusion, GhPIP2 proteins are the primary aquaporin isoforms in fibres. They selectively form hetero-oligomers in order to regulate their activities to meet the requirements for rapid fibre elongation.

  4. 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, 2010 CFR

    2010-07-01

    ... Pollution Control and Ecology Regulation Number 12—Storage Tank Regulation: a. Chapter 1: General Provisions... Section 3: Initial Fund Eligibility Section 4: Loss and Restoration of Initial Fund Eligibility Section 5...) Replenishment of guarantees, letters of credit, or, surety bonds. (w) Suspension of enforcement. (x) 40 CFR...

  5. 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, 2011 CFR

    2011-07-01

    ... Pollution Control and Ecology Regulation Number 12—Storage Tank Regulation: a. Chapter 1: General Provisions... Section 3: Initial Fund Eligibility Section 4: Loss and Restoration of Initial Fund Eligibility Section 5...) Replenishment of guarantees, letters of credit, or, surety bonds. (w) Suspension of enforcement. (x) 40 CFR...

  6. Analysis of genes negatively regulated by phytochrome action in Lemna gibba and identification of a promoter region required for phytochrome responsiveness.

    PubMed Central

    Okubara, P A; Williams, S A; Doxsee, R A; Tobin, E M

    1993-01-01

    As a step to understanding how the photoreceptor phytochrome acts to change the transcription of specific nuclear genes in Lemna gibba, we wish to compare promoter elements involved in negative regulation by phytochrome with those involved in positive regulation. We have isolated three genes negatively regulated by phytochrome, designated NR (negatively phytochrome regulated) genes (P.A. Okubara, E.M. Tobin [1991] Plant Physiol 96:1237-1245), and we have now sequenced two of these. The promoters of both contain some sequence motifs that are identical with motifs from other genes. We used a transient assay in L. gibba to demonstrate that approximately 1.7 kb pairs of the NPR1 promoter and 1.1 kb pairs of the NPR2 promoter could confer negative phytochrome regulation to a luciferase reporter gene. Deletion analysis of the NPR2 promoter showed that sequences between -208 and -82 from the transcription start were necessary for negative phytochrome regulation. However, this region was not sufficient to confer negative regulation by phytochrome to another promoter. Additionally, we noted that this region showed no similarity to a region identified as important for the negative regulation of the oat phyA promoter (W.B. Bruce, X.-W. Deng, P.H. Quail [1991] EMBO J 10:3015-3024), but it does contain a sequence element found in several other kinds of genes, including ones positively regulated by phytochrome. The deduced amino acid sequences of NPR1 and NPR2 were found to share similarities with many abscisic acid-induced or seed-abundant proteins. Thus, these genes, like other phytochrome-regulated genes, might respond to multiple regulatory signals. PMID:8310060

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

    PubMed

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

    1999-08-01

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

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

    PubMed

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

    2016-03-01

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

  9. Regulation of Wnt signaling by the tumor suppressor adenomatous polyposis coli does not require the ability to enter the nucleus or a particular cytoplasmic localization.

    PubMed

    Roberts, David M; Pronobis, Mira I; Poulton, John S; Kane, Eric G; Peifer, Mark

    2012-06-01

    Wnt signaling plays key roles in development and disease. The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling. Its best-characterized role is as part of the destruction complex, targeting the Wnt effector β-catenin (βcat) for phosphorylation and ultimate destruction, but several studies suggested APC also may act in the nucleus at promoters of Wnt-responsive genes or to shuttle βcat out for destruction. Even in its role in the destruction complex, APC's mechanism of action remains mysterious. We have suggested APC positions the destruction complex at the appropriate subcellular location, facilitating βcat destruction. In this study, we directly tested APC's proposed roles in the nucleus or in precisely localizing the destruction complex by generating a series of APC2 variants to which we added tags relocalizing otherwise wild-type APC to different cytoplasmic locations. We tested these for function in human colon cancer cells and Drosophila embryos. Strikingly, all rescue Wnt regulation and down-regulate Wnt target genes in colon cancer cells, and most restore Wnt regulation in Drosophila embryos null for both fly APCs. These data suggest that APC2 does not have to shuttle into the nucleus or localize to a particular subcellular location to regulate Wnt signaling.

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

    PubMed Central

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

    2016-01-01

    Formins comprise a large family of proteins with diverse roles in remodeling the actin cytoskeleton. However, the spatiotemporal mechanisms used by cells to control formin activities are only beginning to be understood. Here we dissected Smy1, which has dual roles in regulating formins and myosin. Using mutagenesis, we identified specific sequences in Smy1 critical for its in vitro inhibitory effects on the FH2 domain of the formin Bnr1. By integrating smy1 alleles targeting those sequences, we genetically uncoupled Smy1’s functions in regulating formins and myosin. Quantitative imaging analysis further demonstrated that the ability of Smy1 to directly control Bnr1 activity is crucial in vivo for proper actin cable length, shape, and velocity and, in turn, efficient secretory vesicle transport. A Smy1-like sequence motif was also identified in a different Bnr1 regulator, Bud14, and found to be essential for Bud14 functions in regulating actin cable architecture and function in vivo. Together these observations reveal unanticipated mechanistic ties between two distinct formin regulators. Further, they emphasize the importance of tightly controlling formin activities in vivo to generate specialized geometries and dynamics of actin structures tailored to their physiological roles. PMID:26764093

  11. Regulation of Wnt signaling by the tumor suppressor adenomatous polyposis coli does not require the ability to enter the nucleus or a particular cytoplasmic localization

    PubMed Central

    Roberts, David M.; Pronobis, Mira I.; Poulton, John S.; Kane, Eric G.; Peifer, Mark

    2012-01-01

    Wnt signaling plays key roles in development and disease. The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling. Its best-characterized role is as part of the destruction complex, targeting the Wnt effector β-catenin (βcat) for phosphorylation and ultimate destruction, but several studies suggested APC also may act in the nucleus at promoters of Wnt-responsive genes or to shuttle βcat out for destruction. Even in its role in the destruction complex, APC's mechanism of action remains mysterious. We have suggested APC positions the destruction complex at the appropriate subcellular location, facilitating βcat destruction. In this study, we directly tested APC's proposed roles in the nucleus or in precisely localizing the destruction complex by generating a series of APC2 variants to which we added tags relocalizing otherwise wild-type APC to different cytoplasmic locations. We tested these for function in human colon cancer cells and Drosophila embryos. Strikingly, all rescue Wnt regulation and down-regulate Wnt target genes in colon cancer cells, and most restore Wnt regulation in Drosophila embryos null for both fly APCs. These data suggest that APC2 does not have to shuttle into the nucleus or localize to a particular subcellular location to regulate Wnt signaling. PMID:22513088

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

    PubMed

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

    2013-11-01

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

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

    PubMed

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

    2007-05-01

    Iron is both essential for bacterial growth and toxic at higher concentrations; thus, iron homeostasis is tightly regulated. In Neisseria meningitidis the majority of iron-responsive gene regulation is mediated by the ferric uptake regulator protein (Fur), a protein classically defined as a transcriptional repressor. Recently, however, microarray studies have identified a number of genes in N. meningitidis that are iron and Fur activated, demonstrating a new role for Fur as a transcriptional activator. Since Fur has been shown to indirectly activate gene transcription through the repression of small regulatory RNA molecules in other organisms, we hypothesized that a similar mechanism could account for Fur-dependent, iron-activated gene transcription in N. meningitidis. In this study, we used a bioinformatics approach to screen for the presence of Fur-regulated small RNA molecules in N. meningitidis MC58. This screen identified one small RNA, herein named NrrF (for neisserial regulatory RNA responsive to iron [Fe]), which was demonstrated to be both iron responsive and Fur regulated and which has a well-conserved orthologue in N. gonorrhoeae. In addition, this screen identified a number of other likely, novel small RNA transcripts. Lastly, we utilized a new bioinformatics approach to predict regulatory targets of the NrrF small RNA. This analysis led to the identification of the sdhA and sdhC genes, which were subsequently demonstrated to be under NrrF regulation in an nrrF mutant. This study is the first report of small RNAs in N. meningitidis and the first to use a bioinformatics approach to identify, a priori, regulatory targets of a small RNA. PMID:17351036

  14. Vibrio parahaemolyticus ToxRS regulator is required for stress tolerance and colonization in a novel orogastric streptomycin-induced adult murine model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vibrio parahaemolyticus, a marine bacterium, is the causative agent of gastroenteritis associated with the consumption of seafood. It contains a homologue of the toxRS operon that in V. cholerae is the key regulator of virulence gene expression. We examined a non-polar mutation in toxRS to determi...

  15. Appetitive Cue-Evoked ERK Signaling in the Nucleus Accumbens Requires NMDA and D1 Dopamine Receptor Activation and Regulates CREB Phosphorylation

    ERIC Educational Resources Information Center

    Kirschmann, Erin K. Z.; Mauna, Jocelyn C.; Willis, Cory M.; Foster, Rebecca L.; Chipman, Amanda M.; Thiels, Edda

    2014-01-01

    Conditioned stimuli (CS) can modulate reward-seeking behavior. This modulatory effect can be maladaptive and has been implicated in excessive reward seeking and relapse to drug addiction. We previously demonstrated that exposure to an appetitive CS causes an increase in the activation of extracellular signal-regulated kinase (ERK) and cyclic-AMP…

  16. The Global Arginine Regulator ArgR Controls Expression of argF in Pseudomonas syringae pv. phaseolicola but Is Not Required for the Synthesis of Phaseolotoxin or for the Regulated Expression of argK†

    PubMed Central

    Hernández-Flores, José Luis; López-López, Karina; Garcidueñas-Piña, Rogelio; Jofre-Garfias, Alba E.; Alvarez-Morales, Ariel

    2004-01-01

    In Pseudomonas syringae pv. phaseolicola the enzyme ornithine carbamoyltransferase (OCTase), encoded by argF, is negatively regulated by argR, similar to what has been reported for Pseudomonas aeruginosa. However, production of the phaseolotoxin-resistant OCTase encoded by argK, synthesis of phaseolotoxin, and infectivity for bean pods occur independently of the ArgR protein. PMID:15150254

  17. Developmentally regulated HEART STOPPER, a mitochondrially targeted L18 ribosomal protein gene, is required for cell division, differentiation, and seed development in Arabidopsis

    PubMed Central

    Zhang, Hongyu; Luo, Ming; Day, Robert C.; Talbot, Mark J.; Ivanova, Aneta; Ashton, Anthony R.; Chaudhury, Abed M.; Macknight, Richard C.; Hrmova, Maria; Koltunow, Anna M.

    2015-01-01

    Evidence is presented for the role of a mitochondrial ribosomal (mitoribosomal) L18 protein in cell division, differentiation, and seed development after the characterization of a recessive mutant, heart stopper (hes). The hes mutant produced uncellularized endosperm and embryos arrested at the late globular stage. The mutant embryos differentiated partially on rescue medium with some forming callus. HES (At1g08845) encodes a mitochondrially targeted member of a highly diverged L18 ribosomal protein family. The substitution of a conserved amino residue in the hes mutant potentially perturbs mitoribosomal function via altered binding of 5S rRNA and/or influences the stability of the 50S ribosomal subunit, affecting mRNA binding and translation. Consistent with this, marker genes for mitochondrial dysfunction were up-regulated in the mutant. The slow growth of the endosperm and embryo indicates a defect in cell cycle progression, which is evidenced by the down-regulation of cell cycle genes. The down-regulation of other genes such as EMBRYO DEFECTIVE genes links the mitochondria to the regulation of many aspects of seed development. HES expression is developmentally regulated, being preferentially expressed in tissues with active cell division and differentiation, including developing embryos and the root tips. The divergence of the L18 family, the tissue type restricted expression of HES, and the failure of other L18 members to complement the hes phenotype suggest that the L18 proteins are involved in modulating development. This is likely via heterogeneous mitoribosomes containing different L18 members, which may result in differential mitochondrial functions in response to different physiological situations during development. PMID:26105995

  18. The histone chaperone Spt6 is required for activation-induced cytidine deaminase target determination through H3K4me3 regulation.

    PubMed

    Begum, Nasim A; Stanlie, Andre; Nakata, Mikiyo; Akiyama, Hideo; Honjo, Tasuku

    2012-09-21

    H3K4me3 plays a critical role in the activation-induced cytidine deaminase (AID)-induced DNA cleavage of switch (S) regions in the immunoglobulin heavy chain (IgH) locus during class-switch recombination (CSR). The histone chaperone complex facilitates chromatin transcription (FACT) is responsible for forming H3K4me3 at AID target loci. Here we show that the histone chaperone suppressor of Ty6 (Spt6) also participates in regulating H3K4me3 for CSR and for somatic hypermutation in AID target loci. We found that H3K4me3 loss was correlated with defects in AID-induced DNA breakage and reduced mutation frequencies in IgH loci in both S and variable regions and in non-IgH loci such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and small nucleolar RNA host gene 3 (SNHG3). Global gene expression analysis revealed that Spt6 can act as both a positive and negative transcriptional regulator in B cells, affecting ∼5% of the genes that includes suppressor of Ty4 (Spt4) and AID. Interestingly, Spt6 regulates CSR and AID expression through two distinct histone modification pathways, H3K4me3 and H3K36me3, respectively. Tandem SH2 domain of Spt6 plays a critical role in CSR and H3K4me3 regulation involving Set1 histone methyltransferase. We conclude that Spt6 is a unique histone chaperone capable of regulating the histone epigenetic state of both AID targets and the AID locus.

  19. The histone chaperone Spt6 is required for activation-induced cytidine deaminase target determination through H3K4me3 regulation.

    PubMed

    Begum, Nasim A; Stanlie, Andre; Nakata, Mikiyo; Akiyama, Hideo; Honjo, Tasuku

    2012-09-21

    H3K4me3 plays a critical role in the activation-induced cytidine deaminase (AID)-induced DNA cleavage of switch (S) regions in the immunoglobulin heavy chain (IgH) locus during class-switch recombination (CSR). The histone chaperone complex facilitates chromatin transcription (FACT) is responsible for forming H3K4me3 at AID target loci. Here we show that the histone chaperone suppressor of Ty6 (Spt6) also participates in regulating H3K4me3 for CSR and for somatic hypermutation in AID target loci. We found that H3K4me3 loss was correlated with defects in AID-induced DNA breakage and reduced mutation frequencies in IgH loci in both S and variable regions and in non-IgH loci such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and small nucleolar RNA host gene 3 (SNHG3). Global gene expression analysis revealed that Spt6 can act as both a positive and negative transcriptional regulator in B cells, affecting ∼5% of the genes that includes suppressor of Ty4 (Spt4) and AID. Interestingly, Spt6 regulates CSR and AID expression through two distinct histone modification pathways, H3K4me3 and H3K36me3, respectively. Tandem SH2 domain of Spt6 plays a critical role in CSR and H3K4me3 regulation involving Set1 histone methyltransferase. We conclude that Spt6 is a unique histone chaperone capable of regulating the histone epigenetic state of both AID targets and the AID locus. PMID:22843687

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

    PubMed Central

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

    2008-01-01

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