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Sample records for dead box rna

  1. From unwinding to clamping - the DEAD box RNA helicase family.

    PubMed

    Linder, Patrick; Jankowsky, Eckhard

    2011-07-22

    RNA helicases of the DEAD box family are present in all eukaryotic cells and in many bacteria and Archaea. These highly conserved enzymes are required for RNA metabolism from transcription to degradation and are therefore important players in gene expression. DEAD box proteins use ATP to unwind short duplex RNA in an unusual fashion and remodel RNA-protein complexes, but they can also function as ATP-dependent RNA clamps to provide nucleation centres that establish larger RNA-protein complexes. Structural, mechanistic and molecular biological studies have started to reveal how these conserved proteins can perform such diverse functions and how accessory proteins have a central role in their regulation.

  2. DEAD-Box RNA Binding Protein DDX5: Not a Black-Box during Reprogramming.

    PubMed

    Nefzger, Christian M; Polo, Jose M

    2017-04-06

    The role of RNA binding proteins (RBPs) during nuclear reprogramming is poorly characterized. In this issue of Cell Stem Cell,Li et al. (2017) show that DEAD-box RBP DDX5 acts as a reprogramming roadblock and give important mechanistic insights into the establishment of pluripotency by characterizing the intricate downstream events.

  3. DEAD-Box Helicase Proteins Disrupt RNA Tertiary Structure Through Helix Capture

    PubMed Central

    Pan, Cynthia; Potratz, Jeffrey P.; Cannon, Brian; Simpson, Zachary B.; Ziehr, Jessica L.; Tijerina, Pilar; Russell, Rick

    2014-01-01

    DEAD-box helicase proteins accelerate folding and rearrangements of highly structured RNAs and RNA–protein complexes (RNPs) in many essential cellular processes. Although DEAD-box proteins have been shown to use ATP to unwind short RNA helices, it is not known how they disrupt RNA tertiary structure. Here, we use single molecule fluorescence to show that the DEAD-box protein CYT-19 disrupts tertiary structure in a group I intron using a helix capture mechanism. CYT-19 binds to a helix within the structured RNA only after the helix spontaneously loses its tertiary contacts, and then CYT-19 uses ATP to unwind the helix, liberating the product strands. Ded1, a multifunctional yeast DEAD-box protein, gives analogous results with small but reproducible differences that may reflect its in vivo roles. The requirement for spontaneous dynamics likely targets DEAD-box proteins toward less stable RNA structures, which are likely to experience greater dynamic fluctuations, and provides a satisfying explanation for previous correlations between RNA stability and CYT-19 unfolding efficiency. Biologically, the ability to sense RNA stability probably biases DEAD-box proteins to act preferentially on less stable misfolded structures and thereby to promote native folding while minimizing spurious interactions with stable, natively folded RNAs. In addition, this straightforward mechanism for RNA remodeling does not require any specific structural environment of the helicase core and is likely to be relevant for DEAD-box proteins that promote RNA rearrangements of RNP complexes including the spliceosome and ribosome. PMID:25350280

  4. The mechanism of ATP-dependent RNA unwinding by DEAD box proteins.

    PubMed

    Hilbert, Manuel; Karow, Anne R; Klostermeier, Dagmar

    2009-12-01

    DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

  5. A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo

    PubMed Central

    Ruminski, Dana J.; Watson, Peter Y.; Mahen, Elisabeth M.; Fedor, Martha J.

    2016-01-01

    RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo. PMID:26759451

  6. A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo.

    PubMed

    Ruminski, Dana J; Watson, Peter Y; Mahen, Elisabeth M; Fedor, Martha J

    2016-03-01

    RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo.

  7. Do DEAD-box proteins promote group II intron splicing without unwinding RNA?

    PubMed

    Del Campo, Mark; Tijerina, Pilar; Bhaskaran, Hari; Mohr, Sabine; Yang, Quansheng; Jankowsky, Eckhard; Russell, Rick; Lambowitz, Alan M

    2007-10-12

    The DEAD-box protein Mss116p promotes group II intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss116p promotes group II intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss116p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group II intron splicing activity in quantitative assays. Additionally, we show that Mss116p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-box protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-box proteins function in group II intron splicing as in other processes by using their unwinding activity to act as RNA chaperones.

  8. Structure of the Yeast DEAD Box Protein Mss116p Reveals Two Wedges that Crimp RNA

    SciTech Connect

    Del Campo, Mark; Lambowitz, Alan M.

    2010-01-12

    The yeast DEAD box protein Mss116p is a general RNA chaperone that functions in mitochondrial group I and II intron splicing, translational activation, and RNA end processing. Here we determined high-resolution X-ray crystal structures of Mss116p complexed with an RNA oligonucleotide and ATP analogs AMP-PNP, ADP-BeF{sub 3}, or ADP-AlF{sub 4}{sup -}. The structures show the entire helicase core acting together with a functionally important C-terminal extension. In all structures, the helicase core is in a closed conformation with a wedge {alpha} helix bending RNA 3' of the central bound nucleotides, as in previous DEAD box protein structures. Notably, Mss116p's C-terminal extension also bends RNA 5' of the central nucleotides, resulting in RNA crimping. Despite reported functional differences, we observe few structural changes in ternary complexes with different ATP analogs. The structures constrain models of DEAD box protein function and reveal a strand separation mechanism in which a protein uses two wedges to act as a molecular crimper.

  9. DbpA: a DEAD box protein specifically activated by 23s rRNA.

    PubMed Central

    Fuller-Pace, F V; Nicol, S M; Reid, A D; Lane, D P

    1993-01-01

    The Escherichia coli protein DbpA is a member of the 'DEAD box' family of putative RNA-dependent ATPases and RNA helicases, so called because they share the highly conserved motif Asp-Glu-Ala-Asp, together with several other conserved elements. We have investigated DbpA expression under conditions where an endogenous promoter is used. In this context, translation initiation does not occur at the previously identified AUG, but at an upstream, in-frame GUG. Mutation of the GUG initiation codon to AUG virtually abolishes DbpA expression, suggesting an unusual translation initiation mechanism. Using an inducible overexpression plasmid, we have purified milligram quantities of DbpA to homogeneity and shown that the purified protein hydrolyses ATP in an RNA-dependent manner. This ATPase activity is interesting in that, unlike that of other DEAD box proteins investigated to date, it absolutely requires a specific bacterial RNA, which we have identified as 23S rRNA. This observation is particularly significant since DbpA will bind other RNAs and DNA, but will only hydrolyse ATP in the presence of 23S rRNA. Images PMID:8253085

  10. SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato.

    PubMed

    Zhu, Mingku; Chen, Guoping; Dong, Tingting; Wang, Lingling; Zhang, Jianling; Zhao, Zhiping; Hu, Zongli

    2015-01-01

    The DEAD-box RNA helicases are involved in almost every aspect of RNA metabolism, associated with diverse cellular functions including plant growth and development, and their importance in response to biotic and abiotic stresses is only beginning to emerge. However, none of DEAD-box genes was well characterized in tomato so far. In this study, we reported on the identification and characterization of two putative DEAD-box RNA helicase genes, SlDEAD30 and SlDEAD31 from tomato, which were classified into stress-related DEAD-box proteins by phylogenetic analysis. Expression analysis indicated that SlDEAD30 was highly expressed in roots and mature leaves, while SlDEAD31 was constantly expressed in various tissues. Furthermore, the expression of both genes was induced mainly in roots under NaCl stress, and SlDEAD31 mRNA was also increased by heat, cold, and dehydration. In stress assays, transgenic tomato plants overexpressing SlDEAD31 exhibited dramatically enhanced salt tolerance and slightly improved drought resistance, which were simultaneously demonstrated by significantly enhanced expression of multiple biotic and abiotic stress-related genes, higher survival rate, relative water content (RWC) and chlorophyll content, and lower water loss rate and malondialdehyde (MDA) production compared to wild-type plants. Collectively, these results provide a preliminary characterization of SlDEAD30 and SlDEAD31 genes in tomato, and suggest that stress-responsive SlDEAD31 is essential for salt and drought tolerance and stress-related gene regulation in plants.

  11. SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato

    PubMed Central

    Zhu, Mingku; Chen, Guoping; Dong, Tingting; Wang, Lingling; Zhang, Jianling; Zhao, Zhiping; Hu, Zongli

    2015-01-01

    The DEAD-box RNA helicases are involved in almost every aspect of RNA metabolism, associated with diverse cellular functions including plant growth and development, and their importance in response to biotic and abiotic stresses is only beginning to emerge. However, none of DEAD-box genes was well characterized in tomato so far. In this study, we reported on the identification and characterization of two putative DEAD-box RNA helicase genes, SlDEAD30 and SlDEAD31 from tomato, which were classified into stress-related DEAD-box proteins by phylogenetic analysis. Expression analysis indicated that SlDEAD30 was highly expressed in roots and mature leaves, while SlDEAD31 was constantly expressed in various tissues. Furthermore, the expression of both genes was induced mainly in roots under NaCl stress, and SlDEAD31 mRNA was also increased by heat, cold, and dehydration. In stress assays, transgenic tomato plants overexpressing SlDEAD31 exhibited dramatically enhanced salt tolerance and slightly improved drought resistance, which were simultaneously demonstrated by significantly enhanced expression of multiple biotic and abiotic stress-related genes, higher survival rate, relative water content (RWC) and chlorophyll content, and lower water loss rate and malondialdehyde (MDA) production compared to wild-type plants. Collectively, these results provide a preliminary characterization of SlDEAD30 and SlDEAD31 genes in tomato, and suggest that stress-responsive SlDEAD31 is essential for salt and drought tolerance and stress-related gene regulation in plants. PMID:26241658

  12. The embryonic RNA helicase gene (ERH): a new member of the DEAD box family of RNA helicases.

    PubMed Central

    Sowden, J; Putt, W; Morrison, K; Beddington, R; Edwards, Y

    1995-01-01

    DEAD box proteins share several highly conserved motifs including the characteristic Asp-Glu-Ala-Asp (D-E-A-D in the amino acid single-letter code) motif and have established or putative ATP-dependent RNA helicase activity. These proteins are implicated in a range of cellular processes that involve regulation of RNA function, including translation initiation, RNA splicing and ribosome assembly. Here we describe the isolation and characterization of an embryonic RNA helicase gene, ERH, which maps to mouse chromosome 1 and encodes a new member of the DEAD box family of proteins. The predicted ERH protein shows high sequence similarity to the testes-specific mouse PL10 and to the maternally acting Xenopus An3 helicase proteins. The ERH expression profile is similar, to that of An3, which localizes to the animal hemisphere of oocytes and is abundantly expressed in the embryo. ERH is expressed in oocytes and is a ubiquitous mRNA in the 9 days-post-conception embryo, and at later stages of development shows a more restricted pattern of expression in brain and kidney. The similarities in sequence and in expression profile suggest that ERH is the murine equivalent of the Xenopus An3 gene, and we propose that ERH plays a role in translational activation of mRNA in the oocyte and early embryo. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8948440

  13. Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p.

    PubMed

    Mallam, Anna L; Del Campo, Mark; Gilman, Benjamin; Sidote, David J; Lambowitz, Alan M

    2012-10-04

    DEAD-box proteins are the largest family of nucleic acid helicases, and are crucial to RNA metabolism throughout all domains of life. They contain a conserved 'helicase core' of two RecA-like domains (domains (D)1 and D2), which uses ATP to catalyse the unwinding of short RNA duplexes by non-processive, local strand separation. This mode of action differs from that of translocating helicases and allows DEAD-box proteins to remodel large RNAs and RNA-protein complexes without globally disrupting RNA structure. However, the structural basis for this distinctive mode of RNA unwinding remains unclear. Here, structural, biochemical and genetic analyses of the yeast DEAD-box protein Mss116p indicate that the helicase core domains have modular functions that enable a novel mechanism for RNA-duplex recognition and unwinding. By investigating D1 and D2 individually and together, we find that D1 acts as an ATP-binding domain and D2 functions as an RNA-duplex recognition domain. D2 contains a nucleic-acid-binding pocket that is formed by conserved DEAD-box protein sequence motifs and accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Upon a conformational change in which the two core domains join to form a 'closed state' with an ATPase active site, conserved motifs in D1 promote the unwinding of duplex substrates bound to D2 by excluding one RNA strand and bending the other. Our results provide a comprehensive structural model for how DEAD-box proteins recognize and unwind RNA duplexes. This model explains key features of DEAD-box protein function and affords a new perspective on how the evolutionarily related cores of other RNA and DNA helicases diverged to use different mechanisms.

  14. An ecdysone-inducible putative "DEAD box" RNA helicase in the spruce budworm (Choristoneura fumiferana).

    PubMed

    Zhang, D-Y; Zheng, S-C; Zheng, Y-P; Ladd, T R; Pang, A S D; Davey, K G; Krell, P J; Arif, B M; Retnakaran, A; Feng, Q-L

    2004-03-01

    RNA helicases are a family of enzymes that unwind nucleic acid duplexes, such as RNA/RNA and RNA/DNA, in a 3' to 5' direction into single-stranded polynucleotides. A putative RNA helicase cDNA (CfrHlc64) was isolated from the spruce budworm, Choristoneura fumiferana. CfrHlc64 was 1998 nucleotides in length, and the deduced protein had 565 amino acids with a predicted molecular mass of 64 kDa. It contained eight functional motifs conserved in the "DEAD box" family of RNA helicases. The deduced amino acid sequence showed 10-50% identities to homologues of other species from bacteria to human. In vitro expression of the cDNA resulted in recombinant proteins of 64 kDa as expected from the deduced amino acid sequence. Northern blotting and RT-PCR analyses revealed the presence of CfrHlc64 mRNA in all developmental stages from embryo to adult. Higher levels of CfrHlc64 mRNA were detected in the fat body and midgut than in the epidermis of sixth instar larvae. The CfrHlc64 protein was distributed mainly in the fat body. Female adults expressed CfrHlc64 mRNA at higher levels than male adults. The nonsteroidal ecdysone agonist, tebufenozide, enhanced the expression of CfrHlc64 in a dose-dependent manner.

  15. Characterization of a DEAD box ATPase/RNA helicase protein of Arabidopsis thaliana.

    PubMed Central

    Okanami, M; Meshi, T; Iwabuchi, M

    1998-01-01

    We have isolated cDNAs encoding a novel member of the DEAD box RNA helicase family from Arabidopsis. The protein, named AtDRH1, is composed of 619 amino acids and the central portion has high similarity with the helicase core region of a prototypic RNA helicase, the human nuclear protein p68. The N- and C-terminal regions are considerably diverged from the animal and yeast p68 homologs at the amino acid sequence level, but like the p68 subfamily members, an RGG box-like domain is present near the C-terminus. RNA blot analysis showed that the AtDRH1 transcript accumulates at a high level and almost equally in every part of the Arabidopsis plant. The purified, recombinant AtDRH1 was capable of unwinding double-stranded RNA in the presence of ATP or dATP and of hydrolyzing ATP. The ATPase activity was stimulated by some single-stranded RNAs and DNAs, including poly(A) and poly(dT), but not by poly(dA). The ability of the polynucleotides to stimulate the ATPase activity was largely consistent with their affinity for AtDRH1. These results show that AtDRH1 is a novel type of ATP/dATP-dependent RNA helicase and polynucleotide-dependent ATPase. PMID:9592148

  16. Duplex unwinding with DEAD-box proteins.

    PubMed

    Jankowsky, Eckhard; Putnam, Andrea

    2010-01-01

    DEAD-box proteins, which comprise the largest helicase family, are involved in virtually all aspects of RNA metabolism. DEAD-box proteins catalyze diverse ATP-driven functions including the unwinding of RNA secondary structures. In contrast to many well-studied DNA and viral RNA helicases, DEAD-box proteins do not rely on translocation on one of the nucleic acid strands for duplex unwinding, but directly load onto helical regions and then locally pry the strands apart in an ATP-dependent fashion. In this chapter, we outline substrate design and unwinding protocols for DEAD-box proteins and focus on the quantitative evaluation of their unwinding activity.

  17. A DEAD-box-family protein is required for nucleocytoplasmic transport of yeast mRNA.

    PubMed Central

    Liang, S; Hitomi, M; Hu, Y H; Liu, Y; Tartakoff, A M

    1996-01-01

    An enormous variety of primary and secondary mRNA structures are compatible with export from the nucleus to the cytoplasm. Therefore, there seems to be a mechanism for RNA export which is independent of sequence recognition. There nevertheless is likely to be some relatively uniform mechanism which allows transcripts to be packaged as ribonucleoprotein particles, to gain access to the periphery of the nucleus and ultimately to translocate across nuclear pores. To study these events, we and others have generated temperature-sensitive recessive mRNA transport (mtr) mutants of Saccharomyces cerevisiae which accumulate poly(A)+ RNA in the nucleus at 37 degrees C. Several of the corresponding genes have been cloned. Upon depletion of one of these proteins, Mtr4p, conspicuous amounts of nuclear poly(A)+ RNA accumulate in association with the nucleolus. Corresponding dense material is also seen by electron microscopy. MTR4 is essential for growth and encodes a novel nuclear protein with a size of approximately 120 kDa. Mtr4p shares characteristic motifs with DEAD-box RNA helicases and associates with RNA. It therefore may well affect RNA conformation. It shows extensive homology to a human predicted gene product and the yeast antiviral protein Ski2p. Critical residues of Mtr4p, including the mtr4-1 point mutation, have been identified. Mtr4p may serve as a chaperone which translocates or normalizes the structure of mRNAs in preparation for export. PMID:8756671

  18. Unwinding by local strand separation is critical for the function of DEAD-box proteins as RNA chaperones

    PubMed Central

    Campo, Mark Del; Mohr, Sabine; Jiang, Yue; Jia, Huijue; Jankowsky, Eckhard

    2009-01-01

    The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements the mitochondrial translation and group I and II intron splicing defects in mss116Δ strains, stimulates the in vitro splicing of group I as well as group II introns, and functions indistinguishably from CYT-19 to resolve different non-native secondary and/or tertiary structures in the Tetrahymena thermophila LSU-ΔP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and vaccinia virus DExH-box protein NPH-II gave little if any group I or II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. By contrast, DbpA binds group I and II intron RNAs non-specifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and II intron splicing correlates primarily with their RNA-unwinding activity, which for the protein preparations used here was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Further, this correlation holds for all group I and II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their

  19. Pathway of ATP utilization and duplex rRNA unwinding by the DEAD-box helicase, DbpA.

    PubMed

    Henn, Arnon; Cao, Wenxiang; Licciardello, Nicholas; Heitkamp, Sara E; Hackney, David D; De La Cruz, Enrique M

    2010-03-02

    DEAD-box RNA helicase proteins use the energy of ATP hydrolysis to drive the unwinding of duplex RNA. However, the mechanism that couples ATP utilization to duplex RNA unwinding is unknown. We measured ATP utilization and duplex RNA unwinding by DbpA, a non-processive bacterial DEAD-box RNA helicase specifically activated by the peptidyl transferase center (PTC) of 23S rRNA. Consumption of a single ATP molecule is sufficient to unwind and displace an 8 base pair rRNA strand annealed to a 32 base pair PTC-RNA "mother strand" fragment. Strand displacement occurs after ATP binding and hydrolysis but before P(i) product release. P(i) release weakens binding to rRNA, thereby facilitating the release of the unwound rRNA mother strand and the recycling of DbpA for additional rounds of unwinding. This work explains how ATPase activity of DEAD-box helicases is linked to RNA unwinding.

  20. Requirement of DDX39 DEAD box RNA helicase for genome integrity and telomere protection.

    PubMed

    Yoo, Hyun Hee; Chung, In Kwon

    2011-08-01

    Human chromosome ends associate with shelterin, a six-protein complex that protects telomeric DNA from being recognized as sites of DNA damage. The shelterin subunit TRF2 has been implicated in the protection of chromosome ends by facilitating their organization into the protective capping structure and by associating with several accessory proteins involved in various DNA transactions. Here we describe the characterization of DDX39 DEAD-box RNA helicase as a novel TRF2-interacting protein. DDX39 directly interacts with the telomeric repeat binding factor homology domain of TRF2 via the FXLXP motif (where X is any amino acid). DDX39 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT but has no effect on telomerase activity. Whereas overexpression of DDX39 in telomerase-positive human cancer cells led to progressive telomere elongation, depletion of endogenous DDX39 by small hairpin RNA (shRNA) resulted in telomere shortening. Furthermore, depletion of DDX39 induced DNA-damage response foci at internal genome as well as telomeres as evidenced by telomere dysfunction-induced foci. Some of the metaphase chromosomes showed no telomeric signal at chromatid ends, suggesting an aberrant telomere structure. Our findings suggest that DDX39, in addition to its role in mRNA splicing and nuclear export, is required for global genome integrity as well as telomere protection and represents a new pathway for telomere maintenance by modulating telomere length homeostasis.

  1. Structure-guided mutational analysis of a yeast DEAD-box protein involved in mitochondrial RNA splicing.

    PubMed

    Bifano, Abby L; Turk, Edward M; Caprara, Mark G

    2010-05-07

    DEAD-box proteins are RNA-dependent ATPase enzymes that have been implicated in nearly all aspects of RNA metabolism. Since many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, to bind nucleic acid, and to promote helix unwinding, DEAD-box proteins are generally thought to modulate RNA structure in vivo. However, the extent to which these enzymatic properties are important for the in vivo functions of DEAD-box proteins remains unclear. To evaluate how these properties influence DEAD-box protein native function, we probed the importance of several highly conserved residues in the yeast DEAD-box protein Mss116p, which is required for the splicing of all mitochondrial catalytic introns in Saccharomyces cerevisiae. Using an MSS116 deletion strain, we have expressed plasmid-borne variants of MSS116 containing substitutions in residues predicted to be important for extensive networks of interactions required for ATP hydrolysis and helix unwinding. We have analyzed the importance of these residues to the splicing functions of Mss116p in vivo and compared these results with the biochemical properties of recombinant proteins determined here and in previously published work. We observed that the efficiency by which an Mss116p variant catalyzes ATP hydrolysis correlates with facilitating mitochondrial splicing, while efficient helix unwinding appears to be insufficient for splicing. In addition, we show that each splicing-defective variant affects the splicing of structurally diverse introns to the same degree. Together, these observations suggest that the efficiency by which Mss116p catalyzes the hydrolysis of ATP is critical for all of its splicing functions in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, these observations support a model in which enzyme turnover is a crucial factor in Mss116p splicing function. These results are discussed in the context

  2. Structure-Guided Mutational Analysis of a Yeast DEAD-box Protein Involved in Mitochondrial RNA Splicing

    PubMed Central

    Bifano, Abby L.; Turk, Edward M.; Caprara, Mark G.

    2010-01-01

    Summary DEAD-box proteins are RNA-dependent ATPase enzymes that have been implicated in nearly all aspects of RNA metabolism. Since many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, bind nucleic acid, and promote helix unwinding, DEAD-box proteins are generally thought to modulate RNA structure in vivo. However, the extent to which these enzymatic properties are important for the in vivo functions of DEAD-box proteins remains unclear. To evaluate how these properties influence DEAD-box protein native function, we probe the importance of several highly conserved residues in the yeast DEAD-box protein, Mss116p, which is required for the splicing of all mitochondrial catalytic introns in Saccharomyces cerevisiae. Using a MSS116 deletion strain, we have expressed plasmid-borne variants of MSS116 containing substitutions in residues predicted to be important for extensive networks of interactions required for ATP hydrolysis and helix unwinding. We have analyzed the importance of these residues to the splicing functions of Mss116p in vivo and compared these results with the biochemical properties of recombinant proteins determined here and in previously published work. We observe that the efficiency by which an Mss116p variant catalyzes ATP hydrolysis correlates with facilitating mitochondrial splicing, while efficient helix unwinding appears to be insufficient for splicing. In addition, we show that each splicing-defective variant affects the splicing of structurally diverse introns to the same degree. Together, these observations suggest that the efficiency by which Mss116p catalyzes the hydrolysis of ATP is critical for all of its splicing functions in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, these observations support a model in which enzyme turnover is a crucial factor in Mss116p splicing function. These results are discussed in the context

  3. Structural Insights into a Unique Dimeric DEAD-Box Helicase CshA that Promotes RNA Decay.

    PubMed

    Huen, Jennifer; Lin, Chia-Liang; Golzarroshan, Bagher; Yi, Wan-Li; Yang, Wei-Zen; Yuan, Hanna S

    2017-03-07

    CshA is a dimeric DEAD-box helicase that cooperates with ribonucleases for mRNA turnover. The molecular mechanism for how a dimeric DEAD-box helicase aids in RNA decay remains unknown. Here, we report the crystal structure and small-angle X-ray scattering solution structure of the CshA from Geobacillus stearothermophilus. In contrast to typical monomeric DEAD-box helicases, CshA is exclusively a dimeric protein with the RecA-like domains of each protomer forming a V-shaped structure. We show that the C-terminal domains protruding outward from the tip of the V-shaped structure is critical for mediating strong RNA binding and is crucial for efficient RNA-dependent ATP hydrolysis. We also show that RNA remains bound with CshA during ATP hydrolysis cycles and thus bulk RNAs could be unwound and degraded in a processive manner through cooperation between exoribonucleases and CshA. A dimeric helicase is hence preserved in RNA-degrading machinery for efficient RNA turnover in prokaryotes and eukaryotes.

  4. Allosteric regulation of helicase core activities of the DEAD-box helicase YxiN by RNA binding to its RNA recognition motif.

    PubMed

    Samatanga, Brighton; Andreou, Alexandra Z; Klostermeier, Dagmar

    2017-01-23

    DEAD-box proteins share a structurally similar core of two RecA-like domains (RecA_N and RecA_C) that contain the conserved motifs for ATP-dependent RNA unwinding. In many DEAD-box proteins the helicase core is flanked by ancillary domains. To understand the regulation of the DEAD-box helicase YxiN by its C-terminal RNA recognition motif (RRM), we investigated the effect of RNA binding to the RRM on its position relative to the core, and on core activities. RRM/RNA complex formation substantially shifts the RRM from a position close to the RecA_C to the proximity of RecA_N, independent of RNA contacts with the core. RNA binding to the RRM is communicated to the core, and stimulates ATP hydrolysis and RNA unwinding. The conformational space of the core depends on the identity of the RRM-bound RNA. Allosteric regulation of core activities by RNA-induced movement of ancillary domains may constitute a general regulatory mechanism of DEAD-box protein activity.

  5. Regulation of Notch Signaling by an Evolutionary Conserved DEAD Box RNA Helicase, Maheshvara in Drosophila melanogaster.

    PubMed

    Surabhi, Satya; Tripathi, Bipin K; Maurya, Bhawana; Bhaskar, Pradeep K; Mukherjee, Ashim; Mutsuddi, Mousumi

    2015-11-01

    Notch signaling is an evolutionary conserved process that influences cell fate determination, cell proliferation, and cell death in a context-dependent manner. Notch signaling is fine-tuned at multiple levels and misregulation of Notch has been implicated in a variety of human diseases. We have characterized maheshvara (mahe), a novel gene in Drosophila melanogaster that encodes a putative DEAD box protein that is highly conserved across taxa and belongs to the largest group of RNA helicase. A dynamic pattern of mahe expression along with the maternal accumulation of its transcripts is seen during early stages of embryogenesis. In addition, a strong expression is also seen in the developing nervous system. Ectopic expression of mahe in a wide range of tissues during development results in a variety of defects, many of which resemble a typical Notch loss-of-function phenotype. We illustrate that ectopic expression of mahe in the wing imaginal discs leads to loss of Notch targets, Cut and Wingless. Interestingly, Notch protein levels are also lowered, whereas no obvious change is seen in the levels of Notch transcripts. In addition, mahe overexpression can significantly rescue ectopic Notch-mediated proliferation of eye tissue. Further, we illustrate that mahe genetically interacts with Notch and its cytoplasmic regulator deltex in trans-heterozygous combination. Coexpression of Deltex and Mahe at the dorso-ventral boundary results in a wing-nicking phenotype and a more pronounced loss of Notch target Cut. Taken together we report identification of a novel evolutionary conserved RNA helicase mahe, which plays a vital role in regulation of Notch signaling.

  6. Characterization of DbpA, an Escherichia coli DEAD box protein with ATP independent RNA unwinding activity.

    PubMed Central

    Böddeker, N; Stade, K; Franceschi, F

    1997-01-01

    DbpA is a putative Escherichia coli ATP dependent RNA helicase belonging to the family of DEAD box proteins. It hydrolyzes ATP in the presence of 23S ribosomal RNA and 93 bases in the peptidyl transferase center of 23S rRNA are sufficient to trigger 100% of the ATPase activity of DbpA. In the present study we characterized the ATPase and RNA unwinding activities of DbpA in more detail. We report that-in contrast to eIF-4A, the prototype of the DEAD box protein family-the ATPase and the helicase activities of DbpA are not coupled. Moreover, the RNA unwinding activity of DbpA is not specific for 23S rRNA, since DbpA is also able to unwind 16S rRNA hybrids. Furthermore, we determined that the ATPase activity of DbpA is triggered to a significant extent not only by the 93 bases of the 23S rRNA previously reported but also by other regions of the 23S rRNA molecule. Since all these regions of 23S rRNA are either part of the 'functional core' of the 50S ribosomal subunit or involved in the 50S assembly, DbpA may play an important role in the ribosomal assembly process. PMID:9016593

  7. Global effects of the DEAD-box RNA helicase DeaD (CsdA) on gene expression over a broad range of temperatures

    PubMed Central

    Vakulskas, Christopher A.; Pannuri, Archana; Cortés-Selva, Diana; Zere, Tesfalem R.; Ahmer, Brian M.; Babitzke, Paul; Romeo, Tony

    2014-01-01

    Summary In Escherichia coli, activity of the global regulatory RNA binding protein CsrA is antagonized by two noncoding sRNAs, CsrB and CsrC, which sequester it away from its lower affinity mRNA targets. Transcription of csrB/C requires the BarA-UvrY two component signal transduction system, which responds to short chain carboxylates. We show that two DEAD-box RNA helicases, DeaD and SrmB, activate csrB/C expression by different pathways. DeaD facilitates uvrY translation by counteracting the inhibitory effect of long distance basepairing between the uvrY mRNA leader and coding region, while SrmB does not affect UvrY or UvrY-phosphate levels. Contrary to the prevailing notion that these helicases act primarily at low temperatures, DeaD and SrmB activated csrB expression over a wide temperature range. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) revealed in vivo interactions of DeaD with 39 mRNAs, including those of uvrY and 9 other regulatory genes. Studies on the expression of several of the identified genes revealed regulatory effects of DeaD in all cases and diverse temperature response patterns. Our findings uncover an expanded regulatory role for DeaD, which is mediated through novel mRNA targets, important global regulators and under physiological conditions that were considered to be incompatible with its function. PMID:24708042

  8. A Cold-Inducible DEAD-Box RNA Helicase from Arabidopsis thaliana Regulates Plant Growth and Development under Low Temperature

    PubMed Central

    Liu, Yuelin; Tabata, Daisuke; Imai, Ryozo

    2016-01-01

    DEAD-box RNA helicases comprise a large family and are involved in a range of RNA processing events. Here, we identified one of the Arabidopsis thaliana DEAD-box RNA helicases, AtRH7, as an interactor of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3), which is an RNA chaperone involved in cold adaptation. Promoter:GUS transgenic plants revealed that AtRH7 is expressed ubiquitously and that its levels of the expression are higher in rapidly growing tissues. Knockout mutant lines displayed several morphological alterations such as disturbed vein pattern, pointed first true leaves, and short roots, which resemble ribosome-related mutants of Arabidopsis. In addition, aberrant floral development was also observed in rh7 mutants. When the mutants were germinated at low temperature (12°C), both radicle and first leaf emergence were severely delayed; after exposure of seedlings to a long period of cold, the mutants developed aberrant, fewer, and smaller leaves. RNA blots and circular RT-PCR revealed that 35S and 18S rRNA precursors accumulated to higher levels in the mutants than in WT under both normal and cold conditions, suggesting the mutants are partially impaired in pre-rRNA processing. Taken together, the results suggest that AtRH7 affects rRNA biogenesis and plays an important role in plant growth under cold. PMID:27116354

  9. A Cold-Inducible DEAD-Box RNA Helicase from Arabidopsis thaliana Regulates Plant Growth and Development under Low Temperature.

    PubMed

    Liu, Yuelin; Tabata, Daisuke; Imai, Ryozo

    2016-01-01

    DEAD-box RNA helicases comprise a large family and are involved in a range of RNA processing events. Here, we identified one of the Arabidopsis thaliana DEAD-box RNA helicases, AtRH7, as an interactor of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3), which is an RNA chaperone involved in cold adaptation. Promoter:GUS transgenic plants revealed that AtRH7 is expressed ubiquitously and that its levels of the expression are higher in rapidly growing tissues. Knockout mutant lines displayed several morphological alterations such as disturbed vein pattern, pointed first true leaves, and short roots, which resemble ribosome-related mutants of Arabidopsis. In addition, aberrant floral development was also observed in rh7 mutants. When the mutants were germinated at low temperature (12°C), both radicle and first leaf emergence were severely delayed; after exposure of seedlings to a long period of cold, the mutants developed aberrant, fewer, and smaller leaves. RNA blots and circular RT-PCR revealed that 35S and 18S rRNA precursors accumulated to higher levels in the mutants than in WT under both normal and cold conditions, suggesting the mutants are partially impaired in pre-rRNA processing. Taken together, the results suggest that AtRH7 affects rRNA biogenesis and plays an important role in plant growth under cold.

  10. Pisum sativum p68 DEAD-box protein is ATP-dependent RNA helicase and unique bipolar DNA helicase.

    PubMed

    Tuteja, Narendra; Tarique, Mohammed; Banu, Mst Sufara Akhter; Ahmad, Moaz; Tuteja, Renu

    2014-08-01

    DEAD-box helicases play essential role in DNA and RNA metabolism such as replication, repair, recombination, transcription, translation, ribosome biogenesis and splicing which regulate plant growth and development. The presence of helicases in the stress-induced ORFs identified by cDNA microarray indicates that helicases might be playing an important role in stabilizing growth in plants under stress. p68 DEAD-box helicase has been identified and characterized from animal systems but the properties and functions of plant p68 are poorly understood. In this study, the identification, purification and characterization of recombinant p68 from Pisum sativum (Psp68) is presented. Psp68 possesses all the characteristic motifs like DEAD-box ATP-binding and helicase C terminal motifs and is structurally similar to human p68 homologue. Psp68 exhibits ATPase activity in the presence of both DNA and RNA and it binds to DNA as well as RNA. It contains the characteristic RNA helicase activity. Interestingly Psp68 also shows the unique DNA helicase activity, which is bipolar in nature (unwinds DNA in both the 5'-3' and 3'-5' directions). The Km values of Psp68 for ATPase are 0.5126 and 0.9142 mM in the presence of DNA and RNA, respectively. The Km values of Psp68 are 1.6129 and 1.14 nM for DNA helicase and RNA helicase, respectively. The unique properties of Psp68 suggest that it could be a multifunctional protein involved in different aspect of DNA and RNA metabolism. This discovery should make an important contribution to better understanding of nucleic acids metabolism plants.

  11. DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function.

    PubMed

    Soltanieh, Sahar; Osheim, Yvonne N; Spasov, Krasimir; Trahan, Christian; Beyer, Ann L; Dragon, François

    2015-03-01

    DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.

  12. DEAD-Box RNA Helicase Dbp4 Is Required for Small-Subunit Processome Formation and Function

    PubMed Central

    Soltanieh, Sahar; Osheim, Yvonne N.; Spasov, Krasimir; Trahan, Christian; Beyer, Ann L.

    2014-01-01

    DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5′ end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA. PMID:25535329

  13. The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA.

    PubMed Central

    Nicol, S M; Fuller-Pace, F V

    1995-01-01

    The Escherichia coli DEAD (Asp-Glu-Ala-Asp) box protein DbpA is a putative RNA helicase and established RNA-dependent ATPase and is the only member of the DEAD box protein family for which a specific RNA substrate, bacterial 23S rRNA, has been identified. We have investigated the nature of this specificity in depth and have localized by deletion mutagenesis and PCR a single region of 93 bases (bases 2496-2588) in 23S rRNA that is both necessary and sufficient for complete activation of ATPase activity of DbpA. This target region forms part of the peptidyltransferase center and includes many bases involved in interaction with the 3' terminal adenosines of both A- and P-site tRNAs. Deletion of stem loops within the 93-base segment abolished ATPase activation. Similarly, point mutations that disrupt base pairing within stem structures ablated stimulation of ATPase activity. These data are consistent with roles for DbpA either in establishing and/or maintaining the correct three-dimensional structure of the peptidyltransferase center in 23S rRNA during ribosome assembly or in the peptidyltransferase reaction. Images Fig. 1 Fig. 2 PMID:8524828

  14. Molecular cloning and characterization of a putative nuclear DEAD box RNA helicase in the spruce budworm, Choristoneura fumiferana.

    PubMed

    Zhang, D-Y; Ampasala, D R; Zheng, S-C; Cusson, M; Cheng, X-W; Krell, P J; Feng, Q-L

    2006-04-01

    RNA helicases play important roles in cellular processes such as pre-mRNA splicing, rRNA processing, ribosomal biogenesis, and translation. A full-length DEAD box RNA helicase cDNA (CfrHlc113) was isolated from the spruce budworm, Choristoneura fumiferana. CfrHlc113 contained the eight functional motifs, which are highly conserved in the DEAD box RNA helicase family, and an arginine-serine-aspartate (RSD) domain at its N-terminal end. CfrHlc113 was highly homologous to Rattus norvegicus HEL117 and human prp5 genes, both of which are suggested to be involved in RNA splicing. The results of Northern and Western blotting showed that expression of the CfrHlc113 gene was low or undetectable in eggs, larvae, pupae, and adults. High levels of expression were, however, detected in the three in vitro cultured cell lines, CF-203, CF-124T, and CF-70, which were developed from the midgut, ovaries, and neonate larvae, respectively. Immunocytochemistry revealed that CfrHlc113 protein was present exclusively in the nuclei of these cell lines.

  15. Transcriptome-wide analysis of DEAD-box RNA helicase gene family in an Antarctic psychrophilic alga Chlamydomonas sp. ICE-L.

    PubMed

    Liu, Chenlin; Huang, Xiaohang

    2015-09-01

    DEAD-box RNA helicase family proteins have been identified in almost all living organisms. Some of them play a crucial role in adaptation to environmental changes and stress response, especially in the low-temperature acclimation in different kinds of organisms. Compared with the full swing study in plants and bacteria, the characters and functions of DEAD-box family proteins had not been surveyed in algae. To identify genes critical for freezing acclimation in algae, we screened DEAD-box RNA helicase genes from the transcriptome sequences of a psychrophilic microalga Chlamydomonas sp. ICE-L which was isolated from Antarctic sea ice. Totally 39 DEAD-box RNA helicase genes had been identified. Most of the DEAD-box RNA helicase have 1:1 homologous relationships in Chlamydomonas reinhardtii and Chlamydomonas sp. ICE-L with several exceptions. The homologous proteins in ICE-L to the helicases critical for cold or freezing tolerance in Arabidopsis thaliana had been identified based on phylogenetic comparison studies. The response of these helicase genes is not always identical in the Chlamydomonas sp. ICE-L and Arabidopsis under the same low-temperature treatment. The expression of several DEAD-box RNA helicase genes including CiRH5, CiRH25, CiRH28, and CiRH55 were significantly up-regulated under freezing treatment of ICE-L and their function in freezing acclimation of ICE-L deserved further investigation.

  16. A role for Gle1, a regulator of DEAD-box RNA helicases, at centrosomes and basal bodies

    PubMed Central

    Jao, Li-En; Akef, Abdalla; Wente, Susan R.

    2017-01-01

    Control of organellar assembly and function is critical to eukaryotic homeostasis and survival. Gle1 is a highly conserved regulator of RNA-dependent DEAD-box ATPase proteins, with critical roles in both mRNA export and translation. In addition to its well-defined interaction with nuclear pore complexes, here we find that Gle1 is enriched at the centrosome and basal body. Gle1 assembles into the toroid-shaped pericentriolar material around the mother centriole. Reduced Gle1 levels are correlated with decreased pericentrin localization at the centrosome and microtubule organization defects. Of importance, these alterations in centrosome integrity do not result from loss of mRNA export. Examination of the Kupffer’s vesicle in Gle1-depleted zebrafish revealed compromised ciliary beating and developmental defects. We propose that Gle1 assembly into the pericentriolar material positions the DEAD-box protein regulator to function in localized mRNA metabolism required for proper centrosome function. PMID:28035044

  17. Association of the cold-shock DEAD-box RNA helicase RhlE to the RNA degradosome in Caulobacter crescentus.

    PubMed

    Aguirre, Angel A; Vicente, Alexandre M; Hardwick, Steven W; Alvelos, Daniela M; Mazzon, Ricardo R; Luisi, Ben F; Marques, Marilis V

    2017-04-10

    In diverse bacterial lineages, multi-enzyme assemblies have evolved that are central elements of RNA metabolism and RNA-mediated regulation. The aquatic, Gram-negative bacteria Caulobacter crescentus, which has been a model system for studying the bacterial cell cycle, has an RNA degradosome assembly that is formed by the endoribonuclease RNase E and includes the DEAD-box RNA helicase RhlB. Immunoprecipitations of extracts from cells expressing an epitope-tagged RNase E reveal that RhlE, another member of the DEAD-box helicase family, associates with the degradosome at temperatures below the optimum for growth. Phenotype analyses of mutant strains for rhlE, rhlB and rhlE/rhlB show that RhlE is important for cell fitness at low temperature, and its role may not be substituted by RhlB. Transcriptional and translational fusions of rhlE to the lacZ reporter gene and immunoblot analysis of an epitope-tagged RhlE indicate that its expression is induced upon temperature decrease mainly through post-transcriptional regulation. RNase E pulldown assays show that other proteins, including the transcription termination factor Rho, a second DEAD-box RNA helicase and ribosomal protein S1 also associate with the degradosome at low temperature. The results suggest that the RNA degradosome assembly can be remodeled with environmental change to alter its repertoire of helicases and other accessory proteins.IMPORTANCE DEAD-box RNA helicases are often present in the RNA degradosome complex, helping to unwind secondary structures to facilitate degradation. Caulobacter crescentus is an interesting organism to investigate degradosome remodeling with temperature, because it thrives in freshwater bodies and withstands low temperature. In this study we show that at low temperature the cold-induced DEAD-box RNA helicase RhlE is recruited to the RNA degradosome, along with other helicases and the Rho protein. RhlE is essential for bacterial fitness at low temperature, and its function may not

  18. When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding.

    PubMed

    Rudolph, Markus G; Klostermeier, Dagmar

    2015-08-01

    DEAD-box helicases catalyze RNA duplex unwinding in an ATP-dependent reaction. Members of the DEAD-box helicase family consist of a common helicase core formed by two RecA-like domains. According to the current mechanistic model for DEAD-box mediated RNA unwinding, binding of RNA and ATP triggers a conformational change of the helicase core, and leads to formation of a compact, closed state. In the closed conformation, the two parts of the active site for ATP hydrolysis and of the RNA binding site, residing on the two RecA domains, become aligned. Closing of the helicase core is coupled to a deformation of the RNA backbone and destabilization of the RNA duplex, allowing for dissociation of one of the strands. The second strand remains bound to the helicase core until ATP hydrolysis and product release lead to re-opening of the core. The concomitant disruption of the RNA binding site causes dissociation of the second strand. The activity of the helicase core can be modulated by interaction partners, and by flanking N- and C-terminal domains. A number of C-terminal flanking regions have been implicated in RNA binding: RNA recognition motifs (RRM) typically mediate sequence-specific RNA binding, whereas positively charged, unstructured regions provide binding sites for structured RNA, without sequence-specificity. Interaction partners modulate RNA binding to the core, or bind to RNA regions emanating from the core. The functional interplay of the helicase core and ancillary domains or interaction partners in RNA binding and unwinding is not entirely understood. This review summarizes our current knowledge on RNA binding to the DEAD-box helicase core and the roles of ancillary domains and interaction partners in RNA binding and unwinding by DEAD-box proteins.

  19. Structure of the second domain of the Bacillus subtilis DEAD-box RNA helicase YxiN

    SciTech Connect

    Caruthers, Jonathan M.; Hu, YaoXiong; McKay, David B.

    2006-12-01

    The structure of the second helicase domain of the B. subtilis YxiN protein, a DEAD-box RNA helicase, is presented. The Bacillus subtilis RNA helicase YxiN is a modular three-domain protein. The first two domains form a conserved helicase core that couples an ATPase activity to an RNA duplex-destabilization activity, while the third domain recognizes a stem-loop of 23S ribosomal RNA with high affinity and specificity. The structure of the second domain, amino-acid residues 207–368, has been solved to 1.95 Å resolution, revealing a parallel αβ-fold. The crystallographic asymmetric unit contains two protomers; superposition shows that they differ substantially in two segments of peptide that overlap the conserved helicase sequence motifs V and VI, while the remainder of the domain is isostructural. The conformational variability of these segments suggests that induced fit is intrinsic to the recognition of ligands (ATP and RNA) and the coupling of the ATPase activity to conformational changes.

  20. Coupling between the DEAD-box RNA helicases Ded1p and eIF4A

    PubMed Central

    Gao, Zhaofeng; Putnam, Andrea A; Bowers, Heath A; Guenther, Ulf-Peter; Ye, Xuan; Kindsfather, Audrey; Hilliker, Angela K; Jankowsky, Eckhard

    2016-01-01

    Eukaryotic translation initiation involves two conserved DEAD-box RNA helicases, eIF4A and Ded1p. Here we show that S. cerevisiae eIF4A and Ded1p directly interact with each other and simultaneously with the scaffolding protein eIF4G. We delineate a comprehensive thermodynamic framework for the interactions between Ded1p, eIF4A, eIF4G, RNA and ATP, which indicates that eIF4A, with and without eIF4G, acts as a modulator for activity and substrate preferences of Ded1p, which is the RNA remodeling unit in all complexes. Our results reveal and characterize an unexpected interdependence between the two RNA helicases and eIF4G, and suggest that Ded1p is an integral part of eIF4F, the complex comprising eIF4G, eIF4A, and eIF4E. DOI: http://dx.doi.org/10.7554/eLife.16408.001 PMID:27494274

  1. The DEAD Box RNA Helicase VBH-1 Is a New Player in the Stress Response in C. elegans

    PubMed Central

    Paz-Gómez, Daniel; Villanueva-Chimal, Emmanuel; Navarro, Rosa E.

    2014-01-01

    For several years, DEAD box RNA helicase Vasa (DDX4) has been used as a bona fide germline marker in different organisms. C. elegans VBH-1 is a close homolog of the Vasa protein, which plays an important role in gametogenesis, germ cell survival and embryonic development. Here, we show that VBH-1 protects nematodes from heat shock and oxidative stress. Using the germline-defective mutant glp-4(bn2) we found that a potential somatic expression of vbh-1 might be important for stress survival. We also show that the VBH-1 paralog LAF-1 is important for stress survival, although this protein is not redundant with its counterpart. Furthermore, we observed that the mRNAs of the heat shock proteins hsp-1 and sip-1 are downregulated when vbh-1 or laf-1 are silenced. Previously, we reported that in C. elegans, VBH-1 was primarily expressed in P granules of germ cells and in the cytoplasm of all blastomeres. Here we show that during stress, VBH-1 co-localizes with CGH-1 in large aggregates in the gonad core and oocytes; however, VBH-1 aggregates do not overlap with CGH-1 foci in early embryos under the same conditions. These data demonstrate that, in addition to the previously described role for this protein in the germline, VBH-1 plays an important role during the stress response in C. elegans through the potential direct or indirect regulation of stress response mRNAs. PMID:24844228

  2. A conformational change in the helicase core is necessary but not sufficient for RNA unwinding by the DEAD box helicase YxiN.

    PubMed

    Karow, Anne R; Klostermeier, Dagmar

    2009-07-01

    Cooperative binding of ATP and RNA to DEAD-box helicases induces the closed conformation of their helicase core, with extensive interactions across the domain interface. The bound RNA is bent, and its distortion may constitute the first step towards RNA unwinding. To dissect the role of the conformational change in the helicase core for RNA unwinding, we characterized the RNA-stimulated ATPase activity, RNA unwinding and the propensity to form the closed conformer for mutants of the DEAD box helicase YxiN. The ATPase-deficient K52Q mutant forms a closed conformer upon binding of ATP and RNA, but is deficient in RNA unwinding. A mutation in motif III slows down the catalytic cycle, but neither affects the propensity for the closed conformer nor its global conformation. Hence, the closure of the cleft in the helicase core is necessary but not sufficient for RNA unwinding. In contrast, the G303A mutation in motif V prevents a complete closure of the inter-domain cleft, affecting ATP binding and hydrolysis and is detrimental to unwinding. Possibly, the K52Q and motif III mutants still introduce a kink into the backbone of bound RNA, whereas G303A fails to kink the RNA substrate.

  3. DDX3 DEAD-box RNA helicase plays a central role in mitochondrial protein quality control in Leishmania

    PubMed Central

    Padmanabhan, Prasad Kottayil; Zghidi-Abouzid, Ouafa; Samant, Mukesh; Dumas, Carole; Aguiar, Bruno Guedes; Estaquier, Jerome; Papadopoulou, Barbara

    2016-01-01

    DDX3 is a highly conserved member of ATP-dependent DEAD-box RNA helicases with multiple functions in RNA metabolism and cellular signaling. Here, we describe a novel function for DDX3 in regulating the mitochondrial stress response in the parasitic protozoan Leishmania. We show that genetic inactivation of DDX3 leads to the accumulation of mitochondrial reactive oxygen species (ROS) associated with a defect in hydrogen peroxide detoxification. Upon stress, ROS production is greatly enhanced, causing mitochondrial membrane potential loss, mitochondrial fragmentation, and cell death. Importantly, this phenotype is exacerbated upon oxidative stress in parasites forced to use the mitochondrial oxidative respiratory machinery. Furthermore, we show that in the absence of DDX3, levels of major components of the unfolded protein response as well as of polyubiquitinated proteins increase in the parasite, particularly in the mitochondrion, as an indicator of mitochondrial protein damage. Consistent with these findings, immunoprecipitation and mass-spectrometry studies revealed potential interactions of DDX3 with key components of the cellular stress response, particularly the antioxidant response, the unfolded protein response, and the AAA-ATPase p97/VCP/Cdc48, which is essential in mitochondrial protein quality control by driving proteosomal degradation of polyubiquitinated proteins. Complementation studies using DDX3 deletion mutants lacking conserved motifs within the helicase core support that binding of DDX3 to ATP is essential for DDX3's function in mitochondrial proteostasis. As a result of the inability of DDX3-depleted Leishmania to recover from ROS damage and to survive various stresses in the host macrophage, parasite intracellular development was impaired. Collectively, these observations support a central role for the Leishmania DDX3 homolog in preventing ROS-mediated damage and in maintaining mitochondrial protein quality control. PMID:27735940

  4. The DEAD-box RNA helicase Vasa functions in embryonic mitotic progression in the sea urchin.

    PubMed

    Yajima, Mamiko; Wessel, Gary M

    2011-06-01

    Vasa is a broadly conserved ATP-dependent RNA helicase that functions in the germ line of organisms from cnidarians to mammals. Curiously, Vasa is also present in the somatic cells of many animals and functions as a regulator of multipotent cells. Here, we report a mitotic function of Vasa revealed in the sea urchin embryo. We found that Vasa protein is present in all blastomeres of the early embryo and that its abundance oscillates with the cell cycle. Vasa associates with the spindle and the separating sister chromatids at metaphase, and then quickly disappears after telophase. Inhibition of Vasa protein synthesis interferes with proper chromosome segregation, arrests cells at M-phase, and delays overall cell cycle progression. Cdk activity is necessary for the proper localization of Vasa, implying that Vasa is involved in the cyclin-dependent cell cycle network, and Vasa is required for the efficient translation of cyclinB mRNA. Our results suggest an evolutionarily conserved role of Vasa that is independent of its function in germ line determination.

  5. A Conserved Phenylalanine of Motif IV in Superfamily 2 Helicases Is Required for Cooperative, ATP-Dependent Binding of RNA Substrates in DEAD-Box Proteins▿ †

    PubMed Central

    Banroques, Josette; Cordin, Olivier; Doère, Monique; Linder, Patrick; Tanner, N. Kyle

    2008-01-01

    We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-π interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding. PMID:18332124

  6. Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA

    PubMed Central

    Yao, Hongjie; Brick, Kevin; Evrard, Yvonne; Xiao, Tiaojiang; Camerini-Otero, R. Daniel; Felsenfeld, Gary

    2010-01-01

    CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, although the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. Here we showed that the DEAD-box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF by binding to both, and is required for proper insulator function. PMID:20966046

  7. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28

    SciTech Connect

    Jacewicz, Agata; Schwer, Beate; Smith, Paul; Shuman, Stewart

    2014-10-10

    Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to the adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. As a result, overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.

  8. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28

    DOE PAGES

    Jacewicz, Agata; Schwer, Beate; Smith, Paul; ...

    2014-10-10

    Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to themore » adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. As a result, overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.« less

  9. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

    SciTech Connect

    Kellner, Julian N.; Meinhart, Anton

    2015-08-25

    The structure of the SPRY domain of the human RNA helicase DDX1 was determined at 2.0 Å resolution. The SPRY domain provides a putative protein–protein interaction platform within DDX1 that differs from other SPRY domains in its structure and conserved regions. The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein–protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein–protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

  10. Recruitment, Duplex Unwinding and Protein-Mediated Inhibition of the Dead-Box RNA Helicase Dbp2 at Actively Transcribed Chromatin.

    PubMed

    Ma, Wai Kit; Paudel, Bishnu P; Xing, Zheng; Sabath, Ivan G; Rueda, David; Tran, Elizabeth J

    2016-03-27

    RNA helicases play fundamental roles in modulating RNA structures and facilitating RNA-protein (RNP) complex assembly in vivo. Previously, our laboratory demonstrated that the DEAD-box RNA helicase Dbp2 in Saccharomyces cerevisiae is required to promote efficient assembly of the co-transcriptionally associated mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A)(+)RNA. We also found that Yra1 associates directly with Dbp2 and functions as an inhibitor of Dbp2-dependent duplex unwinding, suggestive of a cycle of unwinding and inhibition by Dbp2. To test this, we undertook a series of experiments to shed light on the order of events for Dbp2 in co-transcriptional mRNP assembly. We now show that Dbp2 is recruited to chromatin via RNA and forms a large, RNA-dependent complex with Yra1 and Mex67. Moreover, single-molecule fluorescence resonance energy transfer and bulk biochemical assays show that Yra1 inhibits unwinding in a concentration-dependent manner by preventing the association of Dbp2 with single-stranded RNA. This inhibition prevents over-accumulation of Dbp2 on mRNA and stabilization of a subset of RNA polymerase II transcripts. We propose a model whereby Yra1 terminates a cycle of mRNP assembly by Dbp2.

  11. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein.

    PubMed

    Kellner, Julian N; Meinhart, Anton

    2015-09-01

    The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein-protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein-protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

  12. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    PubMed

    Garbelli, Anna; Beermann, Sandra; Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-05-12

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  13. Molecular characterization and expression of buffalo (Bubalus bubalis) DEAD-box family VASA gene and mRNA transcript variants isolated from testis tissue.

    PubMed

    Kaushik, Ramakant; Singh, Karn Pratap; Bahuguna, Vivek; Rameshbabu, K; Singh, Manoj Kumar; Manik, Radhey Shyam; Palta, Prabhat; Singla, Suresh Kumar; Chauhan, Manmohan Singh

    2015-11-01

    VASA is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. In the present study, we isolated, sequenced, and characterized VASA gene in buffalo testis. Here, we demonstrated that VASA mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and four different polyadenylation sites. The TSSs identified by 5'-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5'-RACE) were positioned at 48, 53, 85, and 88 nucleotides upstream relative to the translation initiation codon. 3'-RACE experiment revealed the presence of tandem polyadenylation signals, which lead to the expression of at least four different 3'-untranslated regions (209, 233, 239 and 605 nucleotides). The full-length coding region of VASA was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. VASA variants are highly expressed in testis of adult buffalo. We found five variants, one full length VASA (729 aa) and four splice variants VASA 2, 4, 5, 6 (683, 685, 679, 703 aa). The expression level of VASA 1 was significantly higher than rest of all (P < 0.05) except VASA 6. The relative ratio for VASA 1:2:4:5:6 was 100:1.0:1.6:0.9:48.

  14. DEAD-box helicase DDX27 regulates 3′ end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex

    SciTech Connect

    Kellner, Markus; Rohrmoser, Michaela; Forné, Ignasi; Voss, Kirsten; Burger, Kaspar; Mühl, Bastian; Gruber-Eber, Anita; Kremmer, Elisabeth; Imhof, Axel; Eick, Dirk

    2015-05-15

    PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3′ end formation of 47S rRNA independently of the PeBoW-complex. - Highlights: • DEAD-box helicase DDX27 is a new constituent of the PeBoW-complex. • The N-terminal F×F motif of DDX27 interacts with the PeBoW components Pes1 and Bop1. • Nucleolar anchoring of DDX27 via its basic C-terminal domain is RNA dependent. • Knockdown of DDX27 induces a specific defect in 3′ end formation of 47S rRNA.

  15. The eukaryotic initiation factor eIF4H facilitates loop-binding, repetitive RNA unwinding by the eIF4A DEAD-box helicase.

    PubMed

    Sun, Yingjie; Atas, Evrim; Lindqvist, Lisa; Sonenberg, Nahum; Pelletier, Jerry; Meller, Amit

    2012-07-01

    Eukaryotic translation initiation is a highly regulated process in protein synthesis. The principal translation initiation factor eIF4AI displays helicase activity, unwinding secondary structures in the mRNAs 5'-UTR. Single molecule fluorescence resonance energy transfer (sm-FRET) is applied here to directly observe and quantify the helicase activity of eIF4AI in the presence of the ancillary RNA-binding factor eIF4H. Results show that eIF4H can significantly enhance the helicase activity of eIF4AI by strongly binding both to loop structures within the RNA transcript as well as to eIF4AI. In the presence of ATP, the eIF4AI/eIF4H complex exhibits persistent rapid and repetitive cycles of unwinding and re-annealing. ATP titration assays suggest that this process consumes a single ATP molecule per cycle. In contrast, helicase unwinding activity does not occur in the presence of the non-hydrolysable analog ATP-γS. Based on our sm-FRET results, we propose an unwinding mechanism where eIF4AI/eIF4H can bind directly to loop structures to destabilize duplexes. Since eIF4AI is the prototypical example of a DEA(D/H)-box RNA helicase, it is highly likely that this unwinding mechanism is applicable to a myriad of DEAD-box helicases employed in RNA metabolism.

  16. Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1

    PubMed Central

    Alessi, Amelia F.; Khivansara, Vishal; Han, Ting; Freeberg, Mallory A.; Moresco, James J.; Tu, Patricia G.; Montoye, Eric; Yates, John R.; Karp, Xantha; Kim, John K.

    2015-01-01

    MicroRNAs (miRNAs) play essential, conserved roles in diverse developmental processes through association with the miRNA-induced silencing complex (miRISC). Whereas fundamental insights into the mechanistic framework of miRNA biogenesis and target gene silencing have been established, posttranslational modifications that affect miRISC function are less well understood. Here we report that the conserved serine/threonine kinase, casein kinase II (CK2), promotes miRISC function in Caenorhabditis elegans. CK2 inactivation results in developmental defects that phenocopy loss of miRISC cofactors and enhances the loss of miRNA function in diverse cellular contexts. Whereas CK2 is dispensable for miRNA biogenesis and the stability of miRISC cofactors, it is required for efficient miRISC target mRNA binding and silencing. Importantly, we identify the conserved DEAD-box RNA helicase, CGH-1/DDX6, as a key CK2 substrate within miRISC and demonstrate phosphorylation of a conserved N-terminal serine is required for CGH-1 function in the miRNA pathway. PMID:26669440

  17. The DEAD-Box RNA Helicase DDX3 Interacts with NF-κB Subunit p65 and Suppresses p65-Mediated Transcription

    PubMed Central

    Gao, Yu; Song, Feifei; Guo, Liang; Ma, Li; Sun, Guihong; Liu, Dan; Guo, Deyin

    2016-01-01

    RNA helicase family members exhibit diverse cellular functions, including in transcription, pre-mRNA processing, RNA decay, ribosome biogenesis, RNA export and translation. The RNA helicase DEAD-box family member DDX3 has been characterized as a tumour-associated factor and a transcriptional co-activator/regulator. Here, we demonstrate that DDX3 interacts with the nuclear factor (NF)-κB subunit p65 and suppresses NF-κB (p65/p50)-mediated transcriptional activity. The downregulation of DDX3 by RNA interference induces the upregulation of NF-κB (p65/p50)-mediated transcription. The regulation of NF-κB (p65/p50)-mediated transcriptional activity was further confirmed by the expression levels of its downstream cytokines, such as IL-6 and IL-8. Moreover, the binding of the ATP-dependent RNA helicase domain of DDX3 to the N-terminal Rel homology domain (RHD) of p65 is involved in the inhibition of NF-κB-regulated gene transcription. In summary, the results suggest that DDX3 functions to suppress the transcriptional activity of the NF-κB subunit p65. PMID:27736973

  18. Characterization of the kinetics of RNA annealing and strand displacement activities of the E. coli DEAD-box helicase CsdA.

    PubMed

    Stampfl, Sabine; Doetsch, Martina; Beich-Frandsen, Mads; Schroeder, Renée

    2013-01-01

    CsdA is one of five E. coli DEAD-box helicases and as a cold-shock protein assists RNA structural remodeling at low temperatures. The helicase has been shown to catalyze duplex unwinding in an ATP-dependent way and accelerate annealing of complementary RNAs, but detailed kinetic analyses are missing. Therefore, we performed kinetic measurements using a coupled annealing and strand displacement assay with high temporal resolution to analyze how CsdA balances the two converse activities. We furthermore tested the hypothesis that the unwinding activity of DEAD-box helicases is largely determined by the substrate's thermodynamic stability using full-length CsdA and a set of RNAs with constant length, but increasing GC content. The rate constants for strand displacement did indeed decrease with increasing duplex stability, with a calculated free energy between -31.3 and -40 kcal/mol being the limit for helix unwinding. Thus, our data generally support the above hypothesis, showing that for CsdA substrate thermal stability is an important rate limiting factor.

  19. Dead-box proteins: a family affair—active and passive players in RNP-remodeling

    PubMed Central

    Linder, Patrick

    2006-01-01

    DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family. PMID:16936318

  20. The DEAD-box RNA helicase 51 controls non-small cell lung cancer proliferation by regulating cell cycle progression via multiple pathways

    PubMed Central

    Wang, Xiaojing; Liu, Hongli; Zhao, Chengling; Li, Wei; Xu, Huanbai; Chen, Yuqing

    2016-01-01

    The genetic regulation of cell cycle progression and cell proliferation plays a role in the growth of non-small cell lung cancer (NSCLC), one of the most common causes of cancer-related mortality. Although DEAD-box RNA helicases are known to play a role in cancer development, including lung cancer, the potential involvement of the novel family member DDX51 has not yet been investigated. In the current study we assessed the role of DDX51 in NSCLC using a siRNA-based approach. DDX51 siRNA-expressing cells exhibited a slower cell proliferation rate and underwent arrest in S-phase of the cell cycle compared with control cells. Microarray analyses revealed that DDX51siRNA expression resulted in the dysregulation of a number of cell signalling pathways. Moreover, injection of DDX51 siRNA into an animal model resulted in the formation of smaller tumours compared with the control group. We also assessed the expression of DDX51 in patients with NSCLC, and the data revealed that the expression was correlated with patient age but no other risk factors. Overall, our data suggest for the first time that DDX51 aids cell cancer proliferation by regulating multiple signalling pathways, and that this protein might be a therapeutic target for NSCLC. PMID:27198888

  1. A chicken embryo protein related to the mammalian DEAD box protein p68 is tightly associated with the highly purified protein-RNA complex of 5-MeC-DNA glycosylase.

    PubMed

    Jost, J P; Schwarz, S; Hess, D; Angliker, H; Fuller-Pace, F V; Stahl, H; Thiry, S; Siegmann, M

    1999-08-15

    We have shown previously that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. Amino acid sequences of nine peptides derived from a highly purified 5-MeC-DNA glycosylase complex were identified by Nanoelectrospray ionisation mass spectrometry to be identical to the mammalian nuclear DEAD box protein p68 RNA helicase. Antibodies directed against human p68 helicase cross-reacted with the purified 5-MeC-DNA glycosylase complex and immunoprecipitated the glycosylase activity. A 2690 bp cDNA coding for the chicken homologue of mammalian p68 was isolated and sequenced. Its derived amino acid sequence is almost identical to the human p68 DEAD box protein up to amino acid position 473 (from a total of 595). This sequence contains all the essential conserved motifs from the DEAD box proteins which are the ATPase, RNA unwinding and RNA binding motifs. The rest of the 122 amino acids in the C-terminal region rather diverge from the human p68 RNA helicase sequence. The recombinant chicken DEAD box protein expressed in Escherichia coli cross-reacts with the same p68 antibodies as the purified chicken embryo 5-MeC-DNA glycosylase complex. The recombinant protein has an RNA-dependent ATPase and an ATP-dependent helicase activity. However, in the presence or absence of RNA the recombinant protein had no 5-MeC-DNA glycosylase activity. In situ hybridisation of 5 day-old chicken embryos with antisense probes of the chicken DEAD box protein shows a high abundance of its transcripts in differentiating embryonic tissues.

  2. Characterization of a homolog of DEAD-box RNA helicases in Toxoplasma gondii as a marker of cytoplasmic mRNP stress granules.

    PubMed

    Cherry, Ahmed Adnan; Ananvoranich, Sirinart

    2014-06-10

    Toxoplasma gondii is an obligate intracellular protozoan which infects one-third of the human population. Due to its high infection prevalence, Toxoplasma offers an ideal system for the study of host-parasite interaction. Similar to other eukaryotes, Toxoplasma maintains levels and localization of cytoplasmic mRNAs throughout its life cycle as part of a gene regulation network to meet all cellular and biochemical requirements. More recently, it was reported that the presence of cytoplasmic mRNA granules could contribute to the parasite pathogenesis and viability. Here we identified a novel Toxoplasma DEAD-box RNA helicase, referred to as Toxoplasma gondiiHomolog of DOZI (TgHoDI), because of its high homology (81%) to Plasmodium DOZI. TgHoDI is the functional ortholog of yeast DHH1, and its function was authenticated by complementation studies in Δdhh1 yeast strain. We demonstrated that TgHoDI is a marker of cytoplasmic RNA stress granules, which assemble when the parasites experience cellular stresses and translational arrest.

  3. Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNP

    SciTech Connect

    Del Campo, Mark; Lambowitz, Alan M.

    2009-09-02

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone which functions in mitochondrial group I and group II intron splicing, translation and RNA-end processing. For crystallization trials, full-length Mss116p and a C-terminally truncated protein (Mss116p/{Delta}598-664) were overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited low solubility in standard solutions ({le}1 mg ml{sup -1}), but its solubility could be increased by adding 50 mM L-arginine plus 50 mM L-glutamate and 50% glycerol to achieve concentrations of {approx}10 mg ml{sup -1}. Initial crystals were obtained by the microbatch method in the presence of a U{sub 10} RNA oligonucleotide and the ATP analog AMP-PNP and were then improved by using seeding and sitting-drop vapor diffusion. A cryocooled crystal of Mss116p/{Delta}598-664 in complex with AMP-PNP and U{sub 10} belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 88.54, b = 126.52, c = 55.52 {angstrom}, and diffracted X-rays to beyond 1.9 {angstrom} resolution using synchrotron radiation from sector 21 at the Advanced Photon Source.

  4. DEAD-box helicase DDX27 regulates 3' end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex.

    PubMed

    Kellner, Markus; Rohrmoser, Michaela; Forné, Ignasi; Voss, Kirsten; Burger, Kaspar; Mühl, Bastian; Gruber-Eber, Anita; Kremmer, Elisabeth; Imhof, Axel; Eick, Dirk

    2015-05-15

    PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3' end formation of 47S rRNA independently of the PeBoW-complex.

  5. An Arabidopsis ATP-Dependent, DEAD-Box RNA Helicase Loses Activity upon IsoAsp Formation but Is Restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE[C][W

    PubMed Central

    Nayak, Nihar R.; Putnam, Andrea A.; Addepalli, Balasubrahmanyam; Lowenson, Jonathan D.; Chen, Tingsu; Jankowsky, Eckhard; Perry, Sharyn E.; Dinkins, Randy D.; Limbach, Patrick A.; Clarke, Steven G.; Downie, A. Bruce

    2013-01-01

    Orthodox seeds are capable of withstanding severe dehydration. However, in the dehydrated state, Asn and Asp residues in proteins can convert to succinimide residues that can further react to predominantly form isomerized isoAsp residues upon rehydration (imbibition). IsoAsp residues can impair protein function and can render seeds nonviable, but PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT) can initiate isoAsp conversion to Asp residues. The proteins necessary for translation upon imbibition in orthodox seeds may be particularly important to maintain in an active state. One such protein is the large, multidomain protein, Arabidopsis thaliana PLANT RNA HELICASE75 (PRH75), a DEAD-box helicase known to be susceptible to isoAsp residue accumulation. However, the consequences of such isomerization on PRH75 catalysis and for the plant are unknown. Here, it is demonstrated that PRH75 is necessary for successful seed development. It acquires isoAsp rapidly during heat stress, which eliminates RNA unwinding (but not rewinding) competence. The repair by PIMT is able to restore PRH75’s complex biochemical activity provided isoAsp formation has not led to subsequent, destabilizing conformational alterations. For PRH75, an important enzymatic activity associated with translation would be eliminated unless rapidly repaired by PIMT prior to additional, deleterious conformational changes that would compromise seed vitality and germination. PMID:23903319

  6. DP97, a DEAD box DNA/RNA helicase, is a target gene-selective co-regulator of the constitutive androstane receptor

    SciTech Connect

    Kanno, Yuichiro; Serikawa, Takafumi; Inajima, Jun; Inouye, Yoshio

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer DP97 interacts with nuclear receptor CAR. Black-Right-Pointing-Pointer DP97 enhances CAR-mediated transcriptional activation. Black-Right-Pointing-Pointer DP97 synergistically enhances transactivity of CAR by the co-expression of SRC-1 or PGC1{alpha}. Black-Right-Pointing-Pointer DP97 is a gene-selective co-activator for hCAR. -- Abstract: The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that DP97, a member of the DEAD box DNA/RNA helicase protein family, is a novel CAR-interacting protein. Using HepG2 cells expressing human CAR in the presence of tetracycline, we showed that knockdown of DP97 with small interfering RNAs suppressed tetracycline-inducible mRNA expression of CYP2B6 and UGT1A1 but not CYP3A4. Thus, DP97 was found to be a gene (or promoter)-selective co-activator for hCAR. DP97-mediated CAR transactivation was synergistically enhanced by the co-expression of SRC-1 or PGC1{alpha}, therefore it might act as mediator between hCAR and appropriate co-activators.

  7. Regulation of MicroRNA 183 by Cyclooxygenase 2 in Liver Is DEAD-Box Helicase p68 (DDX5) Dependent: Role in Insulin Signaling

    PubMed Central

    Motiño, Omar; Francés, Daniel E.; Castro-Sánchez, Luis; Fernández-Velasco, María; Boscá, Lisardo; García-Monzón, Carmelo; Brea, Rocío; Casado, Marta

    2015-01-01

    Cyclooxygenase (COX) catalyzes the first step in prostanoid biosynthesis and exists as two isoforms. COX-1 is a constitutive enzyme involved in physiological processes, whereas COX-2 is induced by a variety of stimuli. MicroRNAs (miRNAs) are noncoding RNAs that function as key posttranscriptional regulators of gene expression. Although it is known that COX-2 expression is regulated by miRNAs, there are no data regarding COX-2 involvement in miRNA regulation. Considering our previous results showing that COX-2 expression in hepatocytes protects against insulin resistance, we evaluated the role of COX-2 in the regulation of a specific set of miRNAs implicated in insulin signaling in liver cells. Our results provide evidence of the molecular basis for a novel function of COX-2 in miRNA processing. COX-2 represses miRNA 23b (miR-23b), miR-146b, and miR-183 expression in liver cells by increasing the level of DEAD-box helicase p68 (DDX5) through phosphatidylinositol 3-kinase (PI3K)/p300 signaling and by modulating the enzymatic function of the Drosha (RNase type III) complex through its physical association with DDX5. The decrease of miR-183 expression promotes protection against insulin resistance by increasing insulin receptor substrate 1 (IRS1) levels. These results indicate that the modulation of miRNA processing by COX-2 is a key event in insulin signaling in liver and has potential clinical implications for the management of various hepatic dysfunctions. PMID:25963660

  8. Genome-wide analysis of translational efficiency reveals distinct but overlapping functions of yeast DEAD-box RNA helicases Ded1 and eIF4A

    PubMed Central

    Sen, Neelam Dabas; Zhou, Fujun; Ingolia, Nicholas T.; Hinnebusch, Alan G.

    2015-01-01

    DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5′ end or subsequent scanning of the 5′ UTR, but whether they perform unique or overlapping functions in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in budding yeast Saccharomyces cerevisiae by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A variant encoded by tif1-A79V (in a strain lacking the ortholog TIF2) yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5′ UTR length and propensity for secondary structure, implicating Ded1 in scanning through structured 5′ UTRs. Reporter assays confirmed that cap-distal stem–loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs shows a heightened requirement for eIF4A, dependence on eIF4A is correlated with requirements for Ded1 and 5′ UTR features characteristic of Ded1-dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5′ UTRs, and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs. PMID:26122911

  9. The plastidic DEAD-box RNA helicase 22, HS3, is essential for plastid functions both in seed development and in seedling growth.

    PubMed

    Kanai, Masatake; Hayashi, Makoto; Kondo, Maki; Nishimura, Mikio

    2013-09-01

    Plants accumulate large amounts of storage products in seeds to provide an energy reserve and to supply nutrients for germination and post-germinative growth. Arabidopsis thaliana belongs to the Brassica family, and oil is the main storage product in Arabidopsis seeds. To elucidate the regulatory mechanisms of oil biosynthesis in seeds, we screened for high density seeds (heavy seed) that have a low oil content. HS3 (heavy seed 3) encodes the DEAD-box RNA helicase 22 that is localized to plastids. The triacylglycerol (TAG) content of hs3-1 seeds was 10% lower than that of wild-type (WT) seeds, while the protein content was unchanged. The hs3-1 plants displayed a pale-green phenotype in developing seeds and seedlings, but not in adult leaves. The HS3 expression level was high in developing seeds and seedlings, but was low in stems, rosette leaves and flowers. The plastid gene expression profile of WT developing seeds and seedlings differed from that of hs3-1 developing seeds and seedlings. The expression of several genes was reduced in developing hs3-1 seeds, including accD, a gene that encodes the β subunit of carboxyltransferase, which is one component of acetyl-CoA carboxylase in plastids. In contrast, no differences were observed between the expression profiles of WT and hs3-1 rosette leaves. These results show that HS3 is essential for proper mRNA accumulation of plastid genes during seed development and seedling growth, and suggest that HS3 ensures seed oil biosynthesis by maintaining plastid mRNA levels.

  10. Analog sensitive chemical inhibition of the DEAD-box protein DDX3.

    PubMed

    Floor, Stephen N; Barkovich, Krister J; Condon, Kendall J; Shokat, Kevan M; Doudna, Jennifer A

    2016-03-01

    Proper maintenance of RNA structure and dynamics is essential to maintain cellular health. Multiple families of RNA chaperones exist in cells to modulate RNA structure, RNA-protein complexes, and RNA granules. The largest of these families is the DEAD-box proteins, named after their catalytic Asp-Glu-Ala-Asp motif. The human DEAD-box protein DDX3 is implicated in diverse biological processes including translation initiation and is mutated in numerous cancers. Like many DEAD-box proteins, DDX3 is essential to cellular health and exhibits dosage sensitivity, such that both decreases and increases in protein levels can be lethal. Therefore, chemical inhibition would be an ideal tool to probe the function of DDX3. However, most DEAD-box protein active sites are extremely similar, complicating the design of specific inhibitors. Here, we show that a chemical genetic approach best characterized in protein kinases, known as analog-sensitive chemical inhibition, is viable for DDX3 and possibly other DEAD-box proteins. We present an expanded active-site mutant that is tolerated in vitro and in vivo, and is sensitive to chemical inhibition by a novel bulky inhibitor. Our results highlight a course towards analog sensitive chemical inhibition of DDX3 and potentially the entire DEAD-box protein family.

  11. The Arabidopsis STRESS RESPONSE SUPPRESSOR DEAD-box RNA helicases are nucleolar- and chromocenter-localized proteins that undergo stress-mediated relocalization and are involved in epigenetic gene silencing.

    PubMed

    Khan, Asif; Garbelli, Anna; Grossi, Serena; Florentin, Assa; Batelli, Giorgia; Acuna, Tania; Zolla, Gaston; Kaye, Yuval; Paul, Laju K; Zhu, Jian-Kang; Maga, Giovanni; Grafi, Gideon; Barak, Simon

    2014-07-01

    DEAD-box RNA helicases are involved in many aspects of RNA metabolism and in diverse biological processes in plants. Arabidopsis thaliana mutants of two DEAD-box RNA helicases, STRESS RESPONSE SUPPRESSOR1 (STRS1) and STRS2 were previously shown to exhibit tolerance to abiotic stresses and up-regulated stress-responsive gene expression. Here, we show that Arabidopsis STRS-overexpressing lines displayed a less tolerant phenotype and reduced expression of stress-induced genes confirming the STRSs as attenuators of Arabidopsis stress responses. GFP-STRS fusion proteins exhibited localization to the nucleolus, nucleoplasm and chromocenters and exhibited relocalization in response to abscisic acid (ABA) treatment and various stresses. This relocalization was reversed when stress treatments were removed. The STRS proteins displayed mis-localization in specific gene-silencing mutants and exhibited RNA-dependent ATPase and RNA-unwinding activities. In particular, STRS2 showed mis-localization in three out of four mutants of the RNA-directed DNA methylation (RdDM) pathway while STRS1 was mis-localized in the hd2c mutant that is defective in histone deacetylase activity. Furthermore, heterochromatic RdDM target loci displayed reduced DNA methylation and increased expression in the strs mutants. Taken together, our findings suggest that the STRS proteins are involved in epigenetic silencing of gene expression to bring about suppression of the Arabidopsis stress response.

  12. The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote compartmentalized translation initiation of the HIV-1 genomic RNA

    PubMed Central

    Soto-Rifo, Ricardo; Rubilar, Paulina S.; Ohlmann, Théophile

    2013-01-01

    Here, we show a novel molecular mechanism promoted by the DEAD-box RNA helicase DDX3 for translation of the HIV-1 genomic RNA. This occurs through the adenosine triphosphate-dependent formation of a translation initiation complex that is assembled at the 5′ m7GTP cap of the HIV-1 mRNA. This is due to the property of DDX3 to substitute for the initiation factor eIF4E in the binding of the HIV-1 m7GTP 5′ cap structure where it nucleates the formation of a core DDX3/PABP/eIF4G trimeric complex on the HIV-1 genomic RNA. By using RNA fluorescence in situ hybridization coupled to indirect immunofluorescence, we further show that this viral ribonucleoprotein complex is addressed to compartmentalized cytoplasmic foci where the translation initiation complex is assembled. PMID:23630313

  13. Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the DEAD Box (DDX)-5/17 RNA Helicases.

    PubMed

    Yang, Jun; Zhao, Yingxin; Kalita, Mridul; Li, Xueling; Jamaluddin, Mohammad; Tian, Bing; Edeh, Chukwudi B; Wiktorowicz, John E; Kudlicki, Andrzej; Brasier, Allan R

    2015-10-01

    Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1

  14. Amplification of a DEAD box protein gene in retinoblastoma cell lines.

    PubMed Central

    Godbout, R; Squire, J

    1993-01-01

    DEAD box proteins, characterized by the conserved motif Asp-Glu-Ala-Asp, are putative RNA helicases implicated in a number of cellular processes involving alteration of RNA secondary structure such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. Based on their distribution patterns, some members of this family are believed to be involved in embryogenesis, spermatogenesis, and cellular growth and division. Here, we report that the mRNA encoding a DEAD box protein, designated HuDBP-RB, is present at elevated levels in two of six retinoblastoma (RB) cell lines tested and is preferentially expressed in fetal tissues of neuroectodermal origin. It is not possible to classify HuDBP-RB as a member of any of the DEAD box protein subgroups identified to date since the regions of amino acid similarity between HuDBP-RB and other DEAD box proteins are restricted to the conserved motifs found in all members of this family. The HuDBP-RB gene, which has been mapped to chromosome band 2p24, is amplified in the RB cell lines that overexpress HuDBP-RB RNA. Furthermore, the MYCN gene is also present in multiple copies in these two cell lines, suggesting coamplification of the two genes. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7689221

  15. An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon iosAsp formation but is restored by Protein Isoaspartyl Methltransferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabidopsis thaliana PLANT RNA HELICASE75 (AtPRH75) demonstrated an ATP-dependent, RNA duplex unwinding capacity and an ATP-independent, RNA duplex reforming ability. It is known to accumulate isoAsp, but the consequences of isoAsp formation in AtPRH75 are unknown. Duplex unwinding was abolished by ...

  16. p72: a human nuclear DEAD box protein highly related to p68.

    PubMed Central

    Lamm, G M; Nicol, S M; Fuller-Pace, F V; Lamond, A I

    1996-01-01

    P72, a novel human member of the DEAD box family of putative RNA-dependent ATPases and ATP-dependent RNA helicases was isolated from a HeLa cDNA library. The predicted amino acid sequence of p72 is highly homologous to that of the prototypic DEAD box protein p68. In addition to the conserved core domains characteristic of DEAD box proteins, p72 contains several N-terminal RGG RNA-binding domains and a serine/glycine rich C-terminus likely involved in mediating protein-protein interactions. A p72-specific probe detects two mRNAs of approximately 5300 and 9300 bases which, although ubiquitously expressed, show variability in their expression levels in different tissues. Purified recombinant p72 exhibits ATPase activity in the presence of a range of RNA moieties. Immunocytochemical studies of p68 and p72 show that these proteins localise to similar locations in the nucleus of HeLa cells, suggesting their involvement in a nuclear process. PMID:8871553

  17. Evolution of the DEAD box helicase family in chicken: chickens have no DHX9 ortholog.

    PubMed

    Sato, Haruko; Oshiumi, Hiroyuki; Takaki, Hiromi; Hikono, Hirokazu; Seya, Tsukasa

    2015-10-01

    Viral RNA represents a pattern molecule that can be recognized by RNA sensors in innate immunity. Humans and mice possess cytoplasmic DNA/RNA sensors for detecting viral replication. There are a number of DEAD (Asp-Glu-Ala-Asp; DExD/H) box-type helicases in mammals, among which retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA50) are indispensable for RNA sensing; however, they are functionally supported by a number of sensors that directly bind viral RNA or replicative RNA intermediates to convey signals to RIG-I and MDA5. Some DEAD box helicase members recognize DNA irrespective of the origin. These sensors transmit IFN-inducing signals through adaptors, including mitochondrial antiviral signaling. Viral double-stranded RNAs are reportedly sensed by the helicases DDX1, DDX21, DHX36, DHX9, DDX3, DDX41, LGP2 and DDX60, in addition to RIG-I and MDA5, and induce type I IFNs, thereby blocking viral replication. Humans and mice have all nucleic acid sensors listed here. In the RNA sensing system in chicken, it was found in the present study that most DEAD box helicases are conserved; however, DHX9 is genetically deficient in addition to reported RIG-I. Based on the current genome databases, similar DHX9 deficiency was observed in ducks and several other bird species. Because chicken, but not duck, was found to be deficient in RIG-I, the RNA-sensing system of chicken lacks RIG-I and DHX9 and is thus more fragile than that of duck or mammal. DHX9 may generally compensate for the function of RIG-I and deficiency of DHX9 possibly participates in exacerbations of viral infection such as influenza in chickens.

  18. Factor-dependent processivity in human eIF4A DEAD-box helicase

    PubMed Central

    García-García, Cuauhtémoc; Frieda, Kirsten L.; Feoktistova, Kateryna; Fraser, Christopher S.; Block, Steven M.

    2015-01-01

    During eukaryotic translation initiation, the small ribosomal subunit, assisted by initiation factors, locates the messenger RNA start codon by scanning from the 5′ cap. This process is powered by the eukaryotic initiation factor 4A (eIF4A), a DEAD-box helicase. eIF4A has been thought to unwind structures formed in the untranslated 5′ region via a nonprocessive mechanism. Using a single-molecule assay, we found that eIF4A functions instead as an adenosine triphosphate–dependent processive helicase when complexed with two accessory proteins, eIF4G and eIF4B. Translocation occurred in discrete steps of 11 ± 2 base pairs, irrespective of the accessory factor combination. Our findings support a memory-less stepwise mechanism for translation initiation and suggest that similar factor-dependent processivity may be shared by other members of the DEAD-box helicase family. PMID:26113725

  19. Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo.

    PubMed Central

    Grandori, C; Mac, J; Siëbelt, F; Ayer, D E; Eisenman, R N

    1996-01-01

    The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc antibodies, we have identified a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo. Four of 27 sites recovered by this procedure corresponded to the highest affinity 'canonical' CACGTG sequence. However, the most common in vivo binding sites belonged to the group of 'non-canonical' E box-related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contained transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA was induced following activation of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc-Max. In addition, as for c-Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down-regulated during terminal differentiation of HL60 leukemia cells. Our results indicate that Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences and that Myc is likely to activate multiple target genes including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism. Images PMID:8861962

  20. PRH75, a new nucleus-localized member of the DEAD-box protein family from higher plants.

    PubMed Central

    Lorković, Z J; Herrmann, R G; Oelmüller, R

    1997-01-01

    The putative RNA helicases of the DEAD-box protein family are involved in pre-mRNA splicing, rRNA maturation, ribosome assembly, and translation. Members of this protein family have been identified in organisms from Escherichia coli to humans, but except for the translation initiation factor 4A, there have been no reports on the characterization of other DEAD-box proteins from plants. Here we report on a novel member of the DEAD-box protein family, the plant RNA helicase 75 (PRH75). PRH75 is localized in the nucleus and contains two domains for RNA binding. One is located at the C terminus and is similar to RGG RNA-binding domains of nucleus-localized RNA-binding proteins. The other one is located between amino acids 308 and 622, a region containing the conserved motif VI characteristic of DEAD-box proteins and known as the RNA-binding site of eIF-4A. The N-terminal 81 amino acids are sufficient for nuclear targeting of the protein. Northern and Western blot analyses show that PRH75 is mainly expressed in young and rapidly developing tissues. The purified recombinant PRH75 has a weak ATPase activity which is barely stimulated by RNA ligands. The fractionation of spinach whole-cell extracts by glycerol gradient centrifugation and gel filtration on a Superdex 200 column shows that the protein exists in a complex of about 500 kDa. Possible biological functions of PRH75 as well as structure-function relationships in the context of its modular primary structure are discussed. PMID:9121476

  1. Human DEAD-box ATPase DDX3 shows a relaxed nucleoside substrate specificity.

    PubMed

    Franca, Raffaella; Belfiore, Amalia; Spadari, Silvio; Maga, Giovanni

    2007-06-01

    Human DDX3 (hDDX3) is a DEAD-box protein shown to possess RNA-unwinding and adenosine triphosphatase (ATPase) activities. The hDDX3 protein has been implicated in nuclear mRNA export, cell growth control, and cancer progression. In addition, a role of this protein in the replication of human immunodeficiency virus Type 1 and in the pathogenesis of hepatitis C virus has been recently proposed. Its enzymological properties, however, are largely unknown. In this work, we characterized its ATPase activity. We show that hDDX3 ATPase activity is stimulated by various ribo- and deoxynucleic acids. Comparative analysis with different nucleoside triphosphate analogs showed that the hDDX3 ATPase couples high catalytic efficiency to a rather relaxed substrate specificity, both in terms of base selection and sugar selection. In addition, its ability to recognize the L-stereoisomers of both 3' deoxy- and 2',3' dideoxy-ribose, points to a relaxed stereoselectivity. On the basis of these results, we hypothesize the presence of structural determinants on both the base and the sugar moieties, critical for nucleoside binding to the enzyme. Our results expand the knowledge about the DEAD-box RNA helicases in general and can be used for rational design of selective inhibitors of hDDX3, to be tested as potential antitumor and antiviral agents.

  2. High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-Box Protein Mss116p

    SciTech Connect

    Mohr, Georg; Del Campo, Mark; Turner, Kathryn G.; Gilman, Benjamin; Wolf, Rachel Z.; Lambowitz, Alan M.

    2012-03-15

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions - one the previously noted post II region in the helicase core and the other in the CTE - that may help displace or sequester the opposite RNA strand during RNA unwinding.

  3. High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-box Protein Mss116p

    PubMed Central

    Mohr, Georg; Campo, Mark Del; Turner, Kathryn G.; Gilman, Benjamin; Wolf, Rachel Z.; Lambowitz, Alan M.

    2011-01-01

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins, but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends upon ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the C-terminal extension results primarily from a steric block. Finally, we identified two conserved regions, one the previously noted post-II region in the helicase core and the other in the C-terminal extension, which may help displace or sequester the opposite RNA strand during RNA unwinding. PMID:21945532

  4. Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3.

    PubMed

    Floor, Stephen N; Condon, Kendall J; Sharma, Deepak; Jankowsky, Eckhard; Doudna, Jennifer A

    2016-01-29

    DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.

  5. Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3*

    PubMed Central

    Floor, Stephen N.; Condon, Kendall J.; Sharma, Deepak; Jankowsky, Eckhard; Doudna, Jennifer A.

    2016-01-01

    DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins. PMID:26598523

  6. Genetic interactions of conserved regions in the DEAD-box protein Prp28p.

    PubMed Central

    Chang, T H; Latus, L J; Liu, Z; Abbott, J M

    1997-01-01

    The yeast PRP28 g ene has been implicated in nuclear precursor messenger RNA (pre-mRNA) splicing, a two-step reaction involved in a multitude of RNA structural alterations. Prp28p, the gene product of PRP28 , is a member of the evolutionarily conserved DEAD-box proteins (DBPs). Members of DBPs are involved in a variety of RNA-related biochemical processes, presumably by their putative RNA helicase activities. Prp28p has been speculated to play a role in melting the duplex between U4 and U6 small nuclear RNAs (snRNAs), leading to the formation of an active spliceosome. To study the function of Prp28p and its interactions with other components of the splicing machinery, we have isolated and characterized a large number of prp28 conditional mutants. Strikingly, many of these prp28 mutations are localized in the highly conserved motifs found in all the DBPs. Intragenic reversion analysis suggests that regions of motifs II, III and V, as well as of motifs I and IV, in Prp28p are likely to be in close proximity to each other. Our results thus provide the first hint of the local structural arrangement for Prp28p, and perhaps for other DBPs as well. PMID:9396812

  7. Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

    SciTech Connect

    Möhlmann, Sina; Mathew, Rebecca; Neumann, Piotr; Schmitt, Andreas; Lührmann, Reinhard; Ficner, Ralf

    2014-06-01

    The crystal structure of the helicase domain of the human spliceosomal DEAD-box protein Prp28 was solved by SAD. The binding of ADP and ATP by Prp28 was studied biochemically and analysed with regard to the crystal structure. The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

  8. ATPase activity of the DEAD-box protein Dhh1 controls processing body formation

    PubMed Central

    Mugler, Christopher Frederick; Hondele, Maria; Heinrich, Stephanie; Sachdev, Ruchika; Vallotton, Pascal; Koek, Adriana Y; Chan, Leon Y; Weis, Karsten

    2016-01-01

    Translational repression and mRNA degradation are critical mechanisms of posttranscriptional gene regulation that help cells respond to internal and external cues. In response to certain stress conditions, many mRNA decay factors are enriched in processing bodies (PBs), cellular structures involved in degradation and/or storage of mRNAs. Yet, how cells regulate assembly and disassembly of PBs remains poorly understood. Here, we show that in budding yeast, mutations in the DEAD-box ATPase Dhh1 that prevent ATP hydrolysis, or that affect the interaction between Dhh1 and Not1, the central scaffold of the CCR4-NOT complex and an activator of the Dhh1 ATPase, prevent PB disassembly in vivo. Intriguingly, this process can be recapitulated in vitro, since recombinant Dhh1 and RNA, in the presence of ATP, phase-separate into liquid droplets that rapidly dissolve upon addition of Not1. Our results identify the ATPase activity of Dhh1 as a critical regulator of PB formation. DOI: http://dx.doi.org/10.7554/eLife.18746.001 PMID:27692063

  9. Stress responsive DEAD-box helicases: a new pathway to engineer plant stress tolerance.

    PubMed

    Vashisht, Ajay Amar; Tuteja, Narendra

    2006-08-01

    Abiotic stresses including various environmental factors adversely affect plant growth and limit agricultural production worldwide. Minimizing these losses is a major area of concern for all countries. Therefore, it is desirable to develop multi-stress tolerant varieties. Salinity, drought, and cold are among the major environmental stresses that greatly influence the growth, development, survival, and yield of plants. UV-B radiation of sunlight, which damages the cellular genomes, is another growth-retarding factor. Several genes are induced under the influence of various abiotic stresses. Among these are DNA repair genes, which are induced in response to the DNA damage. Since the stresses affect the cellular gene expression machinery, it is possible that molecules involved in nucleic acid metabolism including helicases are likely to be affected. The light-driven shifts in redox-potential can also initiate the helicase gene expression. Helicases are ubiquitous enzymes that catalyse the unwinding of energetically stable duplex DNA (DNA helicases) or duplex RNA secondary structures (RNA helicases). Most helicases are members of DEAD-box protein superfamily and play essential roles in basic cellular processes such as DNA replication, repair, recombination, transcription, ribosome biogenesis and translation initiation. Therefore, helicases might be playing an important role in regulating plant growth and development under stress conditions by regulating some stress-induced pathways. There are now few reports on the up-regulation of DEAD-box helicases in response to abiotic stresses. Recently, salinity-stress tolerant tobacco plants have already been raised by overexpressing a helicase gene, which suggests a new pathway to engineer plant stress tolerance [N. Sanan-Mishra, X.H. Pham, S.K. Sopory, N. Tuteja, Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield. Proc. Natl. Acad. Sci. USA 102 (2005) 509-514]. Presently the

  10. Association of Human DEAD Box Protein DDX1 with a Cleavage Stimulation Factor Involved in 3′-End Processing of Pre-mRNAV⃞

    PubMed Central

    Bléoo, Stacey; Sun, Xuejun; Hendzel, Michael J.; Rowe, John M.; Packer, Mary; Godbout, Roseline

    2001-01-01

    DEAD box proteins are putative RNA helicases that function in all aspects of RNA metabolism, including translation, ribosome biogenesis, and pre-mRNA splicing. Because many processes involving RNA metabolism are spatially organized within the cell, we examined the subcellular distribution of a human DEAD box protein, DDX1, to identify possible biological functions. Immunofluorescence labeling of DDX1 demonstrated that in addition to widespread punctate nucleoplasmic labeling, DDX1 is found in discrete nuclear foci ∼0.5 μm in diameter. Costaining with anti-Sm and anti-promyelocytic leukemia (PML) antibodies indicates that DDX1 foci are frequently located next to Cajal (coiled) bodies and less frequently, to PML bodies. Most importantly, costaining with anti-CstF-64 antibody indicates that DDX1 foci colocalize with cleavage bodies. By microscopic fluorescence resonance energy transfer, we show that labeled DDX1 resides within a Förster distance of 10 nm of labeled CstF-64 protein in both the nucleoplasm and within cleavage bodies. Coimmunoprecipitation analysis indicates that a proportion of CstF-64 protein resides in the same complex as DDX1. These studies are the first to identify a DEAD box protein associating with factors involved in 3′-end cleavage and polyadenylation of pre-mRNAs. PMID:11598190

  11. Molecular cloning and characterization of a salinity stress-induced gene encoding DEAD-box helicase from the halophyte Apocynum venetum.

    PubMed

    Liu, H H; Liu, J; Fan, S L; Song, M Z; Han, X L; Liu, F; Shen, F F

    2008-01-01

    The genes encoding DEAD-box helicases play a key role in various abiotic stresses, including temperature, light, oxygen, and salt stress. A salt-responsive gene, designated AvDH1, was isolated from the halophyte dogbane (Apocynum venetum) by using suppression subtractive hybridization and RACE (rapid amplification of cDNA ends) PCR. The deduced amino acid sequence has nine conserved helicase motifs of the DEAD-box protein family. The AvDH1 gene is present as a single copy in the dogbane genome. This gene is expressed in response to NaCl and not polyethlene glycol (PEG) nor abscisic acid, and its expression increases with time. The transcription of AvDH1 is also induced by low temperature (4 degrees C), but its accumulation first increases then decreases with time. The purified recombinant protein contains ATP-dependent DNA helicase activity, ATP-independent RNA helicase activity, and DNA- or RNA-dependent ATPase activity. The ATPase activity of AvDH1 is stimulated more by single-stranded DNA than by double-stranded DNA or RNA. These results suggested that AvDH1 belonging to the DEAD-box helicase family is induced by salinity, functions as a typical helicase to unwind DNA and RNA, and may play an important role in salinity tolerance.

  12. Cloning, characterization, and expression analysis of the DEAD-box family genes, Fc-vasa and Fc-PL10a, in Chinese shrimp ( Fenneropenaeus chinensis)

    NASA Astrophysics Data System (ADS)

    Zhou, Qianru; Shao, Mingyu; Qin, Zhenkui; Kyoung, Ho Kang; Zhang, Zhifeng

    2010-01-01

    RNA helicases of the DEAD-box and related families are involved in various cellular processes including DNA replication, DNA repair, and RNA processing. However, the function of DEAD-box proteins in aquaculture species is poorly understood at molecular level. We obtained the full-length cDNA sequences of two genes encoding helicase-related proteins, Fc-vasa and Fc-PL10a, from the testes of Chinese shrimp, Fenneropenaeus chinensis. The two predicted amino acid sequences contain all the conserved motifs characterized by the DEAD-box family and several RGG repeats in the N-terminal regions. Homology and phylogenetic analyses indicate that they belong to the vasa and PL10 subfamilies. The three-dimensional structures of the two proteins were predicted with a homology modeling approach. Both core proteins consist of two tandem RecA-like domains similar to those of the DEAD-box RNA helicase. Using reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR we found that Fc-vasa was expressed specifically in the adult gonads. Transcription decreased in the ovary but increased in the testis during gonadal development. Fc-PL10a expression was widely distributed in the tissues we examined. Using in situ hybridization, we demonstrated that the Fc-vasa transcript is localized to the cytoplasm of the spermatogonia and oocytes. Thus, our results suggest that Fc-vasa plays an important role in germ-line development, and has utility as a germ cell lineage marker which will help to generate new insight into the origin and differentiation of germ cells as well as the regulation of reproduction in F. chinensis.

  13. microRNAs targeting DEAD-box helicases are involved in salinity stress response in rice (Oryza sativa L.)

    PubMed Central

    2012-01-01

    Background Rice (Oryza sativa L.), one of the most important food crop in the world, is considered to be a salt-sensitive crop. Excess levels of salt adversely affect all the major metabolic activities, including cell wall damage, cytoplasmic lysis and genomic stability. In order to cope with salt stress, plants have evolved high degrees of developmental plasticity, including adaptation via cascades of molecular networks and changes in gene expression profiles. Posttranscriptional regulation, through the activity of microRNAs, also plays an important role in the plant response to salinity conditions. MicroRNAs are small endogenous RNAs that modulate gene expression and are involved in the most essential physiological processes, including plant development and adaptation to environmental changes. Results In the present study, we investigated the expression profiles of osa-MIR414, osa-MIR408 and osa-MIR164e along with their targeted genes, under salinity stress conditions in wild type and transgenic rice plants ectopically expressing the PDH45 (Pea DNA Helicase) gene. The present miRNAs were predicted to target the OsABP (ATP-Binding Protein), OsDSHCT (DOB1/SK12/helY-like DEAD-box Helicase) and OsDBH (DEAD-Box Helicase) genes, included in the DEAD-box helicase family. An in silico characterization of the proteins was performed and the miRNAs predicted targets were validated by RLM-5′RACE. The qRT-PCR analysis showed that the OsABP, OsDBH and OsDSHCT genes were up-regulated in response to 100 and 200 mM NaCl treatments. The present study also highlighted an increased accumulation of the gene transcripts in wild type plants, with the exception of the OsABP mRNA which showed the highest level (15.1-fold change compared to control) in the transgenic plants treated with 200 mM NaCl. Salinity treatments also affected the expression of osa-MIR414, osa-MIR164e and osa-MIR408, found to be significantly down-regulated, although the changes in miRNA expression were limited

  14. Structural and biochemical analyses of the DEAD-box ATPase Sub2 in association with THO or Yra1

    PubMed Central

    Ren, Yi; Schmiege, Philip; Blobel, Günter

    2017-01-01

    mRNA is cotranscrptionally processed and packaged into messenger ribonucleoprotein particles (mRNPs) in the nucleus. Prior to export through the nuclear pore, mRNPs undergo several obligatory remodeling reactions. In yeast, one of these reactions involves loading of the mRNA-binding protein Yra1 by the DEAD-box ATPase Sub2 as assisted by the hetero-pentameric THO complex. To obtain molecular insights into reaction mechanisms, we determined crystal structures of two relevant complexes: a THO hetero-pentamer bound to Sub2 at 6.0 Å resolution; and Sub2 associated with an ATP analogue, RNA, and a C-terminal fragment of Yra1 (Yra1-C) at 2.6 Å resolution. We found that the 25 nm long THO clamps Sub2 in a half-open configuration; in contrast, when bound to the ATP analogue, RNA and Yra1-C, Sub2 assumes a closed conformation. Both THO and Yra1-C stimulated Sub2’s intrinsic ATPase activity. We propose that THO surveys common landmarks in each nuclear mRNP to localize Sub2 for targeted loading of Yra1. DOI: http://dx.doi.org/10.7554/eLife.20070.001 PMID:28059701

  15. Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    PubMed Central

    Iwasaki, Shintaro; Floor, Stephen N.; Ingolia, Nicholas T.

    2016-01-01

    Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumor cells and repress translation of specific mRNAs1-4. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its mRNA selectivity is proposed to reflect highly structured 5′ UTRs that depend strongly on eIF4A-mediated unwinding5. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA1,2,6. Here, we show that secondary structure in 5′ UTRs is only a minor determinant for RocA selectivity and RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions. PMID:27309803

  16. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    NASA Astrophysics Data System (ADS)

    Zhang, Yuan; Palla, Mirkó; Sun, Andrew; Liao, Jung-Chi

    2013-09-01

    DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

  17. The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System

    PubMed Central

    Intile, Peter J.; Balzer, Grant J.; Wolfgang, Matthew C.

    2015-01-01

    ABSTRACT The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence factor important for phagocytic avoidance, disruption of host cell signaling, and host cell cytotoxicity. ExsA is the master regulator of T3SS transcription. The expression, synthesis, and activity of ExsA is tightly regulated by both intrinsic and extrinsic factors. Intrinsic regulation consists of the well-characterized ExsECDA partner-switching cascade, while extrinsic factors include global regulators that alter exsA transcription and/or translation. To identify novel extrinsic regulators of ExsA, we conducted a transposon mutagenesis screen in the absence of intrinsic control. Transposon disruptions within gene PA2840, which encodes a homolog of the Escherichia coli RNA-helicase DeaD, significantly reduced T3SS gene expression. Recent studies indicate that E. coli DeaD can promote translation by relieving inhibitory secondary structures within target mRNAs. We report here that PA2840, renamed DeaD, stimulates ExsA synthesis at the posttranscriptional level. Genetic experiments demonstrate that the activity of an exsA translational fusion is reduced in a deaD mutant. In addition, exsA expression in trans fails to restore T3SS gene expression in a deaD mutant. We hypothesized that DeaD relaxes mRNA secondary structure to promote exsA translation and found that altering the mRNA sequence of exsA or the native exsA Shine-Dalgarno sequence relieved the requirement for DeaD in vivo. Finally, we show that purified DeaD promotes ExsA synthesis using in vitro translation assays. Together, these data reveal a novel regulatory mechanism for P. aeruginosa DeaD and add to the complexity of global regulation of T3SS. IMPORTANCE Although members of the DEAD box family of RNA helicases are appreciated for their roles in mRNA degradation and ribosome biogenesis, an additional role in gene regulation is now emerging in bacteria. By relaxing secondary structures in mRNAs, DEAD box

  18. DDX4 (DEAD box polypeptide 4) colocalizes with cancer stem cell marker CD133 in ovarian cancers

    SciTech Connect

    Kim, Ki Hyung; Kang, Yun-Jeong; Jo, Jin-Ok; Ock, Mee Sun; Moon, Soo Hyun; Suh, Dong Soo; Yoon, Man Soo; Park, Eun-Sil; Jeong, Namkung; Eo, Wan-Kyu; Kim, Heung Yeol; Cha, Hee-Jae

    2014-05-02

    Highlights: • Germ cell marker DDX4 was significantly increased in ovarian cancer. • Ovarian cancer stem cell marker CD133 was significantly increased in ovarian cancer. • DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. • CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4. • Germ cell marker DDX4 has the potential of ovarian cancer stem cell marker. - Abstract: DDX4 (DEAD box polypeptide 4), characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is an RNA helicase which is implicated in various cellular processes involving the alteration of RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. DDX4 is known to be a germ cell-specific protein and is used as a sorting marker of germline stem cells for the production of oocytes. A recent report about DDX4 in ovarian cancer showed that DDX4 is overexpressed in epithelial ovarian cancer and disrupts a DNA damage-induced G2 checkpoint. We investigated the relationship between DDX4 and ovarian cancer stem cells by analyzing the expression patterns of DDX4 and the cancer stem cell marker CD133 in ovarian cancers via tissue microarray. Both DDX4 and CD133 were significantly increased in ovarian cancer compared to benign tumors, and showed similar patterns of expression. In addition, DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. Furthermore, almost all CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4, suggesting a strong possibility that DDX4 plays an important role in cancer stem cells, and/or can be used as an ovarian cancer stem cell marker.

  19. DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs

    PubMed Central

    Soto-Rifo, Ricardo; Rubilar, Paulina S; Limousin, Taran; de Breyne, Sylvain; Décimo, Didier; Ohlmann, Théophile

    2012-01-01

    Here, we have characterized a step in translation initiation of viral and cellular mRNAs that contain RNA secondary structures immediately at the vicinity of their m7GTP cap. This is mediated by the DEAD-box helicase DDX3 which can directly bind to the 5′ of the target mRNA where it clamps the entry of eIF4F through an eIF4G and Poly A-binding protein cytoplasmic 1 (PABP) double interaction. This could induce limited local strand separation of the secondary structure to allow 43S pre-initiation complex attachment to the 5′ free extremity of the mRNA. We further demonstrate that the requirement for DDX3 is highly specific to some selected transcripts, cannot be replaced or substituted by eIF4A and is only needed in the very early steps of ribosome binding and prior to 43S ribosomal scanning. Altogether, these data define an unprecedented role for a DEAD-box RNA helicase in translation initiation. PMID:22872150

  20. ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo

    PubMed Central

    Potratz, Jeffrey P.; Campo, Mark Del; Wolf, Rachel Z.; Lambowitz, Alan M.; Russell, Rick

    2011-01-01

    The yeast DEAD-box protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize non-native intronic structure. Nevertheless, Mss116p acceleration of all three constructs depends upon ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mtDNA containing only the single intron aI5γ, we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-box proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing conversion of non-functional to functional RNA structure. PMID:21679717

  1. A novel protein toxin from the deadly box jellyfish (Sea Wasp, Habu-kurage) Chiropsalmus quadrigatus.

    PubMed

    Nagai, Hiroshi; Takuwa-Kuroda, Kyoko; Nakao, Masahiro; Oshiro, Naomasa; Iwanaga, Setsuko; Nakajima, Terumi

    2002-01-01

    The deadly box jellyfish (Sea Wasp, Habu-kurage in Japanese) Chiropsalmus quadrigatus Haeckel (Cubozoa) is distributed widely in the tropical Pacific region. In Japan, three fatal cases due to stings from this species have been reported officially. We successfully isolated C. quadrigatus toxin-A (CqTX-A, 44 kDa), a major proteinaceous toxin, for the first time, from the nematocysts of C. quadrigatus. CqTX-A showed lethal toxicity to crayfish when administered via intraperitoneal injection (LD50 = 80 microg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50 = 160 ng/ml). Furthermore, we sequenced the cDNA encoding CqTX-A. The deduced amino acid sequence of CqTX-A (462 amino acids) showed 25.2% and 21.6% sequence similarity to Carybdea rastoni toxins (CrTXs) and Carybdea alata toxin-A (CrTX-A), respectively, which are Cubozoan jellyfish toxins.

  2. An unanticipated early function of DEAD-box ATPase Prp28 during commitment to splicing is modulated by U5 snRNP protein Prp8

    PubMed Central

    Price, Argenta M.; Görnemann, Janina; Guthrie, Christine; Brow, David A.

    2014-01-01

    The stepwise assembly of the highly dynamic spliceosome is guided by RNA-dependent ATPases of the DEAD-box family, whose regulation is poorly understood. In the canonical assembly model, the U4/U6.U5 triple snRNP binds only after joining of the U1 and, subsequently, U2 snRNPs to the intron-containing pre-mRNA. Catalytic activation requires the exchange of U6 for U1 snRNA at the 5′ splice site, which is promoted by the DEAD-box protein Prp28. Because Prp8, an integral U5 snRNP protein, is thought to be a central regulator of DEAD-box proteins, we conducted a targeted search in Prp8 for cold-insensitive suppressors of a cold-sensitive Prp28 mutant, prp28-1. We identified a cluster of suppressor mutations in an N-terminal bromodomain-like sequence of Prp8. To identify the precise defect in prp28-1 strains that is suppressed by the Prp8 alleles, we analyzed spliceosome assembly in vivo and in vitro. Surprisingly, in the prp28-1 strain, we observed a block not only to spliceosome activation but also to one of the earliest steps of assembly, formation of the ATP-independent commitment complex 2 (CC2). The Prp8 suppressor partially corrected both the early assembly and later activation defects of prp28-1, supporting a role for this U5 snRNP protein in both the ATP-independent and ATP-dependent functions of Prp28. We conclude that the U5 snRNP has a role in the earliest events of assembly, prior to its stable incorporation into the spliceosome. PMID:24231520

  3. Enhanced expression of tandem multimers of the antimicrobial peptide buforin II in Escherichia coli by the DEAD-box protein and trxB mutant.

    PubMed

    Lee, J H; Kim, M S; Cho, J H; Kim, S C

    2002-05-01

    The tandem multimeric expression of various peptides has been explored by many researchers. However, expression levels have usually not been proportional to the degree of multimerization. To increase the expression level in Escherichia coli of tandem multimers of a cationic antimicrobial peptide, buforin II, fused to an anionic peptide, we studied the effect of the DEAD-box protein and the trxB mutant on the expression of tandem multimers. An expression vector with a tac promoter was more effective in directing multimeric expression than one with a T7 promoter. The expression level of large multimers was substantially increased with the tac promoter, possibly through stabilization of long transcripts by synchronization of transcription and translation. Coexpression of the DEAD-box protein, an RNA-binding protein, with the T7 expression system increased the expression level of multimers, especially large multimers, due to protection of the long RNA transcripts. In addition, the use of the trxB mutant also enhanced the expression level of tandem multimers, which contain two cysteine residues at both ends of the monomeric unit. It seems that disulfide bonds formed in the multimers in the trxB mutant might help efficient charge neutralization for inclusion body formation of the multimers, resulting in enhancement of expression. Our results show that the expression of multimers can be improved through the stabilization of the long transcripts by the DEAD-box protein or the expression, under an oxidizing environment, of the trxB mutant in which covalent cross-links through disulfide bonds facilitate inclusion body formation of the multimeric fusion peptide.

  4. DExD/H-box RNA helicases in ribosome biogenesis

    PubMed Central

    Martin, Roman; Straub, Annika U.; Doebele, Carmen; Bohnsack, Markus T.

    2013-01-01

    Ribosome synthesis requires a multitude of cofactors, among them DExD/H-box RNA helicases. Bacterial RNA helicases involved in ribosome assembly are not essential, while eukaryotes strictly require multiple DExD/H-box proteins that are involved in the much more complex ribosome biogenesis pathway. Here, RNA helicases are thought to act in structural remodeling of the RNPs including the modulation of protein binding, and they are required for allowing access or the release of specific snoRNPs from pre-ribosomes. Interestingly, helicase action is modulated by specific cofactors that can regulate recruitment and enzymatic activity. This review summarizes the current knowledge and focuses on recent findings and open questions on RNA helicase function and regulation in ribosome synthesis. PMID:22922795

  5. Isolation and functional characterization of the promoter of a DEAD-box helicase Psp68 using Agrobacterium-mediated transient assay.

    PubMed

    Banu, Sufara Akhter; Huda, Kazi Md Kamrul; Tuteja, Narendra

    2014-04-30

    Helicases are molecular motor proteins that perform a variety of cellular functions including transcription, translation, DNA replication and repair, RNA maturation, ribosome synthesis, nuclear export and splicing processes. The p68 is an evolutionarily conserved protein which plays pivotal roles in all aspect RNA metabolism processes. It is well established that helicases provides abiotic stress adaptation in plants but analysis of cis-regulatory elements present in the upstream regions is still infancy. Here we report isolation and functional characterization of the promoter of a DEAD-box helicase Psp68 in response to abiotic stress and hormonal regulation. The promoter of Psp68 was isolated by gene walking PCR from pea genomic DNA library constructed in BD genome walker kit. In silico analysis revealed that promoter of Psp68 contained a TATA, a CAAT motif and also harbors some important stress and hormone associated cis regulatory elements, including E-box, AGAAA, GATA-box, ACGT, GAAAA and GTCTC. Functional analyses were performed by Agrobacterium-mediated transient assay in tobacco leaves. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the Psp68 promoter::GUS, subjected to abiotic stress and exogenous hormonal treatments. Stress-inducible nature of Psp68 promoter opens possibility for the study of the gene regulation under stress condition. Therefore, may be useful in the field of agriculture and biotechnology.

  6. The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus

    PubMed Central

    Senissar, Meriem; Saux, Agnès Le; Belgareh-Touzé, Naïma; Adam, Céline; Banroques, Josette; Tanner, N. Kyle

    2014-01-01

    The DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5′ 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrometry analyses, genetic experiments, sucrose gradients, in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs. PMID:25013175

  7. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.

    PubMed

    Khoshnevis, Sohail; Askenasy, Isabel; Johnson, Matthew C; Dattolo, Maria D; Young-Erdos, Crystal L; Stroupe, M Elizabeth; Karbstein, Katrin

    2016-06-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.

  8. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation

    PubMed Central

    Khoshnevis, Sohail; Askenasy, Isabel; Dattolo, Maria D.; Young-Erdos, Crystal L.; Stroupe, M. Elizabeth; Karbstein, Katrin

    2016-01-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed “helicases,” their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked. PMID:27280440

  9. Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae.

    PubMed Central

    Kressler, D; de la Cruz, J; Rojo, M; Linder, P

    1997-01-01

    A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation. PMID:9372960

  10. Crystal structure of the human eIF4AIII–CWC22 complex shows how a DEAD-box protein is inhibited by a MIF4G domain

    PubMed Central

    Buchwald, Gretel; Schüssler, Steffen; Basquin, Claire; Le Hir, Hervé; Conti, Elena

    2013-01-01

    DEAD-box proteins are involved in all aspects of RNA processing. They bind RNA in an ATP-dependent manner and couple ATP hydrolysis to structural and compositional rearrangements of ribonucleoprotein particles. Conformational control is a major point of regulation for DEAD-box proteins to act on appropriate substrates and in a timely manner in vivo. Binding partners containing a middle domain of translation initiation factor 4G (MIF4G) are emerging as important regulators. Well-known examples are eIF4G and Gle1, which bind and activate the DEAD-box proteins eIF4A and Dbp5. Here, we report the mechanism of an inhibiting MIF4G domain. We determined the 2.0-Å resolution structure of the complex of human eIF4AIII and the MIF4G domain of the splicing factor Complexed With Cef1 (CWC22), an essential prerequisite for exon junction complex assembly by the splicing machinery. The CWC22 MIF4G domain binds both RecA domains of eIF4AIII. The mode of RecA2 recognition is similar to that observed in the activating complexes, yet is specific for eIF4AIII. The way the CWC22 MIF4G domain latches on the eIF4AIII RecA1 domain is markedly different from activating complexes. In the CWC22–eIF4AIII complex, the RNA-binding and ATP-binding motifs of the two RecA domains do not face each other, as would be required in the active state, but are in diametrically opposite positions. The binding mode of CWC22 to eIF4AIII reveals a facet of how MIF4G domains use their versatile structural frameworks to activate or inhibit DEAD-box proteins. PMID:24218557

  11. DExD/H-box RNA helicase genes are differentially expressed between males and females during the critical period of male sex differentiation in channel catfish.

    PubMed

    Tian, Changxu; Tan, Suxu; Bao, Lisui; Zeng, Qifan; Liu, Shikai; Yang, Yujia; Zhong, Xiaoxiao; Liu, Zhanjiang

    2017-03-01

    DExD/H-box RNA helicases are motor proteins participating in nearly all aspects of cellular processes, especially in RNA metabolism. In this study, a total of 54 DExD/H-box RNA helicase genes including 37 DDX (DEAD-box) and 17 DHX (DEAH-box) genes were characterized in channel catfish (Ictalurus punctatus), and annotated through phylogenetic and syntenic analyses. All the catfish RNA helicases contained conserved helicase signature motifs, demonstrating that the RNA helicase gene family was highly conserved. Analysis of the relative rates of synonymous (dS) and nonsynonymous (dN) substitutions revealed that the RNA helicase genes were subjected to strong negative (purifying) selection. Meta-analysis was conducted to determine expression of the RNA helicase genes during the critical period (90-110days post-fertilization, dpf) of male gonad differentiation. At 90dpf, 24 RNA helicase genes were highly differentially expressed in the gonad tissues between the males and females; similarly, 24 and 18 RNA helicase genes were found highly differentially expressed in the gonad tissues between the males and females at 100 and 110dpf, respectively (p<0.01). In general, the vast majority of the RNA helicase genes (31) were expressed at higher levels in females than in males. In the male gonad, a set of 8 RNA helicases were expressed at a significantly higher level at 110dpf than at 90dpf. These findings suggested that RNA helicases may play important roles in sex development and differentiation in teleosts.

  12. Plasticity of archaeal C/D box sRNA biogenesis.

    PubMed

    Tripp, Vanessa; Martin, Roman; Orell, Alvaro; Alkhnbashi, Omer S; Backofen, Rolf; Randau, Lennart

    2017-01-01

    Archaeal and eukaryotic organisms contain sets of C/D box s(no)RNAs with guide sequences that determine ribose 2'-O-methylation sites of target RNAs. The composition of these C/D box sRNA sets is highly variable between organisms and results in varying RNA modification patterns which are important for ribosomal RNA folding and stability. Little is known about the genomic organization of C/D box sRNA genes in archaea. Here, we aimed to obtain first insights into the biogenesis of these archaeal C/D box sRNAs and analyzed the genetic context of more than 300 archaeal sRNA genes. We found that the majority of these genes do not possess independent promoters but are rather located at positions that allow for co-transcription with neighboring genes and their start or stop codons were frequently incorporated into the conserved boxC and D motifs. The biogenesis of plasmid-encoded C/D box sRNA variants was analyzed in vivo in Sulfolobus acidocaldarius. It was found that C/D box sRNA maturation occurs independent of their genetic context and relies solely on the presence of intact RNA kink-turn structures. The observed plasticity of C/D box sRNA biogenesis is suggested to enable their accelerated evolution and, consequently, allow for adjustments of the RNA modification landscape.

  13. An essential role for the Saccharomyces cerevisiae DEAD-box helicase DHH1 in G1/S DNA-damage checkpoint recovery.

    PubMed Central

    Bergkessel, Megan; Reese, Joseph C

    2004-01-01

    The eukaryotic cell cycle displays a degree of plasticity in its regulation; cell cycle progression can be transiently arrested in response to environmental stresses. While the signaling pathways leading to cell cycle arrest are beginning to be well understood, the regulation of the release from arrest has not been well characterized. Here we show that DHH1, encoding a DEAD-box RNA helicase orthologous to the human putative proto-oncogene p54/RCK, is important in release from DNA-damage-induced cell cycle arrest at the G1/S checkpoint. DHH1 mutants are not defective for DNA repair and recover normally from the G2/M and replication checkpoints, suggesting a specific function for Dhh1p in recovery from G1/S checkpoint arrest. Dhh1p has been suggested to play a role in partitioning mRNAs between translatable and nontranslatable pools, and our results implicate this modulation of mRNA metabolism in the recovery from G1/S cell cycle arrest following DNA damage. Furthermore, the high degree of conservation between DHH1 and its human ortholog suggests that this mechanism is conserved among all eukaryotes and potentially important in human disease. PMID:15166134

  14. Box C/D Small Nucleolar RNA (snoRNA) U60 Regulates Intracellular Cholesterol Trafficking*

    PubMed Central

    Brandis, Katrina A.; Gale, Sarah; Jinn, Sarah; Langmade, Stephen J.; Dudley-Rucker, Nicole; Jiang, Hui; Sidhu, Rohini; Ren, Aileen; Goldberg, Anna; Schaffer, Jean E.; Ory, Daniel S.

    2013-01-01

    Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum is essential for cellular cholesterol homeostasis. The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Because mutant cells with defects in PM to endoplasmic reticulum cholesterol trafficking can be isolated on the basis of resistance to amphotericin B, we conducted an amphotericin B loss-of-function screen in Chinese hamster ovary (CHO) cells using insertional mutagenesis to identify genes that regulate this trafficking mechanism. Mutant line A1 displayed reduced cholesteryl ester formation from PM-derived cholesterol and increased de novo cholesterol synthesis, indicating a deficiency in retrograde cholesterol transport. Genotypic analysis revealed that the A1 cell line contained one disrupted allele of the U60 small nucleolar RNA (snoRNA) host gene, resulting in haploinsufficiency of the box C/D snoRNA U60. Complementation and mutational studies revealed the U60 snoRNA to be the essential feature from this locus that affects cholesterol trafficking. Lack of alteration in predicted U60-mediated site-directed methylation of 28 S rRNA in the A1 mutant suggests that the U60 snoRNA modulates cholesterol trafficking by a mechanism that is independent of this canonical function. Our study adds to a growing body of evidence for participation of small noncoding RNAs in cholesterol homeostasis and is the first to implicate a snoRNA in this cellular function. PMID:24174535

  15. Box C/D small nucleolar RNA (snoRNA) U60 regulates intracellular cholesterol trafficking.

    PubMed

    Brandis, Katrina A; Gale, Sarah; Jinn, Sarah; Langmade, Stephen J; Dudley-Rucker, Nicole; Jiang, Hui; Sidhu, Rohini; Ren, Aileen; Goldberg, Anna; Schaffer, Jean E; Ory, Daniel S

    2013-12-13

    Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum is essential for cellular cholesterol homeostasis. The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Because mutant cells with defects in PM to endoplasmic reticulum cholesterol trafficking can be isolated on the basis of resistance to amphotericin B, we conducted an amphotericin B loss-of-function screen in Chinese hamster ovary (CHO) cells using insertional mutagenesis to identify genes that regulate this trafficking mechanism. Mutant line A1 displayed reduced cholesteryl ester formation from PM-derived cholesterol and increased de novo cholesterol synthesis, indicating a deficiency in retrograde cholesterol transport. Genotypic analysis revealed that the A1 cell line contained one disrupted allele of the U60 small nucleolar RNA (snoRNA) host gene, resulting in haploinsufficiency of the box C/D snoRNA U60. Complementation and mutational studies revealed the U60 snoRNA to be the essential feature from this locus that affects cholesterol trafficking. Lack of alteration in predicted U60-mediated site-directed methylation of 28 S rRNA in the A1 mutant suggests that the U60 snoRNA modulates cholesterol trafficking by a mechanism that is independent of this canonical function. Our study adds to a growing body of evidence for participation of small noncoding RNAs in cholesterol homeostasis and is the first to implicate a snoRNA in this cellular function.

  16. Xenopus U3 snoRNA GAC-Box A′ and Box A Sequences Play Distinct Functional Roles in rRNA Processing

    PubMed Central

    Borovjagin, Anton V.; Gerbi, Susan A.

    2001-01-01

    Mutations in the 5′ portion of Xenopus U3 snoRNA were tested for function in oocytes. The results revealed a new cleavage site (A0) in the 3′ region of vertebrate external transcribed spacer sequences. In addition, U3 mutagenesis uncoupled cleavage at sites 1 and 2, flanking the 5′ and 3′ ends of 18S rRNA, and generated novel intermediates: 19S and 18.5S pre-rRNAs. Furthermore, specific nucleotides in Xenopus U3 snoRNA that are required for cleavages in pre-rRNA were identified: box A is essential for site A0 cleavage, the GAC-box A′ region is necessary for site 1 cleavage, and the 3′ end of box A′ and flanking nucleotides are required for site 2 cleavage. Differences between metazoan and yeast U3 snoRNA-mediated rRNA processing are enumerated. The data support a model where metazoan U3 snoRNA acts as a bridge to draw together the 5′ and 3′ ends of the 18S rRNA coding region within pre-rRNA to coordinate their cleavage. PMID:11509664

  17. Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells

    PubMed Central

    Rumm, Andreas; Trasak, Claudia; Ruckdeschel, Klaus; Alawi, Malik; Grundhoff, Adam; Kikhney, Alexey G.; Koch-Nolte, Friedrich; Buck, Friedrich; Perbandt, Markus; Betzel, Christian; Svergun, Dmitri I.; Hentschke, Moritz; Aepfelbacher, Martin

    2016-01-01

    Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines. PMID:27300509

  18. Identification of the human DEAD-box protein p68 as a substrate of Tlk1

    SciTech Connect

    Kodym, Reinhard . E-mail: reinhard.kodym@meduniwien.ac.at; Henoeckl, Christian; Fuerweger, Christoph

    2005-07-29

    The activity of the human protein kinase Tlk1 is down-regulated within minutes after exposure of cells to ionizing radiation. In order to identify signaling pathways which might be relevant in the radiation response of mammalian cells we screened nuclear proteins for substrates of Tlk1. Among several proteins one could be identified as p68 RNA helicase. Furthermore, it could be shown that Tlk1 phosphorylates immunoprecipitated p68. The phosphorylation of the C-terminal fragment of p68 by rTlk1 reduced its affinity to single stranded RNA in a gel shift assay. In addition, it could be demonstrated that increasing the Tlk1 activity in HT1080 cells by forced Tlk1 overexpression leads to an increased phosphorylation of endogenous p68, arguing that p68 might be a physiological substrate of Tlk1. These findings open the possibility that Tlk1 might participate in diverse biologic functions like cell growth and differentiation, pre-mRNA splicing, and transcriptional coactivation.

  19. A novel B cell epitope in cold-shock DEAD-box protein A from Mycobacterium tuberculosis.

    PubMed

    Wang, Huanan; Zhu, Ting; Yu, Shenye; Liu, Huifang; Wang, Xiumei; Chen, Liping; Si, Wei; Pang, Hai; Liu, Siguo

    2013-06-01

    In this study, a hybridoma-based technique and phage display technology were used to obtain mouse monoclonal antibodies (mAb) against cold-shock DEAD-box protein A (CsdA) from Mycobacterium tuberculosis and to determine the location of the relevant epitope. One highly specific mAb, named A3G5, was developed against the recombinant CsdA protein (rCsdA) and could detect rCsdA protein in enzyme-linked immunosorbent assays (ELISA) and Western blot assays. By screening a phage displayed library of random 12-mers (Ph.D.-12), 10 positive phage clones were randomly selected after three rounds of bio-panning and identified by ELISA. Eight of these clones were sequenced, and their amino acid sequences were deduced. One B-cell epitope (-APDPPLSRR-) in the rCsdA protein was identified with mAb A3G5. A synthetic peptide (-MAPDPPLSRR-) (Cpep) matched well with the CsdA sequence at 443-451 aa and was confirmed by affinity ELISA, competitive inhibition assays and the development of an immune response in mice. These results may be of great potential value in the further analysis of the function and structure of the CsdA protein from M. tuberculosis.

  20. MicroRNA regulation of F-box proteins and its role in cancer.

    PubMed

    Wu, Zhao-Hui; Pfeffer, Lawrence M

    2016-02-01

    MicroRNAs (miRNAs) are small endogenous non-coding RNAs, which play critical roles in cancer development by suppressing gene expression at the post-transcriptional level. In general, oncogenic miRNAs are upregulated in cancer, while miRNAs that act as tumor suppressors are downregulated, leading to decreased expression of tumor suppressors and upregulated oncogene expression, respectively. F-box proteins function as the substrate-recognition components of the SKP1-CUL1-F-box (SCF)-ubiquitin ligase complex for the degradation of their protein targets by the ubiquitin-proteasome system. Therefore F-box proteins and miRNAs both negatively regulate target gene expression post-transcriptionally. Since each miRNA is capable of fine-tuning the expression of multiple target genes, multiple F-box proteins may be suppressed by the same miRNA. Meanwhile, one F-box proteins could be regulated by several miRNAs in different cancer types. In this review, we will focus on miRNA-mediated downregulation of various F-box proteins, the resulting stabilization of F-box protein substrates and the impact of these processes on human malignancies. We provide insight into how the miRNA: F-box protein axis may regulate cancer progression and metastasis. We also consider the broader role of F-box proteins in the regulation of pathways that are independent of the ubiquitin ligase complex and how that impacts on oncogenesis. The area of miRNAs and the F-box proteins that they regulate in cancer is an emerging field and will inform new strategies in cancer treatment.

  1. Second-Site Suppression of RNase E Essentiality by Mutation of the deaD RNA Helicase in Escherichia coli

    PubMed Central

    Tamura, Masaru; Kers, Johan A.

    2012-01-01

    Escherichia coli cells normally require RNase E activity to propagate and form colonies. Using random Tn10 insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) to E. coli cells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of the rne mutant phenotype by inactivation of deaD was partial, as rne deaD doubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration by deaD inactivation requires normal expression of the endogenous rng gene in doubly mutant rne deaD cells. Second-site suppression by deaD mutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality. PMID:22328678

  2. T box riboswitches in Actinobacteria: Translational regulation via novel tRNA interactions

    PubMed Central

    Sherwood, Anna V.; Grundy, Frank J.; Henkin, Tina M.

    2015-01-01

    The T box riboswitch regulates many amino acid-related genes in Gram-positive bacteria. T box riboswitch-mediated gene regulation was shown previously to occur at the level of transcription attenuation via structural rearrangements in the 5′ untranslated (leader) region of the mRNA in response to binding of a specific uncharged tRNA. In this study, a novel group of isoleucyl-tRNA synthetase gene (ileS) T box leader sequences found in organisms of the phylum Actinobacteria was investigated. The Stem I domains of these RNAs lack several highly conserved elements that are essential for interaction with the tRNA ligand in other T box RNAs. Many of these RNAs were predicted to regulate gene expression at the level of translation initiation through tRNA-dependent stabilization of a helix that sequesters a sequence complementary to the Shine–Dalgarno (SD) sequence, thus freeing the SD sequence for ribosome binding and translation initiation. We demonstrated specific binding to the cognate tRNAIle and tRNAIle-dependent structural rearrangements consistent with regulation at the level of translation initiation, providing the first biochemical demonstration, to our knowledge, of translational regulation in a T box riboswitch. PMID:25583497

  3. Biochemical Features and Functional Implications of the RNA-Based T-Box Regulatory Mechanism

    PubMed Central

    Gutiérrez-Preciado, Ana; Henkin, Tina M.; Grundy, Frank J.; Yanofsky, Charles; Merino, Enrique

    2009-01-01

    Summary: The T-box mechanism is a common regulatory strategy used for modulating the expression of genes of amino acid metabolism-related operons in gram-positive bacteria, especially members of the Firmicutes. T-box regulation is usually based on a transcription attenuation mechanism in which an interaction between a specific uncharged tRNA and the 5′ region of the transcript stabilizes an antiterminator structure in preference to a terminator structure, thereby preventing transcription termination. Although single T-box regulatory elements are common, double or triple T-box arrangements are also observed, expanding the regulatory range of these elements. In the present study, we predict the functional implications of T-box regulation in genes encoding aminoacyl-tRNA synthetases, proteins of amino acid biosynthetic pathways, transporters, and regulatory proteins. We also consider the global impact of the use of this regulatory mechanism on cell physiology. Novel biochemical relationships between regulated genes and their corresponding metabolic pathways were revealed. Some of the genes identified, such as the quorum-sensing gene luxS, in members of the Lactobacillaceae were not previously predicted to be regulated by the T-box mechanism. Our analyses also predict an imbalance in tRNA sensing during the regulation of operons containing multiple aminoacyl-tRNA synthetase genes or biosynthetic genes involved in pathways common to more than one amino acid. Based on the distribution of T-box regulatory elements, we propose that this regulatory mechanism originated in a common ancestor of members of the Firmicutes, Chloroflexi, Deinococcus-Thermus group, and Actinobacteria and was transferred into the Deltaproteobacteria by horizontal gene transfer. PMID:19258532

  4. Defective chloroplast development inhibits maintenance of normal levels of abscisic acid in a mutant of the Arabidopsis RH3 DEAD-box protein during early post-germination growth.

    PubMed

    Lee, Kwang-Hee; Park, Jiyoung; Williams, Donna S; Xiong, Yuqing; Hwang, Inhwan; Kang, Byung-Ho

    2013-03-01

    The plastid has its own translation system, and its ribosomes are assembled through a complex process in which rRNA precursors are processed and ribosomal proteins are inserted into the rRNA backbone. DEAD-box proteins have been shown to play roles in multiple steps in ribosome biogenesis. To investigate the cellular and physiological roles of an Arabidopsis DEAD-box protein, RH3, we examined its expression and localization and the phenotypes of rh3-4, a T-DNA insertion mutant allele of RH3. The promoter activity of RH3 is strongest in the greening tissues of 3-day and 1-week-old seedlings but reduced afterwards. Cotyledons were pale and seedling growth was retarded in the mutant. The most obvious abnormality in the mutant chloroplasts was their lack of normal ribosomes. Electron tomography analysis indicated that ribosome density in the 3-day-old mutant chloroplasts is only 20% that of wild-type chloroplasts, and the ribosomes in the mutant are smaller. These chloroplast defects in rh3-4 were alleviated in 2-week-old cotyledons and true leaves. Interestingly, rh3-4 seedlings have lower amounts of abscisic acid prior to recovery of their chloroplasts, and were more sensitive to abiotic stresses. Transcriptomic analysis indicated that nuclear genes for chloroplast proteins are down-regulated, and proteins mediating chloroplast-localized steps of abscisic acid biosynthesis are expressed to a lower extent in 1-week-old rh3-4 seedlings. Taken together, these results suggest that conversion of eoplasts into chloroplasts in young seedlings is critical for the seedlings to start carbon fixation as well as for maintenance of abscisic acid levels for responding to environmental challenges.

  5. T box RNA decodes both the information content and geometry of tRNA to affect gene expression.

    PubMed

    Grigg, Jason C; Chen, Yujie; Grundy, Frank J; Henkin, Tina M; Pollack, Lois; Ke, Ailong

    2013-04-30

    The T box leader sequence is an RNA element that controls gene expression by binding directly to a specific tRNA and sensing its aminoacylation state. This interaction controls expression of amino acid-related genes in a negative feedback loop. The T box RNA structure is highly conserved, but its tRNA binding mechanism is only partially understood. Known sequence elements are the specifier sequence, which recognizes the tRNA anticodon, and the antiterminator bulge, which base pairs with the tRNA acceptor end. Here, we reveal the crucial function of the highly conserved stem I distal region in tRNA recognition and report its 2.65-Å crystal structure. The apex of this region contains an intricately woven loop-loop interaction between two conserved motifs, the Adenine-guanine (AG) bulge and the distal loop. This loop-loop structure presents a base triple on its surface that is optimally positioned for base-stacking interactions. Mutagenesis, cross-linking, and small-angle X-ray scattering data demonstrate that the apical base triple serves as a binding platform to dock the tRNA D- and T-loops. Strikingly, the binding platform strongly resembles the D- and T-loop binding elements from RNase P and the ribosome exit site, suggesting that this loop-loop structure may represent a widespread tRNA recognition platform. We propose a two-checkpoint molecular ruler model for tRNA decoding in which the information content of tRNA is first examined through specifier sequence-anticodon interaction, and the length of the tRNA anticodon arm is then measured by the distal loop-loop platform. When both conditions are met, tRNA is secured, and its aminoacylation state is sensed.

  6. Dynamics and persistence of Dead Sea microbial populations as shown by high-throughput sequencing of rRNA.

    PubMed

    Rhodes, Matthew E; Oren, Aharon; House, Christopher H

    2012-04-01

    16S rRNA amplicon libraries from a haloarchaeal bloom in the hypersaline Dead Sea in 1992 were analyzed together with the 2007 residual population and simulated blooms in experimental mesocosms. Significant population shifts were observed during the bloom, and surprisingly a signature from the bloom was retained 15 years later.

  7. Identification of the RGG Box Motif in Shadoo: RNA-Binding and Signaling Roles?

    PubMed Central

    Corley, Susan M.; Gready, Jill E.

    2008-01-01

    Using comparative genomics and in-silico analyses, we previously identified a new member of the prion-protein (PrP) family, the gene SPRN, encoding the protein Shadoo (Sho), and suggested its functions might overlap with those of PrP. Extended bioinformatics and conceptual biology studies to elucidate Sho’s functions now reveal Sho has a conserved RGG-box motif, a well-known RNA-binding motif characterized in proteins such as FragileX Mental Retardation Protein. We report a systematic comparative analysis of RGG-box containing proteins which highlights the motif’s functional versatility and supports the suggestion that Sho plays a dual role in cell signaling and RNA binding in brain. These findings provide a further link to PrP, which has well-characterized RNA-binding properties. PMID:19812790

  8. Structural insights into the mechanism of the DEAH-box RNA helicase Prp43

    PubMed Central

    Tauchert, Marcel J; Fourmann, Jean-Baptiste; Lührmann, Reinhard; Ficner, Ralf

    2017-01-01

    The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43•ATP-analog•RNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a β-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases. DOI: http://dx.doi.org/10.7554/eLife.21510.001 PMID:28092261

  9. The T box mechanism: tRNA as a regulatory molecule

    PubMed Central

    Green, Nicholas J.; Grundy, Frank J.; Henkin, Tina M.

    2009-01-01

    The T box mechanism is widely used in Gram-positive bacteria to regulate expression of aminoacyl-tRNA synthetase genes and genes involved in amino acid biosynthesis and uptake. Binding of a specific uncharged tRNA to a riboswitch element in the nascent transcript causes a structural change in the transcript that promotes expression of the downstream coding sequence. In most cases, this occurs by stabilization of an antiterminator element that competes with formation of a terminator helix. Specific tRNA recognition by the nascent transcript results in increased expression of genes important for tRNA aminoacylation in response to decreased pools of charged tRNA. PMID:19932103

  10. Overexpression of DEAD box protein pMSS116 promotes ATP-dependent splicing of a yeast group II intron in vitro.

    PubMed Central

    Niemer, I; Schmelzer, C; Börner, G V

    1995-01-01

    The group II intron bI1, the first intron of the mitochondrial cytochrome b gene in yeast is self-splicing in vitro. Genetic evidence suggests that trans-acting factors are required for in vivo splicing of this intron. In accordance with these findings, we present in vitro data showing that splicing of bI1 under physiological conditions depends upon the presence of proteins of a mitochondrial lysate. ATP is an essential component is this reaction. Overexpression of the nuclear-encoded DEAD box protein pMSS-116 results in a marked increase in the ATP-dependent splicing activity of the extract, suggesting that pMSS116 may play an important role in splicing of bI1. Images PMID:7659519

  11. Box C/D sRNA stem ends act as stabilizing anchors for box C/D di-sRNPs

    PubMed Central

    Yip, W. S. Vincent; Shigematsu, Hideki; Taylor, David W.; Baserga, Susan J.

    2016-01-01

    Ribosomal RNA (rRNA) modifications are essential for ribosome function in all cellular organisms. Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze 2′-O-methylation, one rRNA modification type in Eukarya and Archaea. Negatively stained electron microscopy (EM) models of archaeal box C/D sRNPs have demonstrated the dimeric sRNP (di-sRNP) architecture, which has been corroborated by nuclear magnetic resonance (NMR) studies. Due to limitations of the structural techniques, the orientation of the box C/D sRNAs has remained unclear. Here, we have used cryo-EM to elucidate the sRNA orientation in a M. jannaschii box C/D di-sRNP. The cryo-EM reconstruction suggests a parallel orientation of the two sRNAs. Biochemical and structural analyses of sRNPs assembled with mutant sRNAs indicate a potential interaction between the sRNA stem ends. Our results suggest that the parallel arrangement of the sRNAs juxtaposes their stem ends into close proximity to allow for a stabilizing interaction that helps maintain the di-sRNP architecture. PMID:27342279

  12. Translation during cold adaptation does not involve mRNA-rRNA base pairing through the downstream box.

    PubMed Central

    La Teana, A; Brandi, A; O'Connor, M; Freddi, S; Pon, C L

    2000-01-01

    The downstream box (DB) has been proposed to enhance translation of several mRNAs and to be a key element controlling the expression of cold-shocked mRNAs. However, the proposal that the DB exerts its effects through a base pairing interaction with the complementary anti-downstream box (antiDB) sequence (nt 1469-1483) located in the penultimate stem (helix 44) of 16S rRNA remains controversial. The existence of this interaction during initiation of protein synthesis under cold-shock conditions has been investigated in the present work using an Escherichia coli strain whose ribosomes lack the potential to base pair with mRNA because of a 12 bp inversion of the antiDB sequence in helix 44. Our results show that this strain is capable of cold acclimation, withstands cold shock, and its ribosomes translate mRNAs that contain or lack DB sequences with similar efficiency, comparable to that of the wild type. The structure of helix 44 in 30S ribosomal subunits from cells grown at 37 degrees C and from cells subjected to cold shock was also analyzed by binding a 32P-labeled oligonucleotide complementary to the antiDB region and by chemical probing with DMS and kethoxal. Both approaches clearly indicate that this region is in a double-stranded conformation and therefore not available for base pairing with mRNA. PMID:11073215

  13. The RNA polymerase I transcription factor xUBF contains 5 tandemly repeated HMG homology boxes.

    PubMed Central

    Bachvarov, D; Moss, T

    1991-01-01

    The RNA polymerase I transcription factor UBF has been identified in human, mouse, rat and Xenopus and the primary structure of the human protein has been determined. Human UBF was shown to contain four tandem homologies to the folding domains of the HMG1 and 2 proteins and hence to belong to a previously unrecognised family of 'HMG-box' transcription factors. Here, cDNA clones encoding the Xenopus laevis UBF (xUBF) have been isolated and sequenced. Northern and Southern blots revealed that in tissue culture cells, xUBF is coded on a single major mRNA size species by a small number of genes. The deduced primary structure of xUBF is highly homologous with the human protein except for a central deletion which removes most of one HMG-box. This explains the major size difference between the X. laevis and human proteins and may well explain their different transcriptional specificities. It is shown that xUBF contains 5 tandemly repeated HMG-boxes and that by analogy the human protein contains 6. Images PMID:2041774

  14. Synthesis and stereospecificity of 4,5-disubstituted oxazolidinone ligands binding to T-box riboswitch RNA

    PubMed Central

    Orac, Crina M.; Zhou, Shu; Means, John A.; Boehm, David; Bergmeier, Stephen C.; Hines, Jennifer V.

    2012-01-01

    The enantiomers and the cis isomers of two previously studied 4,5-disubstituted oxazolidinones have been synthesized and their binding to the T-box riboswitch antiterminator model RNA investigated in detail. Characterization of ligand affinities and binding site localization indicate that there is little stereospecific discrimination for binding antiterminator RNA alone. This binding similarity between enantiomers is likely due to surface binding, which accommodates ligand conformations that result in comparable ligand-antiterminator contacts. These results have significant implications for T-box antiterminator-targeted drug discovery and, in general, for targeting other medicinally relevant RNA that do not present deep binding pockets. PMID:21812425

  15. Crystal structures of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition.

    PubMed

    Edwards, Thomas E; Ferré-D'Amaré, Adrian R

    2006-09-01

    Riboswitches are noncoding mRNA elements that bind small-molecule metabolites with high affinity and specificity, and they regulate the expression of associated genes. The thi-box riboswitch can exhibit a 1000-fold higher affinity for thiamine pyrophosphate over closely related noncognate compounds such as thiamine monophosphate. To understand the chemical basis of thi-box pyrophosphate specificity, we have determined crystal structures of an E. coli thi-box bound to thiamine pyrophosphate, thiamine monophosphate, and the structural analogs benfotiamine and pyrithiamine. When bound to monophosphorylated compounds, the RNA elements that recognize the thiamine and phosphate moieties of the ligand move closer together. This allows the riboswitch to recognize the monophosphate in a manner similar to how it recognizes the beta-phosphate of thiamine pyrophosphate. In the pyrithiamine complex, the pyrophosphate binding site is largely unstructured. These results show how the riboswitch can bind to various metabolites, and why the thi-box preferentially binds thiamine pyrophosphate.

  16. Overexpression of an Apocynum venetum DEAD-Box Helicase Gene (AvDH1) in Cotton Confers Salinity Tolerance and Increases Yield in a Saline Field

    PubMed Central

    Chen, Jie; Wan, Sibao; Liu, Huaihua; Fan, Shuli; Zhang, Yujuan; Wang, Wei; Xia, Minxuan; Yuan, Rui; Deng, Fenni; Shen, Fafu

    2016-01-01

    Soil salinity is a major environmental stress limiting plant growth and productivity. We have reported previously the isolation of an Apocynum venetum DEAD-box helicase 1 (AvDH1) that is expressed in response to salt exposure. Here, we report that the overexpression of AvDH1 driven by a constitutive cauliflower mosaic virus-35S promoter in cotton plants confers salinity tolerance. Southern and Northern blotting analyses showed that the AvDH1 gene was integrated into the cotton genome and expressed. In this study, the growth of transgenic cotton expressing AvDH1 was evaluated under saline conditions in a growth chamber and in a saline field trial. Transgenic cotton overexpressing AvDH1 was much more resistant to salt than the wild-type plants when grown in a growth chamber. The lower membrane ion leakage, along with increased activity of superoxide dismutase, in AvDH1 transgenic lines suggested that these characteristics may prevent membrane damage, which increases plant survival rates. In a saline field, the transgenic cotton lines expressing AvDH1 showed increased boll numbers, boll weights and seed cotton yields compared with wild-type plants, especially at high soil salinity levels. This study indicates that transgenic cotton expressing AvDH1 is a promising option for increasing crop productivity in saline fields. PMID:26779246

  17. DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked is an immunogenic target of cancer stem cells.

    PubMed

    Koshio, Jun; Kagamu, Hiroshi; Nozaki, Koichiro; Saida, Yu; Tanaka, Tomohiro; Shoji, Satoshi; Igarashi, Natsue; Miura, Satoru; Okajima, Masaaki; Watanabe, Satoshi; Yoshizawa, Hirohisa; Narita, Ichiei

    2013-10-01

    Accumulating evidence suggests that most solid malignancies consist of heterogeneous tumor cells and that a relatively small subpopulation, which shares biological features with stem cells, survives through potentially lethal stresses such as chemotherapy and radiation treatment. Since the survival of this subpopulation of cancer stem cells (CSC) plays a critical role in recurrence, it must be eradicated in order to cure cancer. We previously reported that vaccination with CD133(+) murine melanoma cells exhibiting biological CSC features induced CSC-specific effector T cells. These were capable of eradicating CD133(+) tumor cells in vivo, thereby curing the parental tumor. In the current study, we indicated that DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked (DDX3X) is an immunogenic protein preferentially expressed in CD133(+) tumor cells. Vaccination with DDX3X primed specific T cells, resulting in protective and therapeutic antitumor immunity. The DDX3X-primed CD4(+) T cells produced CD133(+) tumor-specific IFNγ and IL-17 and mediated potent antitumor therapeutic efficacy. DDX3X is expressed in various human cancer cells, including lung, colon, and breast cancer cells. These results suggest that anti-DDX3X immunotherapy is a promising treatment option in efforts to eradicate CSC in the clinical setting.

  18. The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes.

    PubMed Central

    Wagner, J D; Jankowsky, E; Company, M; Pyle, A M; Abelson, J N

    1998-01-01

    Of the proteins required for pre-mRNA splicing, at least four, the DEAH-box proteins, are closely related due to the presence of a central 'RNA helicase-like' region, and extended homology through a large portion of the protein. A major unresolved question is the function of these proteins. Indirect evidence suggests that several of these proteins are catalysts for important structural rearrangements in the spliceosome. However, the mechanism for the proposed alterations is presently unknown. We present evidence that PRP22, a DEAH-box protein required for mRNA release from the spliceosome, unwinds RNA duplexes in a concentration- and ATP-dependent manner. This demonstrates that PRP22 can modify RNA structure directly. We also show that the PRP22-dependent release of mRNA from the spliceosome is an ATP-dependent process and that recombinant PRP22 is an ATPase. Non-hydrolyzable ATP analogs did not substitute for ATP in the RNA-unwinding reaction, suggesting that ATP hydrolysis is required for this reaction. Specific mutation of a putative ATP phosphate-binding motif in the recombinant protein eliminated the ATPase and RNA-unwinding capacity. Significantly, these data suggest that the DEAH-box proteins act directly on RNA substrates within the spliceosome. PMID:9582286

  19. Cocrystal structure of a T-box riboswitch Stem I domain in complex with its cognate tRNA

    PubMed Central

    Zhang, Jinwei; Ferré-D’Amaré, Adrian R.

    2013-01-01

    In Gram-positive bacteria, T-box riboswitches regulate expression of aminoacyl-tRNA synthetases (ARSs) and other proteins in response to fluctuating tRNA aminoacylation levels under various nutritional states1. T-boxes reside in the 5’-untranslated regions (UTRs) of the mRNAs they regulate, and comprise two conserved domains. Stem I harbors the specifier trinucleotide that base-pairs with the anticodon of cognate tRNA. 3’ to Stem I is the antiterminator domain, which base-pairs with the tRNA acceptor end and evaluates its aminoacylation state2. Despite high phylogenetic conservation and widespread occurrence in pathogens, the structural basis of tRNA recognition3,4 by this riboswitch remains ill-defined. Here, we demonstrate that the ~100-nucleotide T-box Stem I is necessary and sufficient for specific, high-affinity (Kd ~150 nM) tRNA binding, and report its structure in complex with cognate tRNA at 3.2 Å resolution. Stem I recognizes the overall architecture of tRNA in addition to its anticodon, something accomplished by large ribonucleoproteins (RNPs) like the ribosome or proteins such as ARSs5, but unprecedented for a compact mRNA domain. The C-shaped Stem I cradles the L-shaped tRNA forming an extended (1604 Å2) intermolecular interface. In addition to the specifier-anticodon interaction, two interdigitated T-loops near the apex of Stem I stack on the tRNA elbow in a manner analogous to those of the J11/12-J12/11 motif6 of RNase P and the L1 stalk7 of the ribosomal E-site. Since these RNPs and T-boxes are unrelated, this strategy to recognize an universal tRNA feature likely evolved convergently. Mutually induced fit of Stem I and the tRNA exploiting the intrinsic flexibility of tRNA and its conserved post-transcriptional modifications results in high shape complementarity, which in addition to providing specificity and affinity, globally organizes the T-box to orchestrate tRNA-dependent transcription regulation. PMID:23892783

  20. Detection of dsRNA-binding domains in RNA helicase A and Drosophila maleless: implications for monomeric RNA helicases.

    PubMed Central

    Gibson, T J; Thompson, J D

    1994-01-01

    Searches with dsRNA-binding domain profiles detected two copies of the domain in each of RNA helicase A, Drosophila maleless and C. elegans ORF T20G5-11 (of unknown function). RNA helicase A is unusual in being one of the few characterised DEAD/DExH helicases that are active as monomers. Other monomeric DEAD/DExH RNA helicases (p68, NPH-II) have domains that match another RNA-binding motif, the RGG repeat. The DEAD/DExH domain appears to be insufficient on its own to promote helicase activity and additional RNA-binding capacity must be supplied either as domains adjacent to the DEAD/DExH-box or by bound partners as in the eIF-4AB dimer. The presence or absence of extra RNA-binding domains should allow classification of DEAD/DExH proteins as monomeric or multimeric helicases. Images PMID:8041617

  1. Analysis of bacteriophage N protein and peptide binding to boxB RNA using polyacrylamide gel coelectrophoresis (PACE).

    PubMed Central

    Cilley, C D; Williamson, J R

    1997-01-01

    The antitermination protein N from bacteriophage lambda (Nlambda) interacts with the nut site in its own mRNA, as well as host factors, to facilitate formation of a termination-resistant transcription complex. The conserved, amino-terminal arginine-rich domain of Nlambda protein is known to interact with a small RNA hairpin (boxB) derived from the nut site RNA. We have examined the binding of Nlambda protein, peptides derived from the amino terminus of Nlambda, and the related phage P22 N protein to lambda boxB RNAs. To facilitate the study of complexes that are not amenable to gel retardation assays, a new polyacrylamide affinity coelectrophoresis technique (PACE) was developed. Using the PACE assay, we have demonstrated that a 19-amino acid peptide from the amino terminus of Nlambda protein binds lambda boxB RNA with a Kd,app of 5.2 nM. PACE was also used to study the binding affinity of a number of Nlambda peptide and lambda boxB RNA mutants. The PACE technique is complementary to the traditional gel retardation assay for direct measurement of binding interactions, and will be useful for any procedure that requires a pool of RNAs to be resolved based on their relative affinities for proteins or peptides. PMID:8990399

  2. Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B.

    PubMed

    Özeş, Ali R; Feoktistova, Kateryna; Avanzino, Brian C; Fraser, Christopher S

    2011-09-30

    Eukaryotic initiation factor (eIF) 4A is a DEAD-box helicase that stimulates translation initiation by unwinding mRNA secondary structure. The accessory proteins eIF4G, eIF4B, and eIF4H enhance the duplex unwinding activity of eIF4A, but the extent to which they modulate eIF4A activity is poorly understood. Here, we use real-time fluorescence assays to determine the kinetic parameters of duplex unwinding and ATP hydrolysis by these initiation factors. To ensure efficient duplex unwinding, eIF4B and eIF4G cooperatively activate the duplex unwinding activity of eIF4A. Our data reveal that eIF4H is much less efficient at stimulating eIF4A unwinding activity than eIF4B, implying that eIF4H is not able to completely substitute for eIF4B in duplex unwinding. By monitoring unwinding and ATPase assays under identical conditions, we demonstrate that eIF4B couples the ATP hydrolysis cycle of eIF4A with strand separation, thereby minimizing nonproductive unwinding events. Using duplex substrates with altered GC contents but similar predicted thermal stabilities, we further show that the rate of formation of productive unwinding complexes is strongly influenced by the local stability per base pair, in addition to the stability of the entire duplex. This finding explains how a change in the GC content of a hairpin is able to influence translation initiation while maintaining the overall predicted thermal stability.

  3. Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.

    PubMed

    Graziadei, Andrea; Masiewicz, Pawel; Lapinaite, Audrone; Carlomagno, Teresa

    2016-05-01

    RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product.

  4. A new regulatory pathway of mRNA export by an F-box protein, Mdm30.

    PubMed

    Durairaj, Geetha; Lahudkar, Shweta; Bhaumik, Sukesh R

    2014-02-01

    Mdm30, an F-box protein in yeast, has been recently shown to promote mRNA export. However, it remains unknown how Mdm30 facilitates mRNA export. Here, we show that Mdm30 targets the Sub2 component of the TREX (Transcription/Export) complex for ubiquitylation and subsequent proteasomal degradation. Such a targeted degradation of Sub2 enhances the recruitment of the mRNA export adaptor, Yra1, to the active genes to promote mRNA export. Together, these results elucidate that Mdm30 promotes mRNA export by lowering Sub2's stability and consequently enhancing Yra1 recruitment, thus illuminating new regulatory mechanisms of mRNA export by Mdm30.

  5. TATA-box DNA binding activity and subunit composition for RNA polymerase III transcription factor IIIB from Xenopus laevis.

    PubMed Central

    McBryant, S J; Meier, E; Leresche, A; Sharp, S J; Wolf, V J; Gottesfeld, J M

    1996-01-01

    The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs). Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription inhibition assays and a gel mobility shift assay. Gel shift competition assays with mutant and nonspecific DNAs demonstrate the specificity of the TFIIIB-TATA box DNA complex. The apparent dissociation constant for this protein-DNA interaction is approximately 0.4 nM, similar to the affinity of yeast TBP for the same sequence. TFIIIB transcriptional activity and TATA-box binding activity cofractionate during a series of four ion-exchange chromatographic steps, and reconstituted transcription reactions demonstrate that the TATA-box DNA-protein complex contains TFIIIB TAF activity. Polypeptides with apparent molecular masses of 75 and 92 kDa are associated with TBP in this complex. These polypeptides were renatured after elution from sodium dodecyl sulfate-gels and tested individually and in combination for TFIIIB TAF activity. Recombinant TBP along with protein fractions containing the 75- and 92-kDa polypeptides were sufficient to reconstitute TFIIIB transcriptional activity and DNA binding activity, suggesting that Xenopus TFIIIB is composed of TBP along with these polypeptides. PMID:8756620

  6. Distinct functions and requirements for the Cys-His boxes of the human immunodeficiency virus type 1 nucleocapsid protein during RNA encapsidation and replication.

    PubMed Central

    Schwartz, M D; Fiore, D; Panganiban, A T

    1997-01-01

    The process of retroviral RNA encapsidation involves interaction between trans-acting viral proteins and cis-acting RNA elements. The encapsidation signal on human immunodeficiency virus type 1 (HIV-1) RNA is a multipartite structure composed of functional stem-loop structures. The nucleocapsid (NC) domain of the Gag polyprotein precursor contains two copies of a Cys-His box motif that have been demonstrated to be important in RNA encapsidation. To further characterize the role of the Cys-His boxes of the HIV-1 NC protein in RNA encapsidation, the relative efficiency of RNA encapsidation for virus particles that contained mutations within the Cys-His boxes was measured. Mutations that disrupted the first Cys-His box of the NC protein resulted in virus particles that encapsidated genomic RNA less efficiently and subgenomic RNA more efficiently than did wild-type virus. Mutations within the second Cys-His box did not significantly affect RNA encapsidation. In addition, a full complement of wild-type NC protein in virus particles is not required for efficient RNA encapsidation or virus replication. Finally, both Cys-His boxes of the NC protein play additional roles in virus replication. PMID:9371588

  7. Arabidopsis root initiation defective1, a DEAH-box RNA helicase involved in pre-mRNA splicing, is essential for plant development.

    PubMed

    Ohtani, Misato; Demura, Taku; Sugiyama, Munetaka

    2013-06-01

    Pre-mRNA splicing is a critical process in gene expression in eukaryotic cells. A multitude of proteins are known to be involved in pre-mRNA splicing in plants; however, the physiological roles of only some of these have been examined. Here, we investigated the developmental roles of a pre-mRNA splicing factor by analyzing root initiation defective1-1 (rid1-1), an Arabidopsis thaliana mutant previously shown to have severe defects in hypocotyl dedifferentiation and de novo meristem formation in tissue culture under high-temperature conditions. Phenotypic analysis in planta indicated that RID1 is differentially required during development and has roles in processes such as meristem maintenance, leaf morphogenesis, and root morphogenesis. RID1 was identified as encoding a DEAH-box RNA helicase implicated in pre-mRNA splicing. Transient expression analysis using intron-containing reporter genes showed that pre-mRNA splicing efficiency was affected by the rid1 mutation, which supported the presumed function of RID1 in pre-mRNA splicing. Our results collectively suggest that robust levels of pre-mRNA splicing are critical for several specific aspects of plant development.

  8. Effect on proliferation and apoptosis of retinoblastoma cell by RNA inhibiting high mobility group protein box-1 expression

    PubMed Central

    Wang, Li-Lun; Feng, Yan-Qin; Cheng, Yu-Hong

    2017-01-01

    AIM To investigate the effect of high mobility group protein box-1 (HMGB1) siRNA on proliferation and apoptosis of retinoblastoma (Rb) cells. METHODS The expression of HMGB1 in Rb cells were detected by real-time polymerase chain reaction (RT-PCR) and Western blot. Chemically synthesized HMGB1 siRNA was transfected into Y79 cells. The inhibitory rate was also examined by RT-PCR and Western blot. After HMGB1 siRNA transfection, the cell proliferation was analyzed by MTT, and cell apoptosis was detected by Caspase-3 active detection kit. Cell cycle distribution and apoptosis were detected by flow cytometry. RESULTS The expression of HMGB1 significantly elevated in Rb cells (P<0.01). After transfected by siRNA, the HMGB1 protein level of Y79 cells was significantly reduced (P<0.01). After siRNA interference HMGB1, the proportion of proliferating cells reduced, and the proportion of quiescent cells increased (P<0.05). In addition, apoptosis rate of Y79 cells increased from 2.03% to 9.10% after interfering with HMGB1 siRNA (P<0.05). CONCLUSION Specific HMGB1 siRNA can inhibit the expression of HMGB1. The effect may be attributed to inhibit the proliferation and promote cell apoptosis. PMID:28149773

  9. Assembly into snoRNP controls 5'-end maturation of a box C/D snoRNA in Saccharomyces cerevisiae

    SciTech Connect

    Preti, Milena; Guffanti, Elisa; Valitutto, Eleonora; Dieci, Giorgio . E-mail: giorgio.dieci@unipr.it

    2006-12-15

    The SNR52 gene, coding for a box C/D snoRNA, is the only snoRNA gene transcribed by RNA polymerase (Pol) III in Saccharomyces cerevisiae. Pol III transcription generates a precisely terminated primary transcript that undergoes extensive 5'-end processing. Here, we show that mutations of the box C/D core motif required for snoRNP assembly compromise 5'-end maturation of the SNR52 snoRNA. Upstream processing was also impaired by specific depletion of either Nop1p or Nop58p snoRNP proteins. We further show that the nuclear exosome is required for 3'-end maturation of SNR52 snoRNA, at variance with all the other known Pol III transcripts. Our data suggest a functional coupling between snoRNP assembly and 5'-end maturation of independently transcribed box C/D snoRNAs.

  10. RNA helicases

    PubMed Central

    Owttrim, George W.

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In addition, RNA helicase expression and/or activity are frequently altered during cellular response to abiotic stress, implying they perform defined roles during cellular adaptation to changes in the growth environment. Specifically, RNA helicases contribute to the formation of cold-adapted ribosomes and RNA degradosomes, implying a role in alleviation of RNA secondary structure stabilization at low temperature. A common emerging theme involves RNA helicases acting as scaffolds for protein-protein interaction and functioning as molecular clamps, holding RNA-protein complexes in specific conformations. This review highlights recent advances in DEAD-box RNA helicase association with cellular response to abiotic stress in prokaryotes. PMID:23093803

  11. Polymorphisms in MicroRNA Target Sites of Forkhead Box O Genes Are Associated with Hepatocellular Carcinoma

    PubMed Central

    Zeng, Xiaoyun; Yu, Hongping; Li, Anhua; Bei, Chunhua; Qiu, Xiaoqiang

    2015-01-01

    The forkhead box O (FOXO) transcription factors play important roles in various cancer development including Hepatocellular Carcinoma (HCC). In this study we conducted a hospital-based case control study including 1049 cases (HCC patients) and 1052 controls (non-tumor patients) to examine whether single nucleotide polymorphisms (SNPs) within microRNA (miRNA) target sites of FOXO genes confer HCC susceptibility. A total of three miRNA target site SNPs in the 3’ untranslated regions (UTR) of FOXO1 (rs17592236), FOXO3 (rs4946936) and FOXO4 (rs4503258) were analyzed. No statistically significant differences were found in genotype distribution for rs17592236, rs4946936, and rs4503258 between the HCC patient group and the tumor-free control group using single factor chi-square analysis (P>0.05). However, multivariate logistic regression analysis showed that the CT/TT genotype in rs17592236 was significantly associated with decreased risk of HCC development (P = 0.010, OR = 0.699, 95% CI: 0.526–0.927) as compared to the CC genotype in rs17592236. Additionally, a genetic interaction was found between rs17592236 and rs4503258 (P = 0.003, OR = 0.755, 95% CI: 0.628–0.908). Functional dual luciferase reporter assays verified that the rs17592236 SNP was a target site of human miRNA miR-137. Together, these results indicate that the rs17592236 polymorphism is associated with decreasing of HCC hereditary susceptibility likely through modulating the binding affinity of miR-137 to the 3’UTR in FOXO1 messenger RNA (mRNA). Further knowledge obtained from this study may provide important evidence for the prevention and targeted therapy of HCC. PMID:25739100

  12. The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development.

    PubMed

    Shiboleth, Yoel Moshe; Haronsky, Elina; Leibman, Diana; Arazi, Tzahi; Wassenegger, Michael; Whitham, Steven A; Gaba, Victor; Gal-On, Amit

    2007-12-01

    The helper component-proteinase (HC-Pro) protein of potyviruses is a suppressor of gene silencing and has been shown to elicit plant developmental-defect-like symptoms. In Zucchini yellow mosaic virus (ZYMV), a mutation in the highly conserved FR180NK box of HC-Pro to FI180NK causes attenuation of these symptoms. At 5 days postinoculation and before symptoms appear, virus accumulation, HC-Pro protein levels, and viral short interfering RNA (siRNA) levels are similar for the severe (FRNK) and attenuated (FINK) strains. At this stage, ZYMV(FRNK) caused greater accumulation of most microRNAs (miRNAs), and especially of their complementary miRNA "passenger" strands (miRNA*s), in systemically infected leaves than the attenuated ZYMV(FINK) did. HC-Pro(FRNK) specifically bound artificial siRNA and miRNA/miRNA* duplexes with a much higher affinity than the mutated HC-Pro(FINK). Further analysis of the mutant and wild-type HC-Pro proteins revealed that suppressor activity of the ZYMV HC(FINK) mutant was not diminished. However, the FINK mutation caused a loss of HC-Pro suppressor function in other potyviruses. Replacement of the second positively charged amino acid in the ZYMV FRNK box to result in FRNA also caused symptom attenuation and reduced small RNA duplex-binding affinity without loss of suppressor activity. Our data suggest that the highly conserved FRNK box in the HC-Pro of potyviruses is a probable point of contact with siRNA and miRNA duplexes. The interaction of the FRNK box with populations of miRNAs directly influences their accumulation levels and regulatory functions, resulting in symptom development.

  13. The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis

    PubMed Central

    Schwarz, Stefan; Grande, Arne V.; Bujdoso, Nora; Saedler, Heinz

    2008-01-01

    Throughout development the Arabidopsis shoot apical meristem successively undergoes several major phase transitions such as the juvenile-to-adult and floral transitions until, finally, it will produce flowers instead of leaves and shoots. Members of the Arabidopsis SBP-box gene family of transcription factors have been implicated in promoting the floral transition in dependence of miR156 and, accordingly, transgenics constitutively over-expressing this microRNA are delayed in flowering. To elaborate their roles in Arabidopsis shoot development, we analysed two of the 11 miR156 regulated Arabidopsis SBP-box genes, i.e. the likely paralogous genes SPL9 and SPL15. Single and double mutant phenotype analysis showed these genes to act redundantly in controlling the juvenile-to-adult phase transition. In addition, their loss-of-function results in a shortened plastochron during vegetative growth, altered inflorescence architecture and enhanced branching. In these aspects, the double mutant partly phenocopies constitutive MIR156b over-expressing transgenic plants and thus a major contribution to the phenotype of these transgenics as a result of the repression of SPL9 and SPL15 is strongly suggested. Electronic supplementary material The online version of this article (doi:10.1007/s11103-008-9310-z) contains supplementary material, which is available to authorized users. PMID:18278578

  14. Coilin association with Box C/D scaRNA suggests a direct role for the Cajal body marker protein in scaRNP biogenesis

    PubMed Central

    Enwerem, Isioma I.; Velma, Venkatramreddy; Broome, Hanna J.; Kuna, Marija; Begum, Rowshan A.; Hebert, Michael D.

    2014-01-01

    ABSTRACT Spliceosomal small nuclear ribonucleoproteins (snRNPs) are enriched in the Cajal body (CB). Guide RNAs, known as small Cajal body-specific RNAs (scaRNAs), direct modification of the small nuclear RNA (snRNA) component of the snRNP. The protein WRAP53 binds a sequence motif (the CAB box) found in many scaRNAs and the RNA component of telomerase (hTR) and targets these RNAs to the CB. We have previously reported that coilin, the CB marker protein, associates with certain non-coding RNAs. For a more comprehensive examination of the RNAs associated with coilin, we have sequenced the RNA isolated from coilin immunocomplexes. A striking preferential association of coilin with the box C/D scaRNAs 2 and 9, which lack a CAB box, was observed. This association varied by treatment condition and WRAP53 knockdown. In contrast, reduction of WRAP53 did not alter the level of coilin association with hTR. Additional studies showed that coilin degrades/processes scaRNA 2 and 9, associates with active telomerase and can influence telomerase activity. These findings suggest that coilin plays a novel role in the biogenesis of box C/D scaRNPs and telomerase. PMID:24659245

  15. The RNA polymerase I transcription factor UBF is a sequence-tolerant HMG-box protein that can recognize structured nucleic acids.

    PubMed Central

    Copenhaver, G P; Putnam, C D; Denton, M L; Pikaard, C S

    1994-01-01

    Upstream Binding Factor (UBF) is important for activation of ribosomal RNA transcription and belongs to a family of proteins containing nucleic acid binding domains, termed HMG-boxes, with similarity to High Mobility Group (HMG) chromosomal proteins. Proteins in this family can be sequence-specific or highly sequence-tolerant binding proteins. We show that Xenopus UBF can be classified among the sequence-tolerant class. Methylation interference assays using enhancer DNA probes failed to reveal any critical nucleotides required for UBF binding. Selection by UBF of optimal binding sites among a population of enhancer oligonucleotides with randomized sequences also failed to reveal any consensus sequence. The minor groove specific drugs chromomycin A3, distamycin A and actinomycin D competed against UBF for enhancer binding, suggesting that UBF, like other HMG-box proteins, probably interacts with the minor groove. UBF also shares with other HMG box proteins the ability to bind synthetic cruciform DNA. However, UBF appears different from other HMG-box proteins in that it can bind both RNA (tRNA) and DNA. The sequence-tolerant nature of UBF-nucleic acid interactions may accommodate the rapid evolution of ribosomal RNA gene sequences. Images PMID:8041627

  16. Antiapoptotic Effect of Recombinant HMGB1 A-box Protein via Regulation of microRNA-21 in Myocardial Ischemia-Reperfusion Injury Model in Rats.

    PubMed

    Han, Qiang; Zhang, Hua-Yong; Zhong, Bei-Long; Zhang, Bing; Chen, Hua

    2016-04-01

    The ~80 amino acid A box DNA-binding domain of high mobility group box 1 (HMGB1) protein antagonizes proinflammatory responses during myocardial ischemia reperfusion (I/R) injury. The exact role of microRNA-21 (miR-21) is unknown, but its altered levels are evident in I/R injury. This study examined the roles of HMGB1 A-box and miR-21 in rat myocardial I/R injury model. Sixty Sprague-Dawley rats were randomly divided into six equal groups: (1) Sham; (2) I/R; (3) Ischemic postconditioning (IPost); (4) AntagomiR-21 post-treatment; (5) Recombinant HMGB1 A-box pretreatment; and (6) Recombinant HMGB1 A-box + antagomiR-21 post-treatment. Hemodynamic indexes, arrhythmia scores, ischemic area and infarct size, myocardial injury, and related parameters were studied. Expression of miR-21 was detected by real-time quantitative polymerase chain reaction (qRT-PCR) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to quantify apoptosis. Left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), maximal rate of pressure rise (+dp/dtmax), and decline (-dp/dtmax) showed clear reduction upon treatment with recombinant HMGB1 A-box. Arrhythmia was relieved and infarct area decreased in the group pretreated with recombinant HMGB1 A-box, compared with other groups. Circulating lactate dehydrogenase (LDH) and malondialdehyde (MDA) levels increased in response to irreversible cellular injury, while creatine kinase MB isoenzymes (CK-MB) and superoxide dismutase (SOD) activities were reduced in the I/R group, which was reversed following recombinant HMGB1 A-box treatment. Interestingly, pretreatment with recombinant HMGB1 A-box showed the most dramatic reductions in miR-21 levels, compared with other groups. Significantly reduced apoptotic index (AI) was seen in recombinant HMGB1 A-box pretreatment group and recombinant HMGB1 A-box + antagomiR-21 post-treatment group, with the former showing a more

  17. microRNA-10a Targets T-box 5 to Inhibit the Development of Cardiac Hypertrophy.

    PubMed

    Wang, Dan; Zhai, Guanqun; Ji, Yangfei; Jing, Haiyun

    2017-02-07

    The mechanism of cardiac hypertrophy involving microRNAs (miRNAs) is attracting increasing attention. Our study aimed to investigate the role of miR-10a in cardiac hypertrophy development and the underlying regulatory mechanism.Transverse abdominal aortic constriction (TAAC) surgery was performed to establish a cardiac hypertrophy rat model, and angiotensin II (AngII) was used to induce cardiac hypertrophy in cultured neonatal rat cardiomyocytes. Expression of T-box 5 (TBX5) and miR-10a was altered by cell transfection of siRNA or miRNA mimic/inhibitor. Leucine incorporation assay, histological and cytological examination, quantitative real-time PCR (qRT-PCR), and Western blot were performed to detect the effects of miR-10a and TBX5 on cardiac hypertrophy. Dual-luciferase reporter assay was conducted to verify the regulation of TBX5 by miR-10a.miR-10a was down-regulated, and TBX5 was up-regulated in the rat model and AngII-stimulated cardiomyocytes. miR-10a inhibited TBX5 expression by directly targeting the binding site in Tbx5 3'UTR. Overexpression of miR-10a in AngII-treated cardiomyocytes decreased relative cell area, and significantly reduced the mRNA levels of natriuretic peptide A (Nppa), myosin heavy chain 7 cardiac muscle beta (Myh7), and leucine incorporation (P < 0.01 or P < 0.001). Knockdown of Tbx5 had similar effects on AngII-induced cardiomyocytes.Our findings indicate that miR-10a may inhibit cardiac hypertrophy via targeting Tbx5. Thus, miR-10a provides promising therapeutic strategies for the treatment of cardiac hypertrophy.

  18. Metalloregulator CueR biases RNA polymerase's kinetic sampling of dead-end or open complex to repress or activate transcription.

    PubMed

    Martell, Danya J; Joshi, Chandra P; Gaballa, Ahmed; Santiago, Ace George; Chen, Tai-Yen; Jung, Won; Helmann, John D; Chen, Peng

    2015-11-03

    Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ(70)-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator-DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)--a Cu(+)-responsive MerR-family metalloregulator--modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR-PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.

  19. Box C/D small nucleolar RNA genes and the Prader-Willi syndrome: a complex interplay.

    PubMed

    Cavaillé, Jérôme

    2017-03-13

    The nucleolus of mammalian cells contains hundreds of box C/D small nucleolar RNAs (SNORDs). Through their ability to base pair with ribosomal RNA precursors, most play important roles in the synthesis and/or activity of ribosomes, either by guiding sequence-specific 2'-O-methylations or by facilitating RNA folding and cleavages. A growing number of SNORD genes with elusive functions have been discovered recently. Intriguingly, the vast majority of them are located in two large, imprinted gene clusters at human chromosome region 15q11q13 (the SNURF-SNRPN domain) and at 14q32 (the DLK1-DIO3 domain) where they are expressed, respectively, only from the paternally and maternally inherited alleles. These placental mammal-specific SNORD genes have many features of the canonical SNORDs that guide 2'-O-methylations, yet they lack obvious complementarity with ribosomal RNAs and, surprisingly, they are processed from large, tandemly repeated genes expressed preferentially in the brain. This review summarizes our understanding of the biology of these peculiar SNORD genes, focusing particularly on SNORD115 and SNORD116 in the SNURF-SNRPN domain. It examines the growing evidence that altered levels of these SNORDs and/or their host-gene transcripts may be a primary cause of Prader-Willi syndrome (PWS; a rare disorder characterized by overeating and obesity) as well as abnormalities in signaling through the 5-HT2C serotonin receptor. Finally, the hypothesis that PWS may be a ribosomopathy (ribosomal disease) is also discussed. For further resources related to this article, please visit the WIREs website.

  20. RNA helicase proteins as chaperones and remodelers

    PubMed Central

    Jarmoskaite, Inga; Russell, Rick

    2014-01-01

    Superfamily 2 helicase proteins are ubiquitous in RNA biology and have an extraordinarily broad set of functional roles. Central among these roles are to promote rearrangements of structured RNAs and to remodel RNA-protein complexes (RNPs), allowing formation of native RNA structure or progression through a functional cycle of structures. While all superfamily 2 helicases share a conserved helicase core, they are divided evolutionarily into several families, and it is principally proteins from three families, the DEAD-box, DEAH/RHA and Ski2-like families, that function to manipulate structured RNAs and RNPs. Strikingly, there are emerging differences in the mechanisms of these proteins, both between families and within the largest family (DEAD-box), and these differences appear to be tuned to their RNA or RNP substrates and their specific roles. This review outlines basic mechanistic features of the three families and surveys individual proteins and the current understanding of their biological substrates and mechanisms. PMID:24635478

  1. PRP16, a DEAH-box RNA helicase, is recruited to the spliceosome primarily via its nonconserved N-terminal domain.

    PubMed Central

    Wang, Y; Guthrie, C

    1998-01-01

    Dynamic rearrangement of RNA structure is crucial for intron recognition and formation of the catalytic core during pre-mRNA splicing. Three of the splicing factors that contain sequence motifs characteristic of the DExD/DExH-box family of RNA-dependent ATPases (Prp16, Prp22, and the human homologue of Brr2) recently have been shown to unwind RNA duplexes in vitro, providing biochemical evidence that they may direct structural rearrangements on the spliceosome. Notably, however, the unwinding activity of these proteins is sequence nonspecific, raising the question of how their functional specificity is determined. Because the highly conserved DExD/DExH-box domain in these proteins is typically flanked by one or more nonconserved domains, we have tested the hypothesis that the nonconserved regions of Prp16 determine the functional specificity of the protein. We found that the nonconserved N-terminal domain of Prp16 is (1) essential for viability, (2) required for the nuclear localization of Prp16, and (3) capable of binding to the spliceosome specifically at the step of Prp16 function. Moreover, this domain can interact with the rest of the protein to allow trans-complementation. Based on these results, we propose that the spliceosomal target of the unwinding activity of Prp16, and possibly other DExD/DExH-box splicing factors as well, is defined by factors that specifically interact with the nonconserved domains of the protein. PMID:9769096

  2. Magical Boxes

    ERIC Educational Resources Information Center

    Costello, Judith

    2005-01-01

    Students get excited when they realize that they can transform a flat sheet of paper into a box. By using different sizes of paper, they can make different sizes of boxes and put a box inside a box, inside a box. These magical boxes within boxes can contain unwanted emotions or special treasures. The project described in this article incorporates…

  3. RNA-Seq analyses reveal the order of tRNA processing events and the maturation of C/D box and CRISPR RNAs in the hyperthermophile Methanopyrus kandleri.

    PubMed

    Su, Andreas A H; Tripp, Vanessa; Randau, Lennart

    2013-07-01

    The methanogenic archaeon Methanopyrus kandleri grows near the upper temperature limit for life. Genome analyses revealed strategies to adapt to these harsh conditions and elucidated a unique transfer RNA (tRNA) C-to-U editing mechanism at base 8 for 30 different tRNA species. Here, RNA-Seq deep sequencing methodology was combined with computational analyses to characterize the small RNome of this hyperthermophilic organism and to obtain insights into the RNA metabolism at extreme temperatures. A large number of 132 small RNAs were identified that guide RNA modifications, which are expected to stabilize structured RNA molecules. The C/D box guide RNAs were shown to exist as circular RNA molecules. In addition, clustered regularly interspaced short palindromic repeats RNA processing and potential regulatory RNAs were identified. Finally, the identification of tRNA precursors before and after the unique C8-to-U8 editing activity enabled the determination of the order of tRNA processing events with termini truncation preceding intron removal. This order of tRNA maturation follows the compartmentalized tRNA processing order found in Eukaryotes and suggests its conservation during evolution.

  4. Association of genetic polymorphism of pre-microRNA-146a rs2910164 and serum high-mobility group box 1 with febrile seizures in Egyptian children.

    PubMed

    Issac, Marianne Samir Makboul; Girgis, Marian; Haroun, Mervat; Shalaby, Amal

    2015-03-01

    Interaction between immune-inflammatory process and genetic factors might be implicated in the pathogenesis of febrile seizures. Pre-microRNA (miR)-146a rs2910164 polymorphism is postulated to modulate expression of miR-146a whose anti-inflammatory role involves regulation of high-mobility group box 1. Our aim is to examine whether rs2910164 polymorphism influences serum high-mobility group box 1 levels and whether an association exists between both and febrile seizures. The study included 136 children, divided into 4 groups. Real-time polymerase chain reaction was used for detection of rs2910164 polymorphism and high-mobility group box 1 was measured using enzyme-linked immunosorbent assay. High-mobility group box 1 levels were higher in febrile seizure patients compared to the other groups. Rs2910164 polymorphism was not associated with increased risk of febrile seizures. Rs2910164 polymorphism might be accompanied by an upregulation of the proinflammatory process as it might be associated with an increase in high-mobility group box 1 and leukocytic count.

  5. RNA polymerase II components and Rrn7 form a preinitiation complex on the HomolD box to promote ribosomal protein gene expression in Schizosaccharomyces pombe.

    PubMed

    Montes, Matías; Moreira-Ramos, Sandra; Rojas, Diego A; Urbina, Fabiola; Käufer, Norbert F; Maldonado, Edio

    2017-02-01

    In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription.

  6. Jeweled Boxes

    ERIC Educational Resources Information Center

    Coy, Mary

    2009-01-01

    While an empty cardboard box from a ream of copy paper may be the most coveted box among teachers in the author's school, for other people, brass boxes from India, Khokhlova lacquer boxes from Russia, and puzzle boxes from Japan are more the type that are collected and admired. Whether it is used for storage or decoration, a box can evoke a sense…

  7. TATA boxes in gene transcription and poly (A) tails in mRNA stability: New perspective on the effects of berberine

    PubMed Central

    Yuan, Zhi-Yi; Lu, Xi; Lei, Fan; Chai, Yu-Shuang; Wang, Yu-Gang; Jiang, Jing-Fei; Feng, Tian-Shi; Wang, Xin-Pei; Yu, Xuan; Yan, Xiao-Jin; Xing, Dong-Ming; Du, Li-Jun

    2015-01-01

    Berberine (BBR) is a natural compound with variable pharmacological effects and a broad panel of target genes. We investigated berberine’s pharmacological activities from the perspective of its nucleotide-binding ability and discovered that BBR directly regulates gene expression by targeting TATA boxes in transcriptional regulatory regions as well as the poly adenine (poly (A)) tail at the mRNA terminus. BBR inhibits gene transcription by binding the TATA boxes in the transcriptional regulatory region, but it promotes higher levels of expression by targeting the poly (A) tails of mRNAs. The present study demonstrates that TATA boxes and poly (A) tails are the first and second primary targets by which BBR regulates gene expression. The final outcome of gene regulation by BBR depends on the structure of the individual gene. This is the first study to reveal that TATA boxes and poly (A) tails are direct targets for BBR in its regulation of gene expression. Our findings provide a novel explanation for the complex activities of a small molecule compound in a biological system and a novel horizon for small molecule-compound pharmacological studies. PMID:26671652

  8. The DExD/H box ATPase Dhh1 functions in translational repression, mRNA decay, and processing body dynamics

    PubMed Central

    Carroll, Johanna S.; Munchel, Sarah E.

    2011-01-01

    Translation, storage, and degradation of messenger ribonucleic acids (mRNAs) are key steps in the posttranscriptional control of gene expression, but how mRNAs transit between these processes remains poorly understood. In this paper, we functionally characterized the DExD/H box adenosine triphosphatase (ATPase) Dhh1, a critical regulator of the cytoplasmic fate of mRNAs. Using mRNA tethering experiments in yeast, we showed that Dhh1 was sufficient to move an mRNA from an active state to translational repression. In actively dividing cells, translational repression was followed by mRNA decay; however, deleting components of the 5′–3′ decay pathway uncoupled these processes. Whereas Dhh1’s ATPase activity was not required to induce translational inhibition and mRNA decay when directly tethered to an mRNA, ATP hydrolysis regulated processing body dynamics and the release of Dhh1 from these RNA–protein granules. Our results place Dhh1 at the interface of translation and decay controlling whether an mRNA is translated, stored, or decayed. PMID:21844211

  9. The RNA polymerase I transactivator upstream binding factor requires its dimerization domain and high-mobility-group (HMG) box 1 to bend, wrap, and positively supercoil enhancer DNA.

    PubMed Central

    Putnam, C D; Copenhaver, G P; Denton, M L; Pikaard, C S

    1994-01-01

    Upstream binding factor (UBF) is an important transactivator of RNA polymerase I and is a member of a family of proteins that contain nucleic acid binding domains named high-mobility-group (HMG) boxes because of their similarity to HMG chromosomal proteins. UBF is a highly sequence-tolerant DNA-binding protein for which no binding consensus sequence has been identified. Therefore, it has been suggested that UBF may recognize preformed structural features of DNA, a hypothesis supported by UBF's ability to bind synthetic DNA cruciforms, four-way junctions, and even tRNA. We show here that full-length UBF can also bend linear DNA to mediate circularization of probes as small as 102 bp in the presence of DNA ligase. Longer probes in the presence of UBF become positively supercoiled when ligated, suggesting that UBF wraps the DNA in a right-handed direction, opposite the direction of DNA wrapping around a nucleosome. The dimerization domain and HMG box 1 are necessary and sufficient to circularize short probes and supercoil longer probes in the presence of DNA ligase. UBF's sequence tolerance coupled with its ability to bend and wrap DNA makes UBF an unusual eukaryotic transcription factor. However, UBF's ability to bend DNA might explain how upstream and downstream rRNA gene promoter domains interact. UBF-induced DNA wrapping could also be a mechanism by which UBF counteracts histone-mediated gene repression. Images PMID:7935371

  10. MYC protein inhibits transcription of the microRNA cluster MC-let-7a-1~let-7d via noncanonical E-box.

    PubMed

    Wang, Zifeng; Lin, Sheng; Li, Julia Jun; Xu, Zhenhua; Yao, Hong; Zhu, Xiao; Xie, Dan; Shen, Zan; Sze, Johnny; Li, Kui; Lu, Gang; Chan, Danny Tat-Ming; Poon, Wai Sang; Kung, Hsiang-fu; Lin, Marie Chia-mi

    2011-11-18

    The human microRNA cluster MC-let-7a-1∼let-7d, with three members let-7a-1, let-7f-1, and let-7d, is an important cluster of the let-7 family. These microRNAs play critical roles in regulating development and carcinogenesis. Therefore, precise control of MC-let-7a-1∼let-7d level is critical for cellular functions. In this study, we first showed that the expression of these three members was significantly reduced in human hepatocellular carcinoma HepG2 cells as compared with the immortalized human liver L02 cells. We demonstrated that the MC-let-7a-1∼let-7d cluster was encoded by a single polycistronic transcript driven by a 10-kb upstream promoter, with two MYC-binding sites. Importantly, MYC inhibited MC-let-7a-1∼let-7d promoter activity via binding to the noncanonical E-box 3 downstream of the transcription start sites, whereas it enhanced promoter activity by binding to the canonical E-box 2 upstream of the transcription start sites. We found that although the binding affinity of MYC to E-box 2 was stronger than E-box 3, the binding quantum of MYC to E-box 3 was significantly higher in cancerous HepG2 cells as compared with the noncancerous L02 cells. In addition, forced expression of let-7 could reverse the MYC-mediated cell proliferation. These findings suggested that in L02 cells with a low level of MYC, MYC binds mainly to E-box 2 to enhance MC-let-7a-1∼let-7d expression. However, in HepG2 cells with an elevated MYC, the extra MYC could bind to E-box 3 to suppress the transcription of MC-let-7a-1∼let-7d and thus enable HepG2 cells to maintain a high level of MYC and a low level of let-7 microRNAs simultaneously.

  11. Wanted DEAD/H or Alive: Helicases Winding Up in Cancers.

    PubMed

    Cai, Wanpei; Xiong Chen, Zhi; Rane, Grishma; Satendra Singh, Shikha; Choo, Zhang'e; Wang, Chao; Yuan, Yi; Zea Tan, Tuan; Arfuso, Frank; Yap, Celestial T; Pongor, Lorinc S; Yang, Henry; Lee, Martin B; Cher Goh, Boon; Sethi, Gautam; Benoukraf, Touati; Tergaonkar, Vinay; Prem Kumar, Alan

    2017-01-01

    Cancer is one of the most studied areas of human biology over the past century. Despite having attracted much attention, hype, and investments, the search to find a cure for cancer remains an uphill battle. Recent discoveries that challenged the central dogma of molecular biology not only further increase the complexity but also demonstrate how various types of noncoding RNAs such as microRNA and long noncoding RNA, as well as their related processes such as RNA editing, are important in regulating gene expression. Parallel to this aspect, an increasing number of reports have focused on a family of proteins known as DEAD/H-box helicases involved in RNA metabolism, regulation of long and short noncoding RNAs, and novel roles as "editing helicases" and their association with cancers. This review summarizes recent findings on the roles of RNA helicases in various cancers, which are broadly classified into adult solid tumors, childhood solid tumors, leukemia, and cancer stem cells. The potential small molecule inhibitors of helicases and their therapeutic value are also discussed. In addition, analyzing next-generation sequencing data obtained from public portals and reviewing existing literature, we provide new insights on the potential of DEAD/H-box helicases to act as pharmacological drug targets in cancers.

  12. Microdiversity of deep-sea Bacillales isolated from Tyrrhenian sea sediments as revealed by ARISA, 16S rRNA gene sequencing and BOX-PCR fingerprinting.

    PubMed

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments.

  13. Microdiversity of Deep-Sea Bacillales Isolated from Tyrrhenian Sea Sediments as Revealed by ARISA, 16S rRNA Gene Sequencing and BOX-PCR Fingerprinting

    PubMed Central

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments. PMID:24005887

  14. The human box C/D snoRNAs U3 and U8 are required for pre-rRNA processing and tumorigenesis

    PubMed Central

    Langhendries, Jean-Louis; Nicolas, Emilien; Doumont, Gilles; Goldman, Serge; Lafontaine, Denis L.J.

    2016-01-01

    Small nucleolar RNAs (snoRNAs) are emerging as a novel class of proto-oncogenes and tumor suppressors; their involvement in tumorigenesis remains unclear. The box C/D snoRNAs U3 and U8 are upregulated in breast cancers. Here we characterize the function of human U3 and U8 in ribosome biogenesis, nucleolar structure, and tumorigenesis. We show in breast (MCF-7) and lung (H1944) cancer cells that U3 and U8 are required for pre-rRNA processing reactions leading, respectively, to synthesis of the small and large ribosomal subunits. U3 or U8 depletion triggers a remarkably potent p53-dependent anti-tumor stress response involving the ribosomal proteins uL5 (RPL11) and uL18 (RPL5). Interestingly, the nucleolar structure is more sensitive to perturbations in lung cancer than in breast cancer cells. We reveal in a mouse xenograft model that the tumorigenic potential of cancer cells is reduced in the case of U3 suppression and totally abolished upon U8 depletion. Tumors derived from U3-knockdown cells displayed markedly lower metabolic volume and activity than tumors derived from aggressive control cancer cells. Unexpectedly, metabolic tracer uptake by U3-suppressed tumors appeared more heterogeneous, indicating distinctive tumor growth properties that may reflect non-conventional regulatory functions of U3 (or fragments derived from it) in mRNA metabolism. PMID:27517747

  15. Genome-wide comparative in silico analysis of the RNA helicase gene family in Zea mays and Glycine max: a comparison with Arabidopsis and Oryza sativa.

    PubMed

    Xu, Ruirui; Zhang, Shizhong; Huang, Jinguang; Zheng, Chengchao

    2013-01-01

    RNA helicases are enzymes that are thought to unwind double-stranded RNA molecules in an energy-dependent fashion through the hydrolysis of NTP. RNA helicases are associated with all processes involving RNA molecules, including nuclear transcription, editing, splicing, ribosome biogenesis, RNA export, and organelle gene expression. The involvement of RNA helicase in response to stress and in plant growth and development has been reported previously. While their importance in Arabidopsis and Oryza sativa has been partially studied, the function of RNA helicase proteins is poorly understood in Zea mays and Glycine max. In this study, we identified a total of RNA helicase genes in Arabidopsis and other crop species genome by genome-wide comparative in silico analysis. We classified the RNA helicase genes into three subfamilies according to the structural features of the motif II region, such as DEAD-box, DEAH-box and DExD/H-box, and different species showed different patterns of alternative splicing. Secondly, chromosome location analysis showed that the RNA helicase protein genes were distributed across all chromosomes with different densities in the four species. Thirdly, phylogenetic tree analyses identified the relevant homologs of DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins in each of the four species. Fourthly, microarray expression data showed that many of these predicted RNA helicase genes were expressed in different developmental stages and different tissues under normal growth conditions. Finally, real-time quantitative PCR analysis showed that the expression levels of 10 genes in Arabidopsis and 13 genes in Zea mays were in close agreement with the microarray expression data. To our knowledge, this is the first report of a comparative genome-wide analysis of the RNA helicase gene family in Arabidopsis, Oryza sativa, Zea mays and Glycine max. This study provides valuable information for understanding the classification and putative functions of

  16. Box C/D RNA guides for the ribose methylation of archaeal tRNAs. The tRNATrp intron guides the formation of two ribose-methylated nucleosides in the mature tRNATrp

    PubMed Central

    d’Orval, Béatrice Clouet; Bortolin, Marie-Line; Gaspin, Christine; Bachellerie, Jean-Pierre

    2001-01-01

    Following a search of the Pyrococcus genomes for homologs of eukaryotic methylation guide small nucleolar RNAs, we have experimentally identified in Pyrococcus abyssi four novel box C/D small RNAs predicted to direct 2′-O-ribose methylations onto the first position of the anticodon in tRNALeu(CAA), tRNALeu(UAA), elongator tRNAMet and tRNATrp, respectively. Remarkably, one of them corresponds to the intron of its presumptive target, pre-tRNATrp. This intron is predicted to direct in cis two distinct ribose methylations within the unspliced tRNA precursor, not only onto the first position of the anticodon in the 5′ exon but also onto position 39 (universal tRNA numbering) in the 3′ exon. The two intramolecular RNA duplexes expected to direct methylation, which both span an exon–intron junction in pre-tRNATrp, are phylogenetically conserved in euryarchaeotes. We have experimentally confirmed the predicted guide function of the box C/D intron in halophile Haloferax volcanii by mutagenesis analysis, using an in vitro splicing/RNA modification assay in which the two cognate ribose methylations of pre-tRNATrp are faithfully reproduced. Euryarchaeal pre-tRNATrp should provide a unique system to further investigate the molecular mechanisms of RNA-guided ribose methylation and gain new insights into the origin and evolution of the complex family of archaeal and eukaryotic box C/D small RNAs. PMID:11713301

  17. Position-dependent interactions of Y-box protein 2 (YBX2) with mRNA enable mRNA storage in round spermatids by repressing mRNA translation and blocking translation-dependent mRNA decay.

    PubMed

    Kleene, Kenneth C

    2016-03-01

    Many mRNAs encoding proteins needed for the construction of the specialized organelles of spermatozoa are stored as translationally repressed, free messenger ribonucleoproteins in round spermatids, to be actively translated in elongating and elongated spermatids. The factors that repress translation in round spermatids, however, have been elusive. Two lines of evidence implicate the highly abundant and well-known translational repressor, Y-box protein 2 (YBX2), as a critical factor: First, protamine 1 (Prm1) and sperm-mitochondria cysteine-rich protein (Smcp) mRNAs are prematurely recruited onto polysomes in Ybx2-knockout mouse round spermatids. Second, mutations in 3' untranslated region (3'UTR) cis-elements that abrogate YBX2 binding activate translation of Prm1 and Smcp mRNAs in round spermatids of transgenic mice. The abundance of YBX2 and its affinity for variable sequences, however, raise questions of how YBX2 targets specific mRNAs for repression. Mutations to the Prm1 and Smcp mRNAs in transgenic mice reveal that strong repression in round spermatids requires YBX2 binding sites located near the 3' ends of their 3'UTRs as locating the same sites in upstream positions produce negligible repression. This location-dependence implies that the assembly of repressive complexes is nucleated by adjacent cis-elements that enable cooperative interactions of YBX2 with co-factors. The available data suggest that, in vertebrates, YBX2 has the important role of coordinating the storage of translationally repressed mRNAs in round spermatids by inhibiting translational activity and the degradation of transcripts via translation-dependent deadenylation. These insights should facilitiate future experiments designed to unravel how YBX2 targets mRNAs for repression in round spermatids and how mutations in the YBX2 gene cause infertility in humans. Mol. Reprod. Dev. 83: 190-207, 2016. © 2016 Wiley Periodicals, Inc.

  18. Bento Boxes

    ERIC Educational Resources Information Center

    Hasio, Cindy

    2010-01-01

    Bento boxes are common objects in Japanese culture, designed to hold enough lunch for one person. They have individual compartments and sometimes multiple tiers for rice, vegetables, and other side dishes. They are made of materials ranging from wood, cloth, aluminum, or plastic. In general, the greater the number of foods, the better the box is…

  19. Long non-coding RNA stabilizes the Y-box-binding protein 1 and regulates the epidermal growth factor receptor to promote lung carcinogenesis

    PubMed Central

    Huang, Yun-Chao; Wang, Gui-Zhen; Zhao, Xin-Chun; Pan, Hong-Li; Qu, Li-Wei; Zhang, Jian; Zhang, Chen; Cheng, Xin; Zhou, Guang-Biao

    2016-01-01

    Indoor and outdoor air pollution has been classified as group I carcinogen in humans, but the underlying tumorigenesis remains unclear. Here, we screened for abnormal long noncoding RNAs (lncRNAs) in lung cancers from patients living in Xuanwei city which has the highest lung cancer incidence in China due to smoky coal combustion-generated air pollution. We reported that Xuanwei patients had much more dysregulated lncRNAs than patients from control regions where smoky coal was not used. The lncRNA CAR intergenic 10 (CAR10) was up-regulated in 39/62 (62.9%) of the Xuanwei patients, which was much higher than in patients from control regions (32/86, 37.2%; p=0.002). A multivariate regression analysis showed an association between CAR10 overexpression and air pollution, and a smoky coal combustion-generated carcinogen dibenz[a,h]anthracene up-regulated CAR10 by increasing transcription factor FoxF2 expression. CAR10 bound and stabilized transcription factor Y-box-binding protein 1 (YB-1), leading to up-regulation of the epidermal growth factor receptor (EGFR) and proliferation of lung cancer cells. Knockdown of CAR10 inhibited cell growth in vitro and tumor growth in vivo. These results demonstrate the role of lncRNAs in environmental lung carcinogenesis, and CAR10-YB-1 represents a potential therapeutic target. PMID:27322209

  20. [Differential expression of genes related to photoperiod-temperature sensitive genic male sterility in wheat, revealed by mRNA differential display using G-box family primer].

    PubMed

    Cao, Shuang-He; Liu, Dong-Cheng; Liu, Li-Ke; Guo, Xiao-Li; Zhang, Ai-Min

    2003-01-01

    mRNA differential display with G-box family primer was used to analyze the differential expression of genes of the photoperiod-temperature sensitive genic male sterile(PTSGMS) line of wheat, BAU3338, between the sterile and fertile conditions. The result indicated that gene expression was significantly different between the two types of condition during the fertility transformation phase. The twelve qualitatively different DNA bands were identified with reverse Northern blot hybridization and five positive clones, HT1-G10, HT1-G3, HT2-G2, HT1-G4 and HT2-G5 were sequenced. The homology search indicated that HT1-G10 was highly homological (96%) to the partial sequences of Triticum aestivum chloroplast genes, rbcL and atpB, HT1-G3 was also homological (88%) to Triticum aestivum histone H2A gene and the other three gene fragments were new sequences in Gen-Bank. The analysis of the candidate gene fragments supplied some effective evidences to reveal the developmental mechanism of PTSGMS.

  1. Interaction of the yeast DExH-box RNA helicase prp22p with the 3' splice site during the second step of nuclear pre-mRNA splicing.

    PubMed

    McPheeters, D S; Schwer, B; Muhlenkamp, P

    2000-03-15

    Using site-specific incorporation of the photo-chemical cross-linking reagent 4-thiouridine, we demonstrate the previously unknown association of two proteins with yeast 3' splice sites. One of these is an unidentified approximately 122 kDa protein that cross-links to 3' splice sites during formation of the pre--spliceosome. The other factor is the DExH-box RNA helicase, Prp22p. With substrates functional in the second step of splicing, only very weak cross-linking of Prp22p to intron sequences at the 3' splice site is observed. In contrast, substrates blocked at the second step exhibit strong cross-linking of Prp22 to intron sequences at the 3' splice site, but not to adjacent exon sequences. In vitro reconstitution experiments also show that the association of Prp22p with intron sequences at the 3' splice site is dependent on Prp16p and does not persist when release of mature mRNA from the spliceosome is blocked. Taken together, these results suggest that the 3' splice site of yeast introns is contacted much earlier than previously envisioned by a protein of approximately 120 kDa, and that a transient association of Prp22p with the 3' splice site occurs between the first and second catalytic steps.

  2. Film Boxes.

    ERIC Educational Resources Information Center

    Osterer, Irv

    2002-01-01

    Presents an art lesson in which students created three-dimensional designs for 35mm film packages to improve graphic arts learning. Describes how the students examined and created film boxes using QuarkXPress software. (CMK)

  3. MicroRNA-17-92, a direct Ap-2α transcriptional target, modulates T-box factor activity in orofacial clefting.

    PubMed

    Wang, Jun; Bai, Yan; Li, Hong; Greene, Stephanie B; Klysik, Elzbieta; Yu, Wei; Schwartz, Robert J; Williams, Trevor J; Martin, James F

    2013-01-01

    Among the most common human congenital anomalies, cleft lip and palate (CL/P) affects up to 1 in 700 live births. MicroRNA (miR)s are small, non-coding RNAs that repress gene expression post-transcriptionally. The miR-17-92 cluster encodes six miRs that have been implicated in human cancers and heart development. We discovered that miR-17-92 mutant embryos had severe craniofacial phenotypes, including incompletely penetrant CL/P and mandibular hypoplasia. Embryos that were compound mutant for miR-17-92 and the related miR-106b-25 cluster had completely penetrant CL/P. Expression of Tbx1 and Tbx3, the DiGeorge/velo-cardio-facial (DGS) and Ulnar-mammary syndrome (UMS) disease genes, was expanded in miR-17-92 mutant craniofacial structures. Both Tbx1 and Tbx3 had functional miR seed sequences that mediated gene repression. Analysis of miR-17-92 regulatory regions uncovered conserved and functional AP-2α recognition elements that directed miR-17-92 expression. Together, our data indicate that miR-17-92 modulates expression of critical T-box transcriptional regulators during midface development and is itself a target of Bmp-signaling and the craniofacial pioneer factor AP-2α. Our data are the first genetic evidence that an individual miR or miR cluster is functionally important in mammalian CL/P.

  4. Investigating Aquatic Dead Zones

    ERIC Educational Resources Information Center

    Testa, Jeremy; Gurbisz, Cassie; Murray, Laura; Gray, William; Bosch, Jennifer; Burrell, Chris; Kemp, Michael

    2010-01-01

    This article features two engaging high school activities that include current scientific information, data, and authentic case studies. The activities address the physical, biological, and chemical processes that are associated with oxygen-depleted areas, or "dead zones," in aquatic systems. Students can explore these dead zones through both…

  5. Day of the Dead

    ERIC Educational Resources Information Center

    Dann, Tammy; Murphy, Amy

    2012-01-01

    Foreign Language in Elementary School (FLES) teachers in the West Des Moines schools incorporate the Day of the Dead into the fourth grade curriculum each year. The teachers discuss the Day of the Dead celebration at the Art Center, and many ask for volunteers from fourth grade to participate in the event. Student presentations include a wide…

  6. In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements.

    PubMed Central

    Zamrod, Z; Tyree, C M; Song, Y; Stumph, W E

    1993-01-01

    Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter. Images PMID:8355718

  7. Exploding Boxes

    ERIC Educational Resources Information Center

    Kinney; Jan

    2011-01-01

    How do you teach the "same old, same old" in an interesting and inexpensive way? Art teachers are forever looking for new angles on the good-old elements and principles. And, as budgets tighten, they are trying to be as frugal as possible while still holding their students' attention. Enter exploding boxes! In conceptualizing the three types of…

  8. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  9. RNA helicases: diverse roles in prokaryotic response to abiotic stress.

    PubMed

    Owttrim, George W

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In addition, RNA helicase expression and/or activity are frequently altered during cellular response to abiotic stress, implying they perform defined roles during cellular adaptation to changes in the growth environment. Specifically, RNA helicases contribute to the formation of cold-adapted ribosomes and RNA degradosomes, implying a role in alleviation of RNA secondary structure stabilization at low temperature. A common emerging theme involves RNA helicases acting as scaffolds for protein-protein interaction and functioning as molecular clamps, holding RNA-protein complexes in specific conformations. This review highlights recent advances in DEAD-box RNA helicase association with cellular response to abiotic stress in prokaryotes.

  10. Dead Sea Scrolls

    NASA Technical Reports Server (NTRS)

    1994-01-01

    A consortium of researchers from Jet Propulsion Laboratory and three other organizations used charged coupled devices (CCDs) and other imaging enhancement technology to decipher previously unreadable portions of the Dead Sea Scrolls. The technique has potentially important implications for archeology.

  11. Silencing of Y-box binding protein-1 by RNA interference inhibits proliferation, invasion, and metastasis, and enhances sensitivity to cisplatin through NF-κB signaling pathway in human neuroblastoma SH-SY5Y cells.

    PubMed

    Wang, Hong; Sun, Ruowen; Chi, Zuofei; Li, Shuang; Hao, Liangchun

    2017-04-05

    Y-box binding protein-1 (YB-1), a member of Y-box protein family binding DNA and RNA, has been proposed as a novel marker in multiple malignant tumors and found to be associated with tumor malignancy. Neuroblastoma is an embryonal tumor arising from neuroblast cells of the autonomic nervous system, which is the most common cancer diagnosed in infants. It has been reported that YB-1 is highly expressing in various human tumors including nasopharynx, thyroid, lung, breast, colon, ovary, and prostate cancers. This study aimed to investigate the functional role of YB-1 in neuroblastoma by silencing YB-1 using RNA interference (shRNA) in neuroblastoma SH-SY5Y cells. We found that silencing of YB-1 decreased the proliferation, migration, and invasion of SH-SY5Y cells. At molecular level, inhibition of YB-1 decreased the expression level of PCNA as well as MMP-2 in neuroblastoma SH-SY5Y cells. Also, we discovered that YB-1 silencing sensitized SH-SY5Y cells to cisplatin and promoted the apoptosis induced by cisplatin due to down-regulation of multidrug resistance (MDR) 1 protein via NF-κB signaling pathway. Therefore, we consider that targeting YB-1 is promising for neuroblastoma treatment and for overcoming its cisplatin resistance in the development of new neuroblastoma therapeutic strategies.

  12. ON HYDRODYNAMIC MOTIONS IN DEAD ZONES

    SciTech Connect

    Oishi, Jeffrey S.; Mac Low, Mordecai-Mark E-mail: mordecai@amnh.or

    2009-10-20

    We investigate fluid motions near the midplane of vertically stratified accretion disks with highly resistive midplanes. In such disks, the magnetorotational instability drives turbulence in thin layers surrounding a resistive, stable dead zone. The turbulent layers in turn drive motions in the dead zone. We examine the properties of these motions using three-dimensional, stratified, local, shearing-box, non-ideal, magnetohydrodynamical simulations. Although the turbulence in the active zones provides a source of vorticity to the midplane, no evidence for coherent vortices is found in our simulations. It appears that this is because of strong vertical oscillations in the dead zone. By analyzing time series of azimuthally averaged flow quantities, we identify an axisymmetric wave mode particular to models with dead zones. This mode is reduced in amplitude, but not suppressed entirely, by changing the equation of state from isothermal to ideal. These waves are too low frequency to affect sedimentation of dust to the midplane, but may have significance for the gravitational stability of the resulting midplane dust layers.

  13. The Bacillus subtilis tyrZ Gene Encodes a Highly Selective Tyrosyl-tRNA Synthetase and Is Regulated by a MarR Regulator and T Box Riboswitch

    PubMed Central

    Williams-Wagner, Rebecca N.; Grundy, Frank J.; Raina, Medha; Ibba, Michael

    2015-01-01

    ABSTRACT Misincorporation of d-tyrosine (d-Tyr) into cellular proteins due to mischarging of tRNATyr with d-Tyr by tyrosyl-tRNA synthetase inhibits growth and biofilm formation of Bacillus subtilis. Furthermore, many B. subtilis strains lack a functional gene encoding d-aminoacyl-tRNA deacylase, which prevents misincorporation of d-Tyr in most organisms. B. subtilis has two genes that encode tyrosyl-tRNA synthetase: tyrS is expressed under normal growth conditions, and tyrZ is known to be expressed only when tyrS is inactivated by mutation. We hypothesized that tyrZ encodes an alternate tyrosyl-tRNA synthetase, expression of which allows the cell to grow when d-Tyr is present. We show that TyrZ is more selective for l-Tyr over d-Tyr than is TyrS; however, TyrZ is less efficient overall. We also show that expression of tyrZ is required for growth and biofilm formation in the presence of d-Tyr. Both tyrS and tyrZ are preceded by a T box riboswitch, but tyrZ is found in an operon with ywaE, which is predicted to encode a MarR family transcriptional regulator. Expression of tyrZ is repressed by YwaE and also is regulated at the level of transcription attenuation by the T box riboswitch. We conclude that expression of tyrZ may allow growth when excess d-Tyr is present. IMPORTANCE Accurate protein synthesis requires correct aminoacylation of each tRNA with the cognate amino acid and discrimination against related compounds. Bacillus subtilis produces d-Tyr, an analog of l-Tyr that is toxic when incorporated into protein, during stationary phase. Most organisms utilize a d-aminoacyl-tRNA deacylase to prevent misincorporation of d-Tyr. This work demonstrates that the increased selectivity of the TyrZ form of tyrosyl-tRNA synthetase may provide a mechanism by which B. subtilis prevents misincorporation of d-Tyr in the absence of a functional d-aminoacyl-tRNA deacylase gene. PMID:25733610

  14. Dead sea water intoxication.

    PubMed

    Levy-Khademi, Floris; Brooks, Rebecca; Maayan, Channa; Tenenbaum, Ariel; Wexler, Isaiah D

    2012-08-01

    Near drowning in the Dead Sea is associated with both respiratory manifestations and severe electrolyte abnormalities. It is often difficult to distinguish between the contributions of sea water aspiration or ingestion to clinical manifestations. We present a unique case of accidental ingestion of a large amount of Dead Sea water through a gastrostomy tube in which a patient with familial dysautonomia presented with severe electrolyte disturbances. Forced diuresis with large amounts of intravenous fluids resulted in clinical and biochemical improvement. Full recovery was achieved after 2 days of treatment.

  15. "Living versus Dead":

    PubMed Central

    Chakrabarti, Pratik

    2010-01-01

    Summary The Semple antirabies vaccine was developed by David Semple in India in 1911. Semple introduced a peculiarly British approach within the Pasteurian tradition by using carbolized dead virus. This article studies this unique phase of vaccine research between 1910 and 1935 to show that in the debates and laboratory experiments around the potency and safety of vaccines, categories like "living" and "dead" were often used as ideological and moral denominations. These abstract and ideological debates were crucial in defining the final configuration of the Semple vaccine, the most popular antirabies vaccine used globally, and also in shaping international vaccination policies. PMID:21037397

  16. The Dead Sea

    NASA Technical Reports Server (NTRS)

    2006-01-01

    The Dead Sea is the lowest point on Earth at 418 meters below sea level, and also one of the saltiest bodies of water on Earth with a salinity of about 300 parts-per-thousand (nine times greater than ocean salinity). It is located on the border between Jordan and Israel, and is fed by the Jordan River. The Dead Sea is located in the Dead Sea Rift, formed as a result of the Arabian tectonic plate moving northward away from the African Plate. The mineral content of the Dead Sea is significantly different from that of ocean water, consisting of approximately 53% magnesium chloride, 37% potassium chloride and 8% sodium chloride. In the early part of the 20th century, the Dead Sea began to attract interest from chemists who deduced that the Sea was a natural deposit of potash and bromine. From the Dead Sea brine, Israel and Jordan produce 3.8 million tons potash, 200,000 tons elemental bromine, 45,000 tons caustic soda, 25, 000 tons magnesium metal, and sodium chloride. Both countries use extensive salt evaporation pans that have essentially diked the entire southern end of the Dead Sea.

    With its 14 spectral bands from the visible to the thermal infrared wavelength region, and its high spatial resolution of 15 to 90 meters (about 50 to 300 feet), ASTER images Earth to map and monitor the changing surface of our planet.

    ASTER is one of five Earth-observing instruments launched December 18, 1999, on NASA's Terra satellite. The instrument was built by Japan's Ministry of Economy, Trade and Industry. A joint U.S./Japan science team is responsible for validation and calibration of the instrument and the data products.

    The broad spectral coverage and high spectral resolution of ASTER provides scientists in numerous disciplines with critical information for surface mapping, and monitoring of dynamic conditions and temporal change. Example applications are: monitoring glacial advances and retreats; monitoring potentially active volcanoes; identifying crop

  17. The Analysis of the Inflorescence miRNome of the Orchid Orchis italica Reveals a DEF-Like MADS-Box Gene as a New miRNA Target

    PubMed Central

    Aceto, Serena; Sica, Maria; De Paolo, Sofia; D'Argenio, Valeria; Cantiello, Piergiuseppe; Salvatore, Francesco; Gaudio, Luciano

    2014-01-01

    Plant microRNAs (miRNAs) are small, regulatory non-coding RNAs involved in a wide range of biological processes, from organ development to response to stimuli. In recent years, an increasing number of studies on model plant species have highlighted the evolutionary conservation of a high number of miRNA families and the existence of taxon-specific ones. However, few studies have examined miRNAs in non-model species such as orchids, which are characterized by highly diversified floral structures and pollination strategies. Therefore, we analysed a small RNA library of inflorescence tissue of the Mediterranean orchid Orchis italica to increase the knowledge on miRNAs in a non-model plant species. The high-throughput sequencing and analysis of a small RNA library of inflorescence of O. italica revealed 23 conserved and 161 putative novel miRNA families. Among the putative miRNA targets, experimental validation demonstrated that a DEF-like MADS-box transcript is cleaved by the homolog of miR5179 of O. italica. The presence of conserved miRNA families in the inflorescence of O. italica indicates that the basic developmental flower regulatory mechanisms mediated by miRNAs are maintained through evolution. Because, according to the “orchid code” theory, DEF-like genes exert a key function in the diversification of tepals and lip, the cleavage-mediated inhibitory activity of miR5179 on a OitaDEF-like transcript suggests that, in orchids, miRNAs play an important role in the diversification of the perianth organs. PMID:24832004

  18. The analysis of the inflorescence miRNome of the orchid Orchis italica reveals a DEF-like MADS-box gene as a new miRNA target.

    PubMed

    Aceto, Serena; Sica, Maria; De Paolo, Sofia; D'Argenio, Valeria; Cantiello, Piergiuseppe; Salvatore, Francesco; Gaudio, Luciano

    2014-01-01

    Plant microRNAs (miRNAs) are small, regulatory non-coding RNAs involved in a wide range of biological processes, from organ development to response to stimuli. In recent years, an increasing number of studies on model plant species have highlighted the evolutionary conservation of a high number of miRNA families and the existence of taxon-specific ones. However, few studies have examined miRNAs in non-model species such as orchids, which are characterized by highly diversified floral structures and pollination strategies. Therefore, we analysed a small RNA library of inflorescence tissue of the Mediterranean orchid Orchis italica to increase the knowledge on miRNAs in a non-model plant species. The high-throughput sequencing and analysis of a small RNA library of inflorescence of O. italica revealed 23 conserved and 161 putative novel miRNA families. Among the putative miRNA targets, experimental validation demonstrated that a DEF-like MADS-box transcript is cleaved by the homolog of miR5179 of O. italica. The presence of conserved miRNA families in the inflorescence of O. italica indicates that the basic developmental flower regulatory mechanisms mediated by miRNAs are maintained through evolution. Because, according to the "orchid code" theory, DEF-like genes exert a key function in the diversification of tepals and lip, the cleavage-mediated inhibitory activity of miR5179 on a OitaDEF-like transcript suggests that, in orchids, miRNAs play an important role in the diversification of the perianth organs.

  19. The RNA Helicase eIF4A Is Required for Sapovirus Translation

    PubMed Central

    Hosmillo, Myra; Sweeney, Trevor R.; Chaudhry, Yasmin; Leen, Eoin; Curry, Stephen

    2016-01-01

    The eukaryotic initiation factor 4A (eIF4A) is a DEAD box helicase that unwinds RNA structure in the 5′ untranslated region (UTR) of mRNAs. Here, we investigated the role of eIF4A in porcine sapovirus VPg-dependent translation. Using inhibitors and dominant-negative mutants, we found that eIF4A is required for viral translation and infectivity, suggesting that despite the presence of a very short 5′ UTR, eIF4A is required to unwind RNA structure in the sapovirus genome to facilitate virus translation. PMID:26937032

  20. Dead Sea rhodopsins revisited.

    PubMed

    Bodaker, Idan; Suzuki, Marcelino T; Oren, Aharon; Béjà, Oded

    2012-12-01

    The Dead Sea is a unique hypersaline ecosystem with near toxic magnesium levels (∼2 M), dominance of divalent cations and a slightly acidic pH. Previously, we reported a haloarchaeon related to Halobacterium salinarum to dominate in a microbial bloom that developed in 1992 in the upper water layers of the lake following massive freshwater runoff. Whether this clade also dominated an earlier bloom in 1980-1982 cannot be ascertained as no samples for cultivation-independent analysis were preserved. The presence of the light-driven proton pump bacteriorhodopsin was reported in the 1980-1982 bloom of prokaryotes that had developed in the Dead Sea. To test the hypothesis that bacteriorhodopsin proton pumping may play a major role in determining what type of haloarchaea may dominate in specific bloom conditions, we compared rhodopsin genes recovered from Dead Sea biomass collected in different periods with genes coding for retinal proteins in isolated haloarchaea. Novel bacteriorhodopsin and sensory rhodopsin genes were found in samples collected in 2007 and 2010. The fact that no rhodopsin genes were recovered from samples collected during the 1992 bloom, which was dominated by a single species, suggests that different clades were present in the 1980-1982 and 1992 blooms, and that bacteriorhodopsin proton pumping did not necessarily play a determinative role in the dominance of specific halophiles in the blooms.

  1. Communicating with the Dead

    NASA Astrophysics Data System (ADS)

    Kurtz, Paul

    2000-03-01

    Communicating with dead persons is popular in the new paranatural (paranormal-spiritual) paradigm that has emerged today. This view violates physicalist principles. Scientists have investigated survival questions for over 150 years. Beginning with the Fox sisters in 1848, they have examined reports of apparitions and ghosts, rappings, table turnings, teleportation, levitation, and other alleged physicalist manifestations in séances. Such reports were discredited by uncovering hoaxes by alleged mediums, e.g. Eusapia Palladino (1910s), Marjorie Crandon (1920s), etc. In recent decades there has been a revival of interest in survival with reports of near-death experiences. Skeptics ask: Are such persons clinically dead? Today, channelers such as James Van Praagh, John Edwards, and Sylvia Browne claim to communicate directly with the dead (the "Sixth Sense"). There is no attempt at physical confirmation or independent eyewitness corroboration so essential for scientific inquiry. Subjective claims are uncritically accepted at their face value, though alternative naturalistic explanations can be given for the alleged phenomena.

  2. Dead Band Controls Guide.

    DTIC Science & Technology

    1978-11-01

    n o m i z e r c on t ro l , damper repair may re quire c onsideration. 2. No tubin g costs are included in the above estima tes . Typically 150 to...guidelines include techniques for estimating construction and maintenance cost , and performing economic analysis for each system . N \\ 0~c...43 Cost Estimate 43 Payback Anal ysis 43 PART I V A P P E N D I C E S A EXAMPLE - COST ESTIMATE OF DEAD BAND RETROFIT 52 B E X A M P L E - P A Y B A

  3. Comparative study of two box H/ACA ribonucleoprotein pseudouridine-synthases: relation between conformational dynamics of the guide RNA, enzyme assembly and activity.

    PubMed

    Fourmann, Jean-Baptiste; Tillault, Anne-Sophie; Blaud, Magali; Leclerc, Fabrice; Branlant, Christiane; Charpentier, Bruno

    2013-01-01

    Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10-L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21.

  4. Immunosuppression during active tuberculosis is characterized by decreased interferon- gamma production and CD25 expression with elevated forkhead box P3, transforming growth factor- beta , and interleukin-4 mRNA levels.

    PubMed

    Roberts, Teri; Beyers, Nulda; Aguirre, Ana; Walzl, Gerhard

    2007-03-15

    The balance between effector and regulatory responses after Mycobacterium tuberculosis infection may dictate outcome and progression to active disease. We investigated effector and regulatory T cell responses in bacille Calmette-Guerin (BCG)-stimulated peripheral blood mononuclear cells and whole blood cultures from persons with active tuberculosis (TB), persons with TB at the end of 6 months of treatment, and healthy control subjects with latent TB infection. All 3 groups displayed BCG-induced increases in effector and regulatory T cell phenotypes as defined by CD4(+)CD25(lo) and CD4(+)CD25(hi) T cells, respectively. In case patients with active disease, BCG stimulation induced the lowest increase of CD25, CD4(+)CD25(hi), CTLA-4, and interferon- gamma . However, these case patients expressed the highest mRNA levels of forkhead box P3, transforming growth factor (TGF)- beta , and interleukin (IL)-4 and a lower T-bet : GATA-3 ratio. There were no significant differences in IL-4 delta 2, IL-10, or TGF- beta receptor-II mRNA expression between groups. Together, these results suggest that immunosuppression seen after mycobacterial stimulation in case patients with active TB is associated with naturally occurring regulatory T cells.

  5. P68 RNA Helicase Is A Nucleocytoplasm Shuttling Protein

    PubMed Central

    Wang, Haizhen; Gao, Xueliang; Huang, Yun; Yang, Jenny; Liu, Zhi-Ren

    2009-01-01

    P68 RNA helicase is a prototypical DEAD box RNA helicase. The protein plays a very important role in early organ development and maturation. In consistence with the function of the protein in transcriptional regulation and pre-mRNA splicing, p68 was found to predominately localize in the cell nucleus. However, recent experiments demonstrate a transient cytoplasmic localization of the protein. We report here that p68 shuttles between the nucleus and the cytoplasm. The nucleocytoplasmic shuttling of p68 is mediated by two nuclear localization signal (NLS) and two nuclear exporting signal (NES) sequence elements. Our experiments reveal that p68 shuttles via a classical RanGTPase dependent pathway. PMID:19786986

  6. Dead of night.

    PubMed

    Balter, Leon

    2010-07-01

    Dead of Night, the first psychoanalytic horror film, was produced in England in 1945, immediately after the end of World War II--that is, after the English population had suffered systematic Nazi terror from imminent invasion, incessant aerial bombing, and rocket-bombs. This film continued the prewar format of horror films based on themes of the supernatural and the hubris and excesses of science. However, it introduced psychoanalysis as the science in question. The film is structured on two levels: a genteel English country weekend to which witty and urbane guests have been invited; and five horror stories told by the guests. Psychoanalytic insights into this film structure are used here to explain how the film induces horror in the audience.

  7. DbpA is a region-specific RNA helicase.

    PubMed

    Moore, Anthony F T; Gentry, Riley C; Koculi, Eda

    2017-03-01

    DbpA is a DEAD-box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA-like catalytic core responsible for double-helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core's grasp. In other words, it is not known if DbpA is a site-specific enzyme or region-specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region-specific or a site-specific enzyme. Our data suggest that DbpA is a region-specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C-terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double-helices in its vicinity. The only requirement for a double-helix to serve as a DbpA substrate is for the double-helix to be positioned within the catalytic core's grasp.

  8. Insights into the mechanism of a G-quadruplex-unwinding DEAH-box helicase.

    PubMed

    Chen, Michael C; Murat, Pierre; Abecassis, Keren; Ferré-D'Amaré, Adrian R; Balasubramanian, Shankar

    2015-02-27

    The unwinding of nucleic acid secondary structures within cells is crucial to maintain genomic integrity and prevent abortive transcription and translation initiation. DHX36, also known as RHAU or G4R1, is a DEAH-box ATP-dependent helicase highly specific for DNA and RNA G-quadruplexes (G4s). A fundamental mechanistic understanding of the interaction between helicases and their G4 substrates is important to elucidate G4 biology and pave the way toward G4-targeted therapies. Here we analyze how the thermodynamic stability of G4 substrates affects binding and unwinding by DHX36. We modulated the stability of the G4 substrates by varying the sequence and the number of G-tetrads and by using small, G4-stabilizing molecules. We found an inverse correlation between the thermodynamic stability of the G4 substrates and rates of unwinding by DHX36. In stark contrast, the ATPase activity of the helicase was largely independent of substrate stability pointing toward a decoupling mechanism akin to what has been observed for many double-stranded DEAD-box RNA helicases. Our study provides the first evidence that DHX36 uses a local, non-processive mechanism to unwind G4 substrates, reminiscent of that of eukaryotic initiation factor 4A (eIF4A) on double-stranded substrates.

  9. New Life From Dead Trees

    ERIC Educational Resources Information Center

    DeGraaf, Richard M.

    1978-01-01

    There are numerous bird species that will nest only in dead or dying trees. Current forestry practices include clearing forests of these snags, or dead trees. This practice is driving many species out of the forests. An illustrated example of bird succession in and on a tree is given. (MA)

  10. Reevaluating the dead donor rule.

    PubMed

    Collins, Mike

    2010-04-01

    The dead donor rule justifies current practice in organ procurement for transplantation and states that organ donors must be dead prior to donation. The majority of organ donors are diagnosed as having suffered brain death and hence are declared dead by neurological criteria. However, a significant amount of unrest in both the philosophical and the medical literature has surfaced since this practice began forty years ago. I argue that, first, declaring death by neurological criteria is both unreliable and unjustified but further, the ethical principles which themselves justify the dead donor rule are better served by abandoning that rule and instead allowing individuals who have suffered severe and irreversible brain damage to become organ donors, even though they are not yet dead and even though the removal of their organs would be the proximal cause of death.

  11. Metagenomic insights into important microbes from the Dead Zone

    NASA Astrophysics Data System (ADS)

    Thrash, C.; Baker, B.; Seitz, K.; Temperton, B.; Gillies, L.; Rabalais, N. N.; Mason, O. U.

    2015-12-01

    Coastal regions of eutrophication-driven oxygen depletion are widespread and increasing in number. Also known as dead zones, these regions take their name from the deleterious effects of hypoxia (dissolved oxygen less than 2 mg/L) on shrimp, demersal fish, and other animal life. Dead zones result from nutrient enrichment of primary production, concomitant consumption by chemoorganotrophic aerobic microorganisms, and strong stratification that prevents ventilation of bottom water. One of the largest dead zones in the world occurs seasonally in the northern Gulf of Mexico (nGOM), where hypoxia can reach up to 22,000 square kilometers. While this dead zone shares many features with more well-known marine oxygen minimum zones, it is nevertheless understudied with regards to the microbial assemblages involved in biogeochemical cycling. We performed metagenomic and metatranscriptomic sequencing on six samples from the 2013 nGOM dead zone from both hypoxic and oxic bottom waters. Assembly and binning led to the recovery of over fifty partial to nearly complete metagenomes from key microbial taxa previously determined to be numerically abundant from 16S rRNA data, such as Thaumarcheaota, Marine Group II Euryarchaeota, SAR406, SAR324, Synechococcus spp., and Planctomycetes. These results provide information about the roles of these taxa in the nGOM dead zone, and opportunities for comparing this region of low oxygen to others around the globe.

  12. The effects of n-3 PUFA and intestinal lymph drainage on high-mobility group box 1 and Toll-like receptor 4 mRNA in rats with intestinal ischaemia-reperfusion injury.

    PubMed

    He, Gui-Zhen; Zhou, Kai-Guo; Zhang, Rui; Chen, Xue-Feng

    2012-09-01

    The aim of the present study was to investigate the impacts of n-3 PUFA and lymph drainage (D) on intestinal ischaemia-reperfusion (I/R) injury in rats. A total of forty-eight Sprague-Dawley male rats were randomly divided into three groups (n 16): normal diet (N), enteral nutrition (EN) and EN plus n-3 PUFA. Each group was further divided into lymph drainage (I/R+D) and non-drainage (I/R) sub-groups (n 8). After 5 d with different nutrition regimens, the rats were subjected to 60 min ischaemia by clamping the superior mesenteric artery, followed by 120 min reperfusion. At the same time, the rats in the I/R+D sub-groups were treated with intestinal lymph drainage for 180 min. Organs were harvested and we detected the cytokine, endotoxin, and expression of Toll-like receptor (TLR) 4 mRNA and its endogenous ligand high-mobility group box 1 (HMGB1). We found that the serum levels of HMGB1, inflammatory cytokine and endotoxin in the three I/R+D sub-groups were significantly lower than those in the N (I/R) and EN (I/R) sub-groups (P < 0·05). The activation of NF-κB and the expression of HMGB1 and TLR4 mRNA significantly increased in the jejunum, ileum, liver and lung after intestinal I/R injury, but notably lower in the I/R+D groups than those in I/R (P < 0·05). The injury degree and HMGB1 expression were decreased in the n-3 PUFA group than in the N and EN groups. We preliminarily concluded that nutrition with n-3 PUFA and/or intestinal lymph drainage may reduce HMGB1 and inflammatory cytokine in serum and lymph and inhibit the expression and signal transmission of TLR4 mRNA, thereby alleviating intestinal I/R injury in rats.

  13. Who are the Unclaimed Dead?

    PubMed

    Quinet, Kenna; Nunn, Samuel; Ballew, Alfarena

    2016-01-01

    Unclaimed dead are deceased persons with no known next of kin (NoK) or NoK was located but did not claim the deceased. Unclaimed dead in Marion County, Indiana, 2004-2011, are examined. Comparisons are provided of the unclaimed to the claimed dead population and county death patterns. Race, gender, marital status, age, location, manner and cause of death, NoK, and days to disposition are analyzed. The unclaimed dead were disproportionately male, slightly more likely to be Black, younger at death, died from natural causes, had unknown marital status, were equally likely as not to have NoK, did not die in a hospital, and were subject to autopsy. Nearly half the unclaimed had NoK who did not claim the body; the other half had no identifiable NoK. Unclaimed were more likely to have an autopsy and to die from external causes. Most unclaimed were identified by means outside fingerprints or DNA.

  14. An Improved Box Theater

    NASA Astrophysics Data System (ADS)

    Huster, Michael E.

    2011-09-01

    While designing an optics lab for a conceptual physics course, I came across a "box theater" activity. The box theater is a pinhole camera obscura made from a box that students put over their heads and shoulders. I use the activity as a capstone experience to explain optical systems. (Classroom demonstrations of the camera obscura have been described by others.2) First, the students build and experiment with a camera obscura made from a plastic cup and a convex lens with a focal length of 7.5 cm, and then "wear" the box theater. The difficulty with the box theater is the dimness of the image. A cloth drape has to be hung from the bottom of the box around the shoulders of the students to prevent light leakage, and the students have to wait a few minutes for their eyes to adjust to the darkness.

  15. Climate change and dead zones.

    PubMed

    Altieri, Andrew H; Gedan, Keryn B

    2015-04-01

    Estuaries and coastal seas provide valuable ecosystem services but are particularly vulnerable to the co-occurring threats of climate change and oxygen-depleted dead zones. We analyzed the severity of climate change predicted for existing dead zones, and found that 94% of dead zones are in regions that will experience at least a 2 °C temperature increase by the end of the century. We then reviewed how climate change will exacerbate hypoxic conditions through oceanographic, ecological, and physiological processes. We found evidence that suggests numerous climate variables including temperature, ocean acidification, sea-level rise, precipitation, wind, and storm patterns will affect dead zones, and that each of those factors has the potential to act through multiple pathways on both oxygen availability and ecological responses to hypoxia. Given the variety and strength of the mechanisms by which climate change exacerbates hypoxia, and the rates at which climate is changing, we posit that climate change variables are contributing to the dead zone epidemic by acting synergistically with one another and with recognized anthropogenic triggers of hypoxia including eutrophication. This suggests that a multidisciplinary, integrated approach that considers the full range of climate variables is needed to track and potentially reverse the spread of dead zones.

  16. Dead Star Rumbles

    NASA Technical Reports Server (NTRS)

    2005-01-01

    [figure removed for brevity, see original site] Composite of Supernova Remnant Cassiopeia A This Spitzer Space Telescope composite shows the supernova remnant Cassiopeia A (white ball) and surrounding clouds of dust (gray, orange and blue). It consists of two processed images taken one year apart. Dust features that have not changed over time appear gray, while those that have changed are colored blue or orange. Blue represents an earlier time and orange, a later time.

    These observations illustrate that a blast of light from Cassiopeia A is waltzing outward through the dusty skies. This dance, called an 'infrared echo,' began when the remnant erupted about 50 years ago.

    Cassiopeia A is the remnant of a once massive star that died in a violent supernova explosion 325 years ago. It consists of a dead star, called a neutron star, and a surrounding shell of material that was blasted off as the star died. This remnant is located 10,000 light-years away in the northern constellation Cassiopeia.

    An infrared echo is created when a star explodes or erupts, flashing light into surrounding clumps of dust. As the light zips through the dust clumps, it heats them up, causing them to glow successively in infrared, like a chain of Christmas bulbs lighting up one by one. The result is an optical illusion, in which the dust appears to be flying outward at the speed of light. This apparent motion can be seen here by the shift in colored dust clumps.

    Echoes are distinct from supernova shockwaves, which are made up material that is swept up and hurled outward by exploding stars.

    This infrared echo is the largest ever seen, stretching more than 50 light-years away from Cassiopeia A. If viewed from Earth, the entire movie frame would take up the same amount of space as two full moons.

    Hints of an older infrared echo from Cassiopeia A's supernova explosion hundreds of years ago can also be seen.

    The earlier Spitzer image was taken on November 30

  17. GLOVE BOX ATTACHMENT

    DOEpatents

    Butts, H.L.

    1962-02-13

    This invention comprises a housing unit to be fitted between a glove box port and a glove so that a slidable plate within the housing seals off the glove box port for evacuation of the glove box without damage to the glove. The housing and the glove may be evacuated without damage to the glove since movement of the glove is restricted during evacuation by the slidable plate. (AEC)

  18. RNA helicase HEL-1 promotes longevity by specifically activating DAF-16/FOXO transcription factor signaling in Caenorhabditis elegans.

    PubMed

    Seo, Mihwa; Seo, Keunhee; Hwang, Wooseon; Koo, Hee Jung; Hahm, Jeong-Hoon; Yang, Jae-Seong; Han, Seong Kyu; Hwang, Daehee; Kim, Sanguk; Jang, Sung Key; Lee, Yoontae; Nam, Hong Gil; Lee, Seung-Jae V

    2015-08-04

    The homeostatic maintenance of the genomic DNA is crucial for regulating aging processes. However, the role of RNA homeostasis in aging processes remains unknown. RNA helicases are a large family of enzymes that regulate the biogenesis and homeostasis of RNA. However, the functional significance of RNA helicases in aging has not been explored. Here, we report that a large fraction of RNA helicases regulate the lifespan of Caenorhabditis elegans. In particular, we show that a DEAD-box RNA helicase, helicase 1 (HEL-1), promotes longevity by specifically activating the DAF-16/forkhead box O (FOXO) transcription factor signaling pathway. We find that HEL-1 is required for the longevity conferred by reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) and is sufficient for extending lifespan. We further show that the expression of HEL-1 in the intestine and neurons contributes to longevity. HEL-1 enhances the induction of a large fraction of DAF-16 target genes. Thus, the RNA helicase HEL-1 appears to promote longevity in response to decreased IIS as a transcription coregulator of DAF-16. Because HEL-1 and IIS are evolutionarily well conserved, a similar mechanism for longevity regulation via an RNA helicase-dependent regulation of FOXO signaling may operate in mammals, including humans.

  19. 46 CFR 171.117 - Dead covers.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Dead covers. 171.117 Section 171.117 Shipping COAST... Dead covers. (a) Except as provided in paragraph (b) of this section, each port light with the sill located below the margin line must have a hinged, inside dead cover. (b) The dead cover on a port...

  20. Structure and mechanism of the T-box riboswitches.

    PubMed

    Zhang, Jinwei; Ferré-D'Amaré, Adrian R

    2015-01-01

    In most Gram-positive bacteria, including many clinically devastating pathogens from genera such as Bacillus, Clostridium, Listeria, and Staphylococcus, T-box riboswitches sense and regulate intracellular availability of amino acids through a multipartite messenger RNA (mRNA)-transfer RNA (tRNA) interaction. The T-box mRNA leaders respond to nutrient starvation by specifically binding cognate tRNAs and sensing whether the bound tRNA is aminoacylated, as a proxy for amino acid availability. Based on this readout, T-boxes direct a transcriptional or translational switch to control the expression of downstream genes involved in various aspects of amino acid metabolism: biosynthesis, transport, aminoacylation, transamidation, and so forth. Two decades after its discovery, the structural and mechanistic underpinnings of the T-box riboswitch were recently elucidated, producing a wealth of insights into how two structured RNAs can recognize each other with robust affinity and exquisite selectivity. The T-box paradigm exemplifies how natural noncoding RNAs can interact not just through sequence complementarity but can add molecular specificity by precisely juxtaposing RNA structural motifs, exploiting inherently flexible elements and the biophysical properties of post-transcriptional modifications, ultimately achieving a high degree of shape complementarity through mutually induced fit. The T-box also provides a proof-of-principle that compact RNA domains can recognize minute chemical changes (such as tRNA aminoacylation) on another RNA. The unveiling of the structure and mechanism of the T-box system thus expands our appreciation of the range of capabilities and modes of action of structured noncoding RNAs, and hints at the existence of networks of noncoding RNAs that communicate through both, structural and sequence specificity.

  1. Thinking outside the Box

    ERIC Educational Resources Information Center

    Fanshawe, Simon; Sriskandarajah, Dhananjayan

    2010-01-01

    Britain is not only more diverse than ever before, but that diversity itself is growing more diverse. Britain's simplistic "tick-box" approach to identity is in danger of inhibiting the very equality it seeks to promote. To question the tick-box is not to accuse local authorities of "political correctness gone mad". The notion…

  2. Math in the Box

    ERIC Educational Resources Information Center

    DeYoung, Mary J.

    2009-01-01

    This article describes how to make an origami paper box and explores the algebra, geometry, and other mathematics that unfolds. A set of origami steps that transforms the paper into an open box can hold mathematical surprises for both students and teachers. An origami lesson can engage students in an open-ended exploration of the relationship…

  3. Straw in a Box

    ERIC Educational Resources Information Center

    Jerrard, Richard; Schneider, Joel; Smallberg, Ralph; Wetzel, John

    2006-01-01

    A problem on a state's high school exit exam asked for the longest straw that would fit in a box. The examiners apparently wanted the length of a diagonal of the box, but the figure accompanying the question suggested otherwise--that the radius of the straw be considered. This article explores that more general problem.

  4. Localization of Components of the RNA-Degrading Machine in Bacillus subtilis

    PubMed Central

    Cascante-Estepa, Nora; Gunka, Katrin; Stülke, Jörg

    2016-01-01

    In bacteria, the control of mRNA stability is crucial to allow rapid adaptation to changing conditions. In most bacteria, RNA degradation is catalyzed by the RNA degradosome, a protein complex composed of endo- and exoribonucleases, RNA helicases, and accessory proteins. In the Gram-positive model organism Bacillus subtilis, the existence of a RNA degradosome assembled around the membrane-bound endoribonuclease RNase Y has been proposed. Here, we have studied the intracellular localization of the protein that have been implicated in the potential B. subtilis RNA degradosome, i.e., polynucleotide phosphorylase, the exoribonucleases J1 and J2, the DEAD-box RNA helicase CshA, and the glycolytic enzymes enolase and phosphofructokinase. Our data suggests that the bulk of these enzymes is located in the cytoplasm. The RNases J1 and J2 as well as the RNA helicase CshA were mainly localized in the peripheral regions of the cell where also the bulk of messenger RNA is localized. We were able to demonstrate active exclusion of these proteins from the transcribing nucleoid. Taken together, our findings suggest that the interactions of the enzymes involved in RNA degradation in B. subtilis are rather transient. PMID:27708634

  5. [Parasitic dead-end: update].

    PubMed

    Magnaval, J F

    2006-08-01

    Parasitic dead-ends occur when a parasite is unable to establish a permanent interaction in an unnatural host. Although the likelihood of successful reproduction by the pathogenic agent is nul, parasitic dead-end heralds capture of new parasites and therefore expansion of the host range. Angiostrongyliasis due to A. cantonensis or A. costaricensis, anisakiasis, Ancylostoma caninum infection, gnathostomiasis and sparganosis are undoubtedly emerging zoonoses of particular medical interest. Prevention of these diseases relies on abstinence from eating raw meat from invertebrates or cold-blooded (poikilotherm) vertebrates (e.g. used in exotic dishes). These guidelines must be included in recommendations to travelers.

  6. RNA chaperones buffer deleterious mutations in E. coli

    PubMed Central

    Rudan, Marina; Schneider, Dominique; Warnecke, Tobias; Krisko, Anita

    2015-01-01

    Both proteins and RNAs can misfold into non-functional conformations. Protein chaperones promote native folding of nascent polypeptides and refolding of misfolded species, thereby buffering mutations that compromise protein structure and function. Here, we show that RNA chaperones can also act as mutation buffers that enhance organismal fitness. Using competition assays, we demonstrate that overexpression of select RNA chaperones, including three DEAD box RNA helicases (DBRHs) (CsdA, SrmB, RhlB) and the cold shock protein CspA, improves fitness of two independently evolved Escherichia coli mutator strains that have accumulated deleterious mutations during short- and long-term laboratory evolution. We identify strain-specific mutations that are deleterious and subject to buffering when introduced individually into the ancestral genotype. For DBRHs, we show that buffering requires helicase activity, implicating RNA structural remodelling in the buffering process. Our results suggest that RNA chaperones might play a fundamental role in RNA evolution and evolvability. DOI: http://dx.doi.org/10.7554/eLife.04745.001 PMID:25806682

  7. Glove box shield

    DOEpatents

    Brackenbush, Larry W.; Hoenes, Glenn R.

    1981-01-01

    According to the present invention, a shield for a glove box housing radioactive material is comprised of spaced apart clamping members which maintain three overlapping flaps in place therebetween. There is a central flap and two side flaps, the side flaps overlapping at the interior edges thereof and the central flap extending past the intersection of the side flaps in order to insure that the shield is always closed when the user withdraws his hand from the glove box. Lead loaded neoprene rubber is the preferred material for the three flaps, the extent of lead loading depending upon the radiation levels within the glove box.

  8. Glove box shield

    DOEpatents

    Brackenbush, L.W.; Hoenes, G.R.

    A shield for a glove box housing radioactive material is comprised of spaced apart clamping members which maintain three overlapping flaps in place therebetween. There is a central flap and two side flaps, the side flaps overlapping at the interior edges thereof and the central flap extending past the intersection of the side flaps in order to insure that the shield is always closed when the user wthdraws his hand from the glove box. Lead loaded neoprene rubber is the preferred material for the three flaps, the extent of lead loading depending upon the radiation levels within the glove box.

  9. Structure and mechanism of the T-box riboswitches

    PubMed Central

    Zhang, Jinwei

    2015-01-01

    In most Gram-positive bacteria, including many clinically devastating pathogens from genera such as Bacillus, Clostridium, Listeria and Staphylococcus, T-box riboswitches sense and regulate intracellular availability of amino acids through a multipartite mRNA-tRNA interaction. The T-box mRNA leaders respond to nutrient starvation by specifically binding cognate tRNAs and sensing whether the bound tRNA is aminoacylated, as a proxy for amino acid availability. Based on this readout, T-boxes direct a transcriptional or translational switch to control the expression of downstream genes involved in various aspects of amino acid metabolism: biosynthesis, transport, aminoacylation, transamidation, etc. Two decades after its discovery, the structural and mechanistic underpinnings of the T-box riboswitch were recently elucidated, producing a wealth of insights into how two structured RNAs can recognize each other with robust affinity and exquisite selectivity. The T-box paradigm exemplifies how natural non-coding RNAs can interact not just through sequence complementarity, but can add molecular specificity by precisely juxtaposing RNA structural motifs, exploiting inherently flexible elements and the biophysical properties of post-transcriptional modifications, ultimately achieving a high degree of shape complementarity through mutually induced fit. The T-box also provides a proof-of-principle that compact RNA domains can recognize minute chemical changes (such as tRNA aminoacylation) on another RNA. The unveiling of the structure and mechanism of the T-box system thus expands our appreciation of the range of capabilities and modes of action of structured non-coding RNAs, and hints at the existence of networks of non-coding RNAs that communicate through both, structural and sequence specificity. PMID:25959893

  10. Climate in a Box

    NASA Video Gallery

    NASA's Climate in a Box Project is exploring the utility of supercomputers in providing a complete, pre-packaged, ready-to-use toolkit of climate research products and on-demand access to a high-pe...

  11. Voice box (image)

    MedlinePlus

    The larynx, or voice box, is located in the neck and performs several important functions in the body. The larynx is involved in swallowing, breathing, and voice production. Sound is produced when the air which ...

  12. [Boxing: traumatology and prevention].

    PubMed

    Cabanis, Emmanuel-Alain; Iba-Zizen, Marie-Thérèse; Perez, Georges; Senegas, Xavier; Furgoni, Julien; Pineau, Jean-Claude; Louquet, Jean-Louis; Henrion, Roger

    2010-10-01

    In 1986, a surgeon who, as an amateur boxer himself was concerned with boxers' health, approached a pioneering Parisian neuroimaging unit. Thus began a study in close cooperation with the French Boxing Federation, spanning 25 years. In a first series of 52 volunteer boxers (13 amateurs and 39 professionals), during which MRI gradually replaced computed tomography, ten risk factors were identified, which notably included boxing style: only one of 40 "stylists" with a good boxing technique had cortical atrophy (4.5 %), compared to 15 % of "sloggers". Changes to the French Boxing Federation rules placed the accent on medical prevention. The second series, of 247 boxers (81 amateurs and 266 professionals), showed a clear improvement, as lesions were suspected in 14 individuals, of which only 4 (1.35 %) were probably due to boxing. The third and fourth series were part of a protocol called "Brain-Boxing-Ageing", which included 76 boxers (11 having suffered KOs) and 120 MRI scans, with reproducible CT and MRI acquisitions (9 sequences with 1.5 T then 3 T, and CT). MRI anomalies secondary to boxing were found in 11 % of amateurs and 38 % of professionals (atrophy, high vascular T2 signal areas, 2 cases of post-KO subdural bleeding). CT revealed sinus damage in 13 % of the amateurs and 19 % of the professionals. The risk of acute and chronic facial and brain damage was underline, along with detailed precautionary measures (organization of bouts, role of the referee and ringside doctor, and application of French Boxing Federation rules).

  13. Nonneurologic emergencies in boxing.

    PubMed

    Coletta, Domenic F

    2009-10-01

    Professional boxing has done an admirable job in promoting safety standards in its particular sport. However, injuries occur during the normal course of competition and, unfortunately, an occasional life-threatening emergency may arise. Although most common medical emergencies in boxing are injuries from closed head trauma, in this article those infrequent but potentially catastrophic nonneurologic conditions are reviewed along with some less serious emergencies that the physician must be prepared to address.

  14. Infectious disease and boxing.

    PubMed

    King, Osric S

    2009-10-01

    There are no unique boxing diseases but certain factors contributing to the spread of illnesses apply strongly to the boxer, coach, and the training facility. This article examines the nature of the sport of boxing and its surrounding environment, and the likelihood of spread of infection through airborne, contact, or blood-borne routes of transmission. Evidence from other sports such as running, wrestling, and martial arts is included to help elucidate the pathophysiologic elements that could be identified in boxers.

  15. Automatic box loader

    DOEpatents

    Eldridge, Harry H.; Jones, Robert A.; Lindner, Gordon M.; Hight, Paul H.

    1976-01-01

    This invention relates to a system for repetitively forming an assembly consisting of a single layer of tubes and a row of ferromagnetic armatures underlying the same, electromagnetically conveying the resulting assembly to a position overlying a storage box, and depositing the assembly in the box. The system includes means for simultaneously depositing a row of the armatures on the inclined surface of a tube retainer. Tubes then are rolled down the surface to form a single tube layer bridging the armatures. A magnet assembly carrying electromagnets respectively aligned with the armatures is advanced close to the tube layer, and in the course of this advance is angularly displaced to bring the pole pieces of the electromagnets into parallelism with the tube layer. The magnets then are energized to pick up the assembly. The loaded magnet assembly is retracted to a position overlying the box, and during this retraction is again displaced to bring the pole pieces of the electromagnets into a horizontal plane. Means are provided for inserting the loaded electromagnets in the box and then de-energizing the electromagnets to deposit the assembly therein. The system accomplishes the boxing of fragile tubes at relatively high rates. Because the tubes are boxed as separated uniform layers, subsequent unloading operations are facilitated.

  16. FAQ: West Nile Virus and Dead Birds

    MedlinePlus

    ... Education Public Service Videos West Nile Virus & Dead Birds Recommend on Facebook Tweet Share Compartir On This ... dead bird sightings to local authorities. How do birds get infected with West Nile virus? West Nile ...

  17. Cable Tester Box

    NASA Technical Reports Server (NTRS)

    Lee, Jason H.

    2011-01-01

    Cables are very important electrical devices that carry power and signals across multiple instruments. Any fault in a cable can easily result in a catastrophic outcome. Therefore, verifying that all cables are built to spec is a very important part of Electrical Integration Procedures. Currently, there are two methods used in lab for verifying cable connectivity. (1) Using a Break-Out Box and an ohmmeter this method is time-consuming but effective for custom cables and (2) Commercial Automated Cable Tester Boxes this method is fast, but to test custom cables often requires pre-programmed configuration files, and cables used on spacecraft are often uniquely designed for specific purposes. The idea is to develop a semi-automatic continuity tester that reduces human effort in cable testing, speeds up the electrical integration process, and ensures system safety. The JPL-Cable Tester Box is developed to check every single possible electrical connection in a cable in parallel. This system indicates connectivity through LED (light emitting diode) circuits. Users can choose to test any pin/shell (test node) with a single push of a button, and any other nodes that are shorted to the test node, even if they are in the same connector, will light up with the test node. The JPL-Cable Tester Boxes offers the following advantages: 1. Easy to use: The architecture is simple enough that it only takes 5 minutes for anyone to learn how operate the Cable Tester Box. No pre-programming and calibration are required, since this box only checks continuity. 2. Fast: The cable tester box checks all the possible electrical connections in parallel at a push of a button. If a cable normally takes half an hour to test, using the Cable Tester Box will improve the speed to as little as 60 seconds to complete. 3. Versatile: Multiple cable tester boxes can be used together. As long as all the boxes share the same electrical potential, any number of connectors can be tested together.

  18. The C-terminal region of the RNA helicase CshA is required for the interaction with the degradosome and turnover of bulk RNA in the opportunistic pathogen Staphylococcus aureus.

    PubMed

    Giraud, Caroline; Hausmann, Stéphane; Lemeille, Sylvain; Prados, Julien; Redder, Peter; Linder, Patrick

    2015-01-01

    Staphylococcus aureus is a versatile opportunistic pathogen that adapts readily to a variety of different growth conditions. This adaptation requires a rapid regulation of gene expression including the control of mRNA abundance. The CshA DEAD-box RNA helicase was previously shown to be required for efficient turnover of the agr quorum sensing mRNA. Here we show by transcriptome-wide RNA sequencing and microarray analyses that CshA is required for the degradation of bulk mRNA. Moreover a subset of mRNAs is significantly stabilised in absence of CshA. Deletion of the C-terminal extension affects RNA turnover similar to the full deletion of the cshA gene. In accordance with RNA decay data, the C-terminal region of CshA is required for an RNA-independent interaction with components of the RNA degradation machinery. The C-terminal truncation of CshA reduces its ATPase activity and this reduction cannot be compensated at high RNA concentrations. Finally, the deletion of the C-terminal extension does affect growth at low temperatures, but to a significantly lesser degree than the full deletion, indicating that the core of the helicase can assume a partial function and opening the possibility that CshA is involved in different cellular processes.

  19. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 5 2012-01-01 2012-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  20. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 5 2013-01-01 2013-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  1. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 5 2011-01-01 2011-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  2. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 5 2010-01-01 2010-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  3. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 5 2014-01-01 2014-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  4. 32 CFR 632.4 - Deadly force.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 4 2010-07-01 2010-07-01 true Deadly force. 632.4 Section 632.4 National... INVESTIGATIONS USE OF FORCE BY PERSONNEL ENGAGED IN LAW ENFORCEMENT AND SECURITY DUTIES § 632.4 Deadly force. (a) Deadly force is destructive physical force directed against a person or persons (e.g., firing a...

  5. 32 CFR 632.4 - Deadly force.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 4 2011-07-01 2011-07-01 false Deadly force. 632.4 Section 632.4 National... INVESTIGATIONS USE OF FORCE BY PERSONNEL ENGAGED IN LAW ENFORCEMENT AND SECURITY DUTIES § 632.4 Deadly force. (a) Deadly force is destructive physical force directed against a person or persons (e.g., firing a...

  6. Electronic fingerprinting of the dead.

    PubMed

    Rutty, G N; Stringer, K; Turk, E E

    2008-01-01

    To date, a number of methods exist for the capture of fingerprints from cadavers that can then be used in isolation as a primary method for the identification of the dead. We report the use of a handheld, mobile wireless unit used in conjunction with a personal digital assistant (PDA) device for the capture of fingerprints from the dead. We also consider a handheld single-digit fingerprint scanner that utilises a USB laptop connection for the electronic capture of cadaveric fingerprints. Both are single-operator units that, if ridge detail is preserved, can collect a 10-set of finger pad prints in approximately 45 and 90 s, respectively. We present our observations on the restrictions as to when such devices can be used with cadavers. We do, however, illustrate that the images are of sufficient quality to allow positive identification from finger pad prints of the dead. With the development of mobile, handheld, biometric, PDA-based units for the police, we hypothesize that, under certain circumstances, devices such as these could be used for the accelerated acquisition of fingerprint identification data with the potential for rapid near-patient identification in the future.

  7. Single-molecule FRET reveals nucleotide-driven conformational changes in molecular machines and their link to RNA unwinding and DNA supercoiling.

    PubMed

    Klostermeier, Dagmar

    2011-04-01

    Many complex cellular processes in the cell are catalysed at the expense of ATP hydrolysis. The enzymes involved bind and hydrolyse ATP and couple ATP hydrolysis to the catalysed process via cycles of nucleotide-driven conformational changes. In this review, I illustrate how smFRET (single-molecule fluorescence resonance energy transfer) can define the underlying conformational changes that drive ATP-dependent molecular machines. The first example is a DEAD-box helicase that alternates between two different conformations in its catalytic cycle during RNA unwinding, and the second is DNA gyrase, a topoisomerase that undergoes a set of concerted conformational changes during negative supercoiling of DNA.

  8. Upstream box/TATA box order is the major determinant of the direction of transcription.

    PubMed Central

    Xu, L C; Thali, M; Schaffner, W

    1991-01-01

    Mammalian gene promoters for transcription by RNA polymerase II are typically organized in the following order: upstream sequence motif(s)/TATA box/initiation site. Here we report studies in which the order, orientation and DNA sequences of these three elements are varied to determine how these affect polarity of transcription. We have constructed promoters with an 'octamer' upstream sequence ATTTGCAT (or its complement ATGCAAAT) in combination with several different TATA boxes and initiation (cap) sites, and tested these promoters in transfection experiments with cultured cells. TATA boxes derived from the adenovirus major late promoter (TATAAAA), immunoglobulin kappa light chain (TTATATA) and heavy chain (TAAATATA) promoter functioned equally well or even better when inverted. Only the beta-globin TATA box (CATAAAA) was poorly active when inverted. In addition, a symmetrical TATA box (TATATATA) derived from a casein gene was very active. Our results suggest that the asymmetry of most TATA boxes (consensus TATAAAA) is not a primary determinant of the polarity of transcription. We also found that the initiation (cap) site, which usually consists of an adenine embedded in a pyrimidine-rich region (PyPyCAPyPyPyPyPy), was permissive towards sequence alterations; even a randomly composed sequence worked well. However, an inverted, hence purine-rich, cap site reduced transcript levels to 1/7th, as did an oligo G sequence. Irrespective of the presence of a cap site, the configuration: 'TATA box/octamer' yielded a strong leftward, rather than rightward transcription. From this, we conclude that the polarity of transcription is primarily determined by the linear order of an upstream sequence relative to a TATA box, rather than by the individual orientations of either of these two elements. Images PMID:1762900

  9. Thinking "Inside" the Box

    ERIC Educational Resources Information Center

    Jeffries, Carolyn

    2011-01-01

    The authors conducted a test to determine whether they could incorporate a discovery box into a preschool setting was successful. It stimulated the students' natural inquiry processes while promoting understanding of healthy foods and allowing for practice of fine-motor skills. It was easily incorporated into the curriculum and classroom space.…

  10. Drawing inside the Box

    ERIC Educational Resources Information Center

    Franklin, Ranella

    2007-01-01

    When working with very young children and/or students with special needs, it is beneficial for teachers to think "outside the box" in order to preserve and enhance a child's natural curiosity. In an effort to teach young children to control their drawing tools, they are often presented with coloring book-type pages and instructed to "stay inside…

  11. EPA ExpoBox

    EPA Pesticide Factsheets

    EPA ExpoBox is a toolbox for exposure assessors. Its purpose is to provide a compendium of exposure assessment and risk characterization tools that will present comprehensive step-by-step guidance and links to relevant exposure assessment data bases, mode

  12. Mystery Box Marvels

    ERIC Educational Resources Information Center

    Santos, Joel; Centurio, Tina

    2012-01-01

    What happens in the first week of school could very well set the stage for the rest of the school year. Setting high standards for science activities based in inquiry can start on the first day of science class and develop as the year unfolds. With the use of simple, readily available, inexpensive materials, an efficient mystery box lesson can be…

  13. The Idea Box.

    ERIC Educational Resources Information Center

    National Association for the Education of Young Children, Washington, DC.

    Five pamphlets offer helpful ideas and instructions on teacher planning, learning environments, teaching with nature, a creative curriculum, and ideas for administrators in "The Idea Box," compiled by members of the Austin Association for the Education of Young Children. Each pamphlet contains useful information for working with young children.…

  14. Shoe Box Circuits

    ERIC Educational Resources Information Center

    Sandifer, Cody

    2009-01-01

    Students' eyes grow wide with wonder as they get a motor to work or make a bulb light for the first time. As these daunting feats of electrical engineering remind us, teaching electricity is invariably rewarding and worthwhile. In this inquiry-based science project, elementary students work in pairs to design and wire a shoe box "room" that meets…

  15. Teaching with Box Tops.

    ERIC Educational Resources Information Center

    Raiser, Lynne; D'Zamko, Mary Elizabeth

    1984-01-01

    Using environmental materials (such as the phone book and placemats from fast food restaurants) can be a motivating way to teach learning disabled students skills and concepts, as shown in an approach to reading, math, science and nutrition, and social studies instruction using a JELL-O brand gelatin box. (CL)

  16. Hydrophobic, Porous Battery Boxes

    NASA Technical Reports Server (NTRS)

    Bragg, Bobby J.; Casey, John E., Jr.

    1995-01-01

    Boxes made of porous, hydrophobic polymers developed to contain aqueous potassium hydroxide electrolyte solutions of zinc/air batteries while allowing air to diffuse in as needed for operation. Used on other types of batteries for in-cabin use in which electrolytes aqueous and from which gases generated during operation must be vented without allowing electrolytes to leak out.

  17. Cereal Box Totems.

    ERIC Educational Resources Information Center

    Jones, AnnMarie

    2002-01-01

    Presents a multicultural project used with fourth-grade students in which they created a three-dimensional totem pole using leftover cereal boxes. Discusses in detail how to create the totem pole. Explains that students learned about Northwest American Indians in class. (CMK)

  18. "Can" the Black Box

    ERIC Educational Resources Information Center

    Lestingi, Francis S.

    1975-01-01

    Describes the use of the "Arcane (mysterious) Can" which is a "tin" can which is permanently sealed, both air- and water-tight, by means of a home canning device. The canning procedure permits the use of a large variety of materials which can not be utilized in the ordinary mystery box. This Can activity is valuable for…

  19. Looking Southwest at Reactor Box Furnaces With Reactor Boxes and ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Looking Southwest at Reactor Box Furnaces With Reactor Boxes and Repossessed Uranium in Recycle Recovery Building - Hematite Fuel Fabrication Facility, Recycle Recovery Building, 3300 State Road P, Festus, Jefferson County, MO

  20. 6. VIEW OF INTERIOR GLOVE BOX DURING CONSTRUCTION. GLOVE BOXES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. VIEW OF INTERIOR GLOVE BOX DURING CONSTRUCTION. GLOVE BOXES CONTAINED ALL PRODUCTION OPERATIONS AND WERE INTERCONNECTED BY CONVEYORS. (9/21/59) - Rocky Flats Plant, Plutonium Fabrication, Central section of Plant, Golden, Jefferson County, CO

  1. A gating mechanism for Pi release governs the mRNA unwinding by eIF4AI during translation initiation.

    PubMed

    Lu, Junyan; Jiang, Chenxiao; Li, Xiaojing; Jiang, Lizhi; Li, Zengxia; Schneider-Poetsch, Tilman; Liu, Jianwei; Yu, Kunqian; Liu, Jun O; Jiang, Hualiang; Luo, Cheng; Dang, Yongjun

    2015-12-02

    Eukaryotic translation initiation factor eIF4AI, the founding member of DEAD-box helicases, undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during translation initiation. However, the mechanism of its coupled enzymatic activities remains unclear. Here we report that a gating mechanism for Pi release controlled by the inter-domain linker of eIF4AI regulates the coupling between ATP hydrolysis and RNA unwinding. Molecular dynamic simulations and experimental results revealed that, through forming a hydrophobic core with the conserved SAT motif of the N-terminal domain and I357 from the C-terminal domain, the linker gated the release of Pi from the hydrolysis site, which avoided futile hydrolysis cycles of eIF4AI. Further mutagenesis studies suggested this linker also plays an auto-inhibitory role in the enzymatic activity of eIF4AI, which may be essential for its function during translation initiation. Overall, our results reveal a novel regulatory mechanism that controls eIF4AI-mediated mRNA unwinding and can guide further mechanistic studies on other DEAD-box helicases.

  2. A gating mechanism for Pi release governs the mRNA unwinding by eIF4AI during translation initiation

    PubMed Central

    Lu, Junyan; Jiang, Chenxiao; Li, Xiaojing; Jiang, Lizhi; Li, Zengxia; Schneider-Poetsch, Tilman; Liu, Jianwei; Yu, Kunqian; Liu, Jun O.; Jiang, Hualiang; Luo, Cheng; Dang, Yongjun

    2015-01-01

    Eukaryotic translation initiation factor eIF4AI, the founding member of DEAD-box helicases, undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during translation initiation. However, the mechanism of its coupled enzymatic activities remains unclear. Here we report that a gating mechanism for Pi release controlled by the inter-domain linker of eIF4AI regulates the coupling between ATP hydrolysis and RNA unwinding. Molecular dynamic simulations and experimental results revealed that, through forming a hydrophobic core with the conserved SAT motif of the N-terminal domain and I357 from the C-terminal domain, the linker gated the release of Pi from the hydrolysis site, which avoided futile hydrolysis cycles of eIF4AI. Further mutagenesis studies suggested this linker also plays an auto-inhibitory role in the enzymatic activity of eIF4AI, which may be essential for its function during translation initiation. Overall, our results reveal a novel regulatory mechanism that controls eIF4AI-mediated mRNA unwinding and can guide further mechanistic studies on other DEAD-box helicases. PMID:26464436

  3. "Dead quasars" in nearby galaxies?

    PubMed

    Rees, M J

    1990-02-16

    The nuclei of some galaxies undergo violent activity, quasars being the most extreme instances of this phenomenon. Such activity is probably short-lived compared to galactic lifetimes, and was most prevalent when the universe was only about one-fifth of its present age. A massive black hole seems the inevitable end point of such activity, and dead quasars should greatly outnumber active ones. In recent years, studies of stellar motions in the cores of several nearby galaxies indicate the presence of central dark masses which could be black holes. This article discusses how such evidence might be corroborated, and the potential implications for our understanding of active galaxies and black holes.

  4. Ocular complications of boxing

    PubMed Central

    Bianco, M; Vaiano, A; Colella, F; Coccimiglio, F; Moscetti, M; Palmieri, V; Focosi, F; Zeppilli, P; Vinger, P

    2005-01-01

    Objectives: To investigate the prevalence of ocular injuries in a large population of boxers over a period of 16 years, in particular, the most severe lesions that may be vision threatening. Methods: Clinical records of the medical archive of the Italian Boxing Federation were analysed. A total of 1032 boxers were examined from February 1982 to October 1998. A complete ophthalmological history was available for 956, who formed the study population (a total of 10 697 examinations). The following data were collected: age when started boxing; duration of competitive boxing career (from the date of the first bout); weight category; a thorough ocular history. The following investigations were carried out: measurement of visual acuity and visual fields, anterior segment inspection, applanation tonometry, gonioscopy, and examination of ocular fundus. Eighty age matched healthy subjects, who had never boxed, formed the control group. Results: Of the 956 boxers examined, 428 were amateur (44.8%) and 528 professional (55.2%). The median age at first examination was 23.1 (4.3) years (range 15–36). The prevalence of conjunctival, corneal, lenticular, vitreal, ocular papilla, and retinal alterations in the study population was 40.9% compared with 3.1% in the control group (p⩽0.0001). The prevalence of serious ocular findings (angle, lens, macula, and peripheral retina alterations) was 5.6% in boxers and 3.1% in controls (NS). Conclusions: Boxing does not result in a higher prevalence of severe ocular lesions than in the general population. However, the prevalence of milder lesions (in particular with regard to the conjunctiva and cornea) is noteworthy, justifying the need for adequate ophthalmological surveillance. PMID:15665199

  5. Unzippers, Resolvers and Sensors: A Structural and Functional Biochemistry Tale of RNA Helicases

    PubMed Central

    Leitão, Ana Lúcia; Costa, Marina C.; Enguita, Francisco J.

    2015-01-01

    The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes. PMID:25622248

  6. Hermit Points on a Box

    ERIC Educational Resources Information Center

    Hess, Richard; Grinstead, Charles; Grindstead, Marshall; Bergstrand, Deborah

    2008-01-01

    Suppose that we are given a rectangular box in 3-space. Given any two points on the surface of this box, we can define the surface distance between them to be the length of the shortest path between them on the surface of the box. This paper determines the pairs of points of maximum surface distance for all boxes. It is often the case that these…

  7. Making Connections with Memory Boxes.

    ERIC Educational Resources Information Center

    Whatley, April

    2000-01-01

    Addresses the use of children's literature within the social studies classroom on the topic of memory boxes. Includes discussions of four books: (1) "The Littlest Angel" (Charles Tazewell); (2) "The Hundred Penny Box" (Sharon Bell Mathis); (3) "Wilfrid Gordon McDonald Partridge" (Mem Fox); and (4) "The Memory Box" (Mary Bahr). (CMK)

  8. Multicultural and Nonsexist Prop Boxes.

    ERIC Educational Resources Information Center

    Boutte, Gloria S.; And Others

    1996-01-01

    Discusses how prop boxes enhance learning and are resources in multicultural and nonsexist primary education, focusing on play, experimentation, and cooperation. Examines integration of prop boxes into the curricula and activities, and presents examples of generic and specific multicultural prop boxes that incorporate art, music, foods,…

  9. Learning with Box Kernels.

    PubMed

    Melacci, Stefano; Gori, Marco

    2013-04-12

    Supervised examples and prior knowledge on regions of the input space have been profitably integrated in kernel machines to improve the performance of classifiers in different real-world contexts. The proposed solutions, which rely on the unified supervision of points and sets, have been mostly based on specific optimization schemes in which, as usual, the kernel function operates on points only. In this paper, arguments from variational calculus are used to support the choice of a special class of kernels, referred to as box kernels, which emerges directly from the choice of the kernel function associated with a regularization operator. It is proven that there is no need to search for kernels to incorporate the structure deriving from the supervision of regions of the input space, since the optimal kernel arises as a consequence of the chosen regularization operator. Although most of the given results hold for sets, we focus attention on boxes, whose labeling is associated with their propositional description. Based on different assumptions, some representer theorems are given which dictate the structure of the solution in terms of box kernel expansion. Successful results are given for problems of medical diagnosis, image, and text categorization.

  10. Learning with box kernels.

    PubMed

    Melacci, Stefano; Gori, Marco

    2013-11-01

    Supervised examples and prior knowledge on regions of the input space have been profitably integrated in kernel machines to improve the performance of classifiers in different real-world contexts. The proposed solutions, which rely on the unified supervision of points and sets, have been mostly based on specific optimization schemes in which, as usual, the kernel function operates on points only. In this paper, arguments from variational calculus are used to support the choice of a special class of kernels, referred to as box kernels, which emerges directly from the choice of the kernel function associated with a regularization operator. It is proven that there is no need to search for kernels to incorporate the structure deriving from the supervision of regions of the input space, because the optimal kernel arises as a consequence of the chosen regularization operator. Although most of the given results hold for sets, we focus attention on boxes, whose labeling is associated with their propositional description. Based on different assumptions, some representer theorems are given that dictate the structure of the solution in terms of box kernel expansion. Successful results are given for problems of medical diagnosis, image, and text categorization.

  11. Structural dynamics of the box C/D RNA kink-turn and its complex with proteins: the role of the A-minor 0 interaction, long-residency water bridges, and structural ion-binding sites revealed by molecular simulations.

    PubMed

    Spacková, Nad'a; Réblová, Kamila; Sponer, Jirí

    2010-08-19

    Kink-turns (K-turns) are recurrent elbow-like RNA motifs that participate in protein-assisted RNA folding and contribute to RNA dynamics. We carried out a set of molecular dynamics (MD) simulations using parm99 and parmbsc0 force fields to investigate structural dynamics of the box C/D RNA and its complexes with two proteins: native archaeal L7ae protein and human 15.5 kDa protein, originally bound to very similar structure of U4 snRNA. The box C/D RNA forms K-turn with A-minor 0 tertiary interaction between its canonical (C) and noncanonical (NC) stems. The local K-turn architecture is thus different from the previously studied ribosomal K-turns 38 and 42 having A-minor I interaction. The simulations reveal visible structural dynamics of this tertiary interaction involving altogether six substates which substantially contribute to the elbow-like flexibility of the K-turn. The interaction can even temporarily shift to the A-minor I type pattern; however, this is associated with distortion of the G/A base pair in the NC-stem of the K-turn. The simulations show reduction of the K-turn flexibility upon protein binding. The protein interacts with the apex of the K-turn and with the NC-stem. The protein-RNA interface includes long-residency hydration sites. We have also found long-residency hydration sites and major ion-binding sites associated with the K-turn itself. The overall topology of the K-turn remains stable in all simulations. However, in simulations of free K-turn, we observed instability of the key C16(O2')-A7(N1) H-bond, which is a signature interaction of K-turns and which was visibly more stable in simulations of K-turns possessing A-minor I interaction. It may reflect either some imbalance of the force field or it may be a correct indication of early stages of unfolding since this K-turn requires protein binding for its stabilization. Interestingly, the 16(O2')-7(N1) H- bond is usually not fully lost since it is replaced by a water bridge with a tightly

  12. Molecular Mechanism of MicroRNA-200c Regulating Transforming Growth Factor-β (TGF-β)/SMAD Family Member 3 (SMAD3) Pathway by Targeting Zinc Finger E-Box Binding Homeobox 1 (ZEB1) in Hypospadias in Rats

    PubMed Central

    Qian, Chong; Dang, Xiangyang; Wang, Xianglin; Xu, Wei; Pang, Guijian; Chen, Yifeng; Liu, Chengbei

    2016-01-01

    Background The aim of this study was to explore effects of microRNA-200c regulating TGF-β/Smad3 pathway by targeting Zeb1 on the occurrence and development of hypospadias and to evaluate the relationship between microRNA-200c and occurrence of hypospadias. Material/Methods Pregnant rats with a gestational age of 12 days were allocated into 2 groups; one received gavage of DEHP-contained soybean oil (1 ml/day, 8 days; Group A) and the other had gavage of normal soybean oil (1 ml/day, 8 days; Group B). Baby rats with hypospadias from Group A were assigned to the model group (n=20) and healthy baby rats from Group B were assigned to the control group (n=20). Real-time quantitative polymerase chain reaction (qRT-PCR), immunohistochemistry and Western blot analysis were performed to detect microRNA-200c, Zeb1, TGF-β, and Smad3 mRNA and protein expressions in the model group (n=20) and the control group (n=20). The relationship between microRNA-200c and Zeb1 was detected using a dual-luciferase reporter gene experiment. After the in vitro intervention experiment in fetal rat penises, Western blot was used to detect the expression of Zeb1, TGF-β, and Smad3. Results In the model group, microRNA-200c was expressed at a low level, and microRNA-200c expression in control group was 2.1 times higher than in the model group (P<0.05). When compared with the control group, mRNA expressions, protein expressions, and positive rates of Zeb1, TGF-β, and Smad3 were higher in the model group (all P<0.01). Luciferase gene report determined that Zeb1 is a target gene of microRNA-200c. The in vitro intervention experiment in fetal rat penises found that a high concentration of microRNA-200c inhibited hypospadias occurrence by suppressing the expression of Zeb1, TGF-β, and Smad3. Conclusions MicroRNA-200c was expressed in hypospadias penis tissues at low levels and was negatively correlated with Zeb1 expression. MicroRNA-200c up-regulated Zeb1 expression to regulate the TGF-β/Smad3

  13. More Dead than Dead: Perceptions of Persons in the Persistent Vegetative State

    ERIC Educational Resources Information Center

    Gray, Kurt; Knickman, T. Anne; Wegner, Daniel M.

    2011-01-01

    Patients in persistent vegetative state (PVS) may be biologically alive, but these experiments indicate that people see PVS as a state curiously more dead than dead. Experiment 1 found that PVS patients were perceived to have less mental capacity than the dead. Experiment 2 explained this effect as an outgrowth of afterlife beliefs, and the…

  14. Mortality due to infectious hematopoietic necrosis of sockeye salmon (Oncorhynchus nerka) fry in streamside egg incubation boxes

    USGS Publications Warehouse

    Mulcahy, D.; Pascho, R.J.; Jenes, C.K.

    1983-01-01

    Infectious hematopoietic necrosis virus caused mortality of sockeye salmon (Oncorhynchus nerka) in streamside egg incubation boxes. Virus was not detectable in eggs or alevins; its first isolation coincided with the appearance of dead fish in a trap on the outflow from the box. Mortality due to the virus did not occur in every egg box studied. However, when fry from the boxes were held in the laboratory, epizootics began as much as 3 wk later, with total mortality exceeding 90%. More than 96% of the dead fry had titers exceeding 105 plaque-forming units per gram. The peak incidence of virus in fry migrating in the river coincided with the arrival of hatchery-produced fry, although some fry believed to have been produced by natural spawning were also infected.Englis

  15. There's Life in Those Dead Logs!

    ERIC Educational Resources Information Center

    Biggs, Devin; Miller, Todd; Hall, Dee

    2006-01-01

    Although it is unspectacular in appearance, dead wood is one of the most ecologically important resources in forests. Fallen logs, dead standing trees, stumps, and even cavities in live trees fulfill a wide range of roles. Prominent among these is that they provide habitat for many organisms, especially insects. Fourth-grade students at Fox…

  16. 42 CFR 71.55 - Dead bodies.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Dead bodies. 71.55 Section 71.55 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES QUARANTINE, INSPECTION, LICENSING FOREIGN QUARANTINE Importations § 71.55 Dead bodies. The remains of a person who died of a communicable...

  17. 49 CFR 236.798 - Section, dead.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 4 2013-10-01 2013-10-01 false Section, dead. 236.798 Section 236.798 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... Section, dead. A section of track, either within a track circuit or between two track circuits, the...

  18. 49 CFR 236.798 - Section, dead.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Section, dead. 236.798 Section 236.798 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... Section, dead. A section of track, either within a track circuit or between two track circuits, the...

  19. 49 CFR 236.798 - Section, dead.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 4 2012-10-01 2012-10-01 false Section, dead. 236.798 Section 236.798 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... Section, dead. A section of track, either within a track circuit or between two track circuits, the...

  20. 49 CFR 236.798 - Section, dead.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 4 2011-10-01 2011-10-01 false Section, dead. 236.798 Section 236.798 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... Section, dead. A section of track, either within a track circuit or between two track circuits, the...

  1. 42 CFR 71.55 - Dead bodies.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Dead bodies. 71.55 Section 71.55 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES QUARANTINE, INSPECTION, LICENSING FOREIGN QUARANTINE Importations § 71.55 Dead bodies. The remains of a person who died of a communicable...

  2. 42 CFR 71.55 - Dead bodies.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Dead bodies. 71.55 Section 71.55 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES QUARANTINE, INSPECTION, LICENSING FOREIGN QUARANTINE Importations § 71.55 Dead bodies. The remains of a person who died of a communicable...

  3. 42 CFR 71.55 - Dead bodies.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Dead bodies. 71.55 Section 71.55 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES QUARANTINE, INSPECTION, LICENSING FOREIGN QUARANTINE Importations § 71.55 Dead bodies. The remains of a person who died of a communicable...

  4. 49 CFR 236.798 - Section, dead.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 4 2014-10-01 2014-10-01 false Section, dead. 236.798 Section 236.798 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... Section, dead. A section of track, either within a track circuit or between two track circuits, the...

  5. 42 CFR 71.55 - Dead bodies.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Dead bodies. 71.55 Section 71.55 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES QUARANTINE, INSPECTION, LICENSING FOREIGN QUARANTINE Importations § 71.55 Dead bodies. The remains of a person who died of a communicable...

  6. 49 CFR 236.55 - Dead section; maximum length.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 4 2011-10-01 2011-10-01 false Dead section; maximum length. 236.55 Section 236... Instructions: All Systems Track Circuits § 236.55 Dead section; maximum length. Where dead section exceeds 35... over such dead section is less than 35 feet, the maximum length of the dead section shall not...

  7. 49 CFR 236.55 - Dead section; maximum length.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 4 2013-10-01 2013-10-01 false Dead section; maximum length. 236.55 Section 236... Instructions: All Systems Track Circuits § 236.55 Dead section; maximum length. Where dead section exceeds 35... over such dead section is less than 35 feet, the maximum length of the dead section shall not...

  8. 49 CFR 236.55 - Dead section; maximum length.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Dead section; maximum length. 236.55 Section 236... Instructions: All Systems Track Circuits § 236.55 Dead section; maximum length. Where dead section exceeds 35... over such dead section is less than 35 feet, the maximum length of the dead section shall not...

  9. 49 CFR 236.55 - Dead section; maximum length.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 4 2014-10-01 2014-10-01 false Dead section; maximum length. 236.55 Section 236... Instructions: All Systems Track Circuits § 236.55 Dead section; maximum length. Where dead section exceeds 35... over such dead section is less than 35 feet, the maximum length of the dead section shall not...

  10. 49 CFR 236.55 - Dead section; maximum length.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 4 2012-10-01 2012-10-01 false Dead section; maximum length. 236.55 Section 236... Instructions: All Systems Track Circuits § 236.55 Dead section; maximum length. Where dead section exceeds 35... over such dead section is less than 35 feet, the maximum length of the dead section shall not...

  11. Small, Lightweight, Collapsible Glove Box

    NASA Technical Reports Server (NTRS)

    James, Jerry

    2009-01-01

    A small, lightweight, collapsible glove box enables its user to perform small experiments and other tasks. Originally intended for use aboard a space shuttle or the International Space Station (ISS), this glove box could also be attractive for use on Earth in settings in which work space or storage space is severely limited and, possibly, in which it is desirable to minimize weight. The development of this glove box was prompted by the findings that in the original space-shuttle or ISS setting, (1) it was necessary to perform small experiments in a large general-purpose work station, so that, in effect, they occupied excessive space; and it took excessive amounts of time to set up small experiments. The design of the glove box reflects the need to minimize the space occupied by experiments and the time needed to set up experiments, plus the requirement to limit the launch weight of the box and the space needed to store the box during transport into orbit. To prepare the glove box for use, the astronaut or other user has merely to insert hands through the two fabric glove ports in the side walls of the box and move two hinges to a locking vertical position (see figure). The user could do this while seated with the glove box on the user fs lap. When stowed, the glove box is flat and has approximately the thickness of two pieces of 8-in. (.20 cm) polycarbonate.

  12. MicroRNAs as regulators and mediators of forkhead box transcription factors function in human cancers.

    PubMed

    Li, Chen; Zhang, Kai; Chen, Jing; Chen, Longbang; Wang, Rui; Chu, Xiaoyuan

    2016-12-16

    Evidence has shown that microRNAs are widely implicated as indispensable components of tumor suppressive and oncogenic pathways in human cancers. Thus, identification of microRNA targets and their relevant pathways will contribute to the development of microRNA-based therapeutics. The forkhead box transcription factors regulate numerous processes including cell cycle progression, metabolism, metastasis and angiogenesis, thereby facilitating tumor initiation and progression. A complex network of protein and non-coding RNAs mediates the expression and activity of forkhead box transcription factors. In this review, we summarize the current knowledge and concepts concerning the involvement of microRNAs and forkhead box transcription factors and describe the roles of microRNAs-forkhead box axis in various disease states including tumor initiation and progression. Additionally, we describe some of the technical challenges in the use of the microRNA-forkhead box signaling pathway in cancer treatment.

  13. Raising the Dead without a Red Sea-Dead Sea project? Hydro-economics and governance

    NASA Astrophysics Data System (ADS)

    Rosenberg, D. E.

    2011-04-01

    Seven decades of extractions have dramatically reduced Jordan River flows, lowered the Dead Sea level, opened sink holes, and caused other environmental problems. The fix Jordan, Israel, and the Palestinians propose would build an expensive multipurpose conveyance project from the Red Sea to the Dead Sea that would also generate hydropower and desalinate water. This paper compares the Red-Dead project to alternatives that may also raise the Dead Sea level. Hydro-economic model results for the Jordan-Israel-Palestinian inter-tied water systems show two restoration alternatives are more economically viable than the proposed Red-Dead project. Many decentralized new supply, wastewater reuse, conveyance, conservation, and leak reduction projects and programs in each country can together increase economic benefits and reliably deliver up to 900 MCM yr-1 to the Dead Sea. Similarly, a smaller Red-Dead project that only generates hydropower can deliver large flows to the Dead Sea when the sale price of generated electricity is sufficiently high. However, for all restoration options, net benefits fall and water scarcity rises as flows to the Dead Sea increase. This finding suggests (i) each country has no individual incentive to return water to the Dead Sea, and (ii) outside institutions that seek to raise the Dead must also offer countries direct incentives to deliver water to the Sea besides building the countries new infrastructure.

  14. Raising the dead without a Red Sea-Dead Sea canal? Hydro-economics and governance

    NASA Astrophysics Data System (ADS)

    Rosenberg, D. E.

    2010-12-01

    Seven decades of extractions have dramatically reduced Jordan River flows, lowered the Dead Sea level, opened sink holes, and caused other environmental problems. The fix Jordan, Israel, and the Palestinians propose would build an expensive multipurpose canal from the Red Sea to the Dead Sea that would also generate hydropower and desalinated water. This paper compares the Red-Dead project to alternatives that may also raise the Dead Sea level. Hydro-economic model results for the Jordan-Israel-Palestinian inter-tied water systems show two restoration alternatives are more economically viable than the proposed Red-Dead project. Many decentralized new supply, wastewater reuse, conveyance, conservation, and leak reduction projects and programs in each country can together increase economic benefits and reliably deliver up to 900 MCM/year to the Dead Sea. Similarly, a smaller Red-Dead project that only generates hydropower can deliver large flows to the Dead Sea when the sale price of generated electricity is sufficiently high. However, for all restoration options, net benefits fall and water scarcity rises as flows to the Dead Sea increase. This finding suggests (i) each country has no individual incentive to return water to the Dead Sea, and (ii) outside institutions that seek to raise the Dead must also offer countries direct incentives to deliver water to the Sea besides building the countries new infrastructure.

  15. Projection optics box

    DOEpatents

    Hale, Layton C.; Malsbury, Terry; Hudyma, Russell M.; Parker, John M.

    2000-01-01

    A projection optics box or assembly for use in an optical assembly, such as in an extreme ultraviolet lithography (EUVL) system using 10-14 nm soft x-ray photons. The projection optics box utilizes a plurality of highly reflective optics or mirrors, each mounted on a precision actuator, and which reflects an optical image, such as from a mask, in the EUVL system onto a point of use, such as a target or silicon wafer, the mask, for example, receiving an optical signal from a source assembly, such as a developed from laser system, via a series of highly reflective mirrors of the EUVL system. The plurality of highly reflective optics or mirrors are mounted in a housing assembly comprised of a series of bulkheads having wall members secured together to form a unit construction of maximum rigidity. Due to the precision actuators, the mirrors must be positioned precisely and remotely in tip, tilt, and piston (three degrees of freedom), while also providing exact constraint.

  16. 46 CFR 111.81-1 - Outlet boxes and junction boxes; general.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Outlet boxes and junction boxes; general. 111.81-1... ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Outlet Boxes and Junction Boxes § 111.81-1 Outlet boxes and junction boxes; general. (a) The requirements of this subpart apply to each outlet box used with a...

  17. The Classroom Animal: Box Turtles.

    ERIC Educational Resources Information Center

    Kramer, David C.

    1986-01-01

    Provides basic information on the anatomy, physiology, behaviors, and distribution patterns of the box turtle. Offers suggestions for the turtle's care and maintenance in a classroom environment. (ML)

  18. 2. UPPER NOTTINGHAM MINE, WOODEN BOXES. BOXES ARE LOCATED APPROXIMATELY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. UPPER NOTTINGHAM MINE, WOODEN BOXES. BOXES ARE LOCATED APPROXIMATELY 10 YARDS TO THE RIGHT AND DOWNSLOPE OF THE ADIT IN ID-31-F-1. CAMERA IS POINTED EAST-SOUTHEAST. - Florida Mountain Mining Sites, Upper Nottingham Mine, West face of Florida Mountain, head of Jacobs Gulch, Silver City, Owyhee County, ID

  19. Amino acid signatures of salinity on an environmental scale with a focus on the Dead Sea.

    PubMed

    Rhodes, Matthew E; Fitz-Gibbon, Sorel T; Oren, Aharon; House, Christopher H

    2010-09-01

    The increase of the acidic nature of proteins as an adaptation to hypersalinity has been well documented within halophile isolates. Here we explore the effect of salinity on amino acid preference on an environmental scale. Via pyrosequencing, we have obtained two distinct metagenomic data sets from the Dead Sea, one from a 1992 archaeal bloom and one from the modern Dead Sea. Our data, along with metagenomes from environments representing a range of salinities, show a strong linear correlation (R(2) = 0.97) between the salinity of an environment and the ratio of acidic to basic amino acids encoded by its inhabitants. Using the amino acid composition of putative protein-encoding reads and the results of 16S rRNA amplicon sequencing, we differentiate recovered sequences representing microorganisms indigenous to the Dead Sea from lateral gene transfer events and foreign DNA. Our methods demonstrate lateral gene transfer events between a halophilic archaeon and relatives of the thermophilic bacterial genus Thermotoga and suggest the presence of indigenous Dead Sea representatives from 10 traditionally non-hyperhalophilic bacterial lineages. The work suggests the possibility that amino acid bias of hypersaline environments might be preservable in fossil DNA or fossil amino acids, serving as a proxy for the salinity of an ancient environment. Finally, both the amino acid profile of the 2007 Dead Sea metagenome and the V9 amplicon library support the conclusion that the dominant microorganism inhabiting the Dead Sea is most closely related to a thus far uncultured relative of an alkaliphilic haloarchaeon.

  20. ACYSYS in a box

    SciTech Connect

    Briegel, C.; Finstrom, D.; Hendricks, B.; King, C.; Lackey, S.; Neswold, R.; Nicklaus, D.; Patrick, J.; Petrov, A.; Rechenmacher, R.; Schumann, C.; /Fermilab

    2011-11-01

    The Accelerator Control System at Fermilab has evolved to enable this relatively large control system to be encapsulated into a 'box' such as a laptop. The goal was to provide a platform isolated from the 'online' control system. This platform can be used internally for making major upgrades and modifications without impacting operations. It also provides a standalone environment for research and development including a turnkey control system for collaborators. Over time, the code base running on Scientific Linux has enabled all the salient features of the Fermilab's control system to be captured in an off-the-shelf laptop. The anticipated additional benefits of packaging the system include improved maintenance, reliability, documentation, and future enhancements.

  1. More box codes

    NASA Technical Reports Server (NTRS)

    Solomon, G.

    1992-01-01

    A new investigation shows that, starting from the BCH (21,15;3) code represented as a 7 x 3 matrix and adding a row and column to add even parity, one obtains an 8 x 4 matrix (32,15;8) code. An additional dimension is obtained by specifying odd parity on the rows and even parity on the columns, i.e., adjoining to the 8 x 4 matrix, the matrix, which is zero except for the fourth column (of all ones). Furthermore, any seven rows and three columns will form the BCH (21,15;3) code. This box code has the same weight structure as the quadratic residue and BCH codes of the same dimensions. Whether there exists an algebraic isomorphism to either code is as yet unknown.

  2. Impedance Measurement Box

    ScienceCinema

    Christophersen, Jon

    2016-07-12

    Energy storage devices, primarily batteries, are now more important to consumers, industries and the military. With increasing technical complexity and higher user expectations, there is also a demand for highly accurate state-of-health battery assessment techniques. IMB incorporates patented, proprietary, and tested capabilities using control software and hardware that can be part of an embedded monitoring system. IMB directly measures the wideband impedance spectrum in seconds during battery operation with no significant impact on service life. It also can be applied to batteries prior to installation, confirming health before entering active service, as well as during regular maintenance. For more information about this project, visit http://www.inl.gov/rd100/2011/impedance-measurement-box/

  3. Single-molecule kinetics of the eukaryotic initiation factor 4AI upon RNA unwinding.

    PubMed

    Sun, Yingjie; Atas, Evrim; Lindqvist, Lisa M; Sonenberg, Nahum; Pelletier, Jerry; Meller, Amit

    2014-07-08

    The eukaryotic translation initiation factor 4AI (eIF4AI) is the prototypical DEAD-box RNA helicase. It has a "dumbbell" structure consisting of two domains connected by a flexible linker. Previous studies demonstrated that eIF4AI, in conjunction with eIF4H, bind to loop structures and repetitively unwind RNA hairpins. Here, we probe the conformational dynamics of eIF4AI in real time using single-molecule FRET. We demonstrate that eIF4AI/eIF4H complex can repetitively unwind RNA hairpins by transitioning between an eIF4AI "open" and a "closed" conformation using the energy derived from ATP hydrolysis. Our experiments directly track the conformational changes in the catalytic cycle of eIF4AI and eIF4H, and this correlates precisely with the kinetics of RNA unwinding. Furthermore, we show that the small-molecule eIF4A inhibitor hippuristanol locks eIF4AI in the closed conformation, thus efficiently inhibiting RNA unwinding. These results indicate that the large conformational changes undertaken by eIF4A during the helicase catalytic cycle are rate limiting.

  4. DDX5 and its associated lncRNA Rmrp modulate Th17 cell effector functions

    PubMed Central

    Huang, Wendy; Thomas, Benjamin; Flynn, Ryan A.; Gavzy, Samuel J.; Wu, Lin; Kim, Sangwon V.; Hall, Jason A.; Miraldi, Emily R.; Ng, Charles P.; Rigo, Frank; Meadows, Sarah; Montoya, Nina R.; Herrera, Natalia G.; Domingos, Ana I.; Rastinejad, Fraydoon; Myers, Richard M.; Fuller-Pace, Frances V.; Bonneau, Richard; Chang, Howard Y.; Acuto, Oreste; Littman, Dan R.

    2016-01-01

    Th17 lymphocytes protect mucosal barriers from infections, but also contribute to multiple chronic inflammatory diseases. Their differentiation is controlled by RORγt, a ligand-regulated nuclear receptor. We identified the DEAD-box RNA helicase DDX5 as a RORγt partner that coordinates transcription of selective Th17 genes and is required for Th17-mediated inflammatory pathologies. Surprisingly, the ability of DDX5 to interact with RORγt and co-activate its targets depends on its intrinsic RNA helicase activity and binding of a conserved nuclear long noncoding RNA (lncRNA), Rmrp, which is mutated in Cartilage-Hair Hypoplasia (CHH) patients. A targeted Rmrp mutation in mice, corresponding to one in CHH patients, abrogated the lncRNA’s chromatin recruitment, ability to potentiate DDX5-RORγt interaction and RORγt target gene transcription. Elucidation of the link between Rmrp and the DDX5-RORγt complex reveals a role for RNA helicases and lncRNAs in tissue-specific transcriptional regulation and promises new opportunities for therapeutic intervention in Th17-dependent diseases. PMID:26675721

  5. The dead donor rule: a defense.

    PubMed

    Birch, Samuel C M

    2013-08-01

    Miller, Truog, and Brock have recently argued that the "dead donor rule," the requirement that donors be determined to be dead before vital organs are procured for transplantation, cannot withstand ethical scrutiny. In their view, the dead donor rule is inconsistent with existing life-saving practices of organ transplantation, lacks a cogent ethical rationale, and is not necessary for maintenance of public trust in organ transplantation. In this paper, the second of these claims will be evaluated. (The first and third are not addressed.) The claim that the dead donor rule lacks a cogent ethical rationale will be shown to be an expression of the contemporary rejection of the moral significance of the traditional distinction between killing and allowing to die. The moral significance of this traditional distinction, and the associated norm that doctors should not kill their patients, will be defended, and this critique of it shown to be unsuccessful.

  6. The Dead Sea transform fault system

    NASA Astrophysics Data System (ADS)

    Girdler, R. W.

    1990-08-01

    A new map showing the major features of the Dead Sea transform fault system based on seismicity, satellite imagery, geological maps and bathymetric charts is presented. Special attention is given to the possible northward continuation of the transform system beneath the Mediterranean Sea near Ed Damur, south of Beirut. The map shows the Dead Sea transform system to be a series of offset, overlapping, left-lateral transform faults with a rhombochasm between each pair. The system has similarities with the equatorial fracture zones in the Atlantic Ocean. Throughout, the Dead Sea transform system is considered in its regional setting, i.e. as extending from the Red Sea spreading centre in the south to the Eurasian collision zone in the north. It is suggested that it may intersect the latter somewhere east of Cyprus making that area the northernmost termination of the Dead Sea transform system.

  7. Dead pixel replacement in LWIR microgrid polarimeters.

    PubMed

    Ratliff, Bradley M; Tyo, J Scott; Boger, James K; Black, Wiley T; Bowers, David L; Fetrow, Matthew P

    2007-06-11

    LWIR imaging arrays are often affected by nonresponsive pixels, or "dead pixels." These dead pixels can severely degrade the quality of imagery and often have to be replaced before subsequent image processing and display of the imagery data. For LWIR arrays that are integrated with arrays of micropolarizers, the problem of dead pixels is amplified. Conventional dead pixel replacement (DPR) strategies cannot be employed since neighboring pixels are of different polarizations. In this paper we present two DPR schemes. The first is a modified nearest-neighbor replacement method. The second is a method based on redundancy in the polarization measurements.We find that the redundancy-based DPR scheme provides an order-of-magnitude better performance for typical LWIR polarimetric data.

  8. How dead ends undermine power grid stability.

    PubMed

    Menck, Peter J; Heitzig, Jobst; Kurths, Jürgen; Joachim Schellnhuber, Hans

    2014-06-09

    The cheapest and thus widespread way to add new generators to a high-voltage power grid is by a simple tree-like connection scheme. However, it is not entirely clear how such locally cost-minimizing connection schemes affect overall system performance, in particular the stability against blackouts. Here we investigate how local patterns in the network topology influence a power grid's ability to withstand blackout-prone large perturbations. Employing basin stability, a nonlinear concept, we find in numerical simulations of artificially generated power grids that tree-like connection schemes--so-called dead ends and dead trees--strongly diminish stability. A case study of the Northern European power system confirms this result and demonstrates that the inverse is also true: repairing dead ends by addition of a few transmission lines substantially enhances stability. This may indicate a topological design principle for future power grids: avoid dead ends.

  9. Dead space: the physiology of wasted ventilation.

    PubMed

    Robertson, H Thomas

    2015-06-01

    An elevated physiological dead space, calculated from measurements of arterial CO2 and mixed expired CO2, has proven to be a useful clinical marker of prognosis both for patients with acute respiratory distress syndrome and for patients with severe heart failure. Although a frequently cited explanation for an elevated dead space measurement has been the development of alveolar regions receiving no perfusion, evidence for this mechanism is lacking in both of these disease settings. For the range of physiological abnormalities associated with an increased physiological dead space measurement, increased alveolar ventilation/perfusion ratio (V'A/Q') heterogeneity has been the most important pathophysiological mechanism. Depending on the disease condition, additional mechanisms that can contribute to an elevated physiological dead space measurement include shunt, a substantial increase in overall V'A/Q' ratio, diffusion impairment, and ventilation delivered to unperfused alveolar spaces.

  10. For Men, Ignoring Diabetes Can Be Deadly

    MedlinePlus

    ... of this page please turn Javascript on. Feature: Diabetes For Men, Ignoring Diabetes Can Be Deadly Past Issues / Fall 2009 Table ... Man's Guide to Living Well with Diabetes. Simpler Diabetes Care: Estimated Average Glucose (eAG) The American Diabetes ...

  11. Spirit Boxes: Expressions of Culture.

    ERIC Educational Resources Information Center

    DeMuro, Ted

    1984-01-01

    After studying the culture and art of the ancient civilizations of South America, Mesopotamia, Greece, and Egypt, secondary level art students made spirit boxes as expressions of the various cultures. How to make the boxes and how to prepare the face molds are described. (RM)

  12. Being Creative "Inside the Box"

    ERIC Educational Resources Information Center

    Tomascoff, Rocky

    2011-01-01

    Artist Joseph Cornell (1903-1972) created wonderful environments inside boxes using mostly found objects. They were often Surrealistic in nature. Some boxes were designed with glass fronts, and others were meant to be interactive with the viewer, wherein the objects could be handled. With Joseph Cornell in mind, the author introduces an art…

  13. What Makes a Better Box?

    ERIC Educational Resources Information Center

    Moyer, Richard; Everett, Susan

    2010-01-01

    Every morning, many Americans start their day with a bowl of cereal. Some spend time while they eat breakfast reading the back of the cereal box, but few consider its size, shape, and construction, or realize that it was designed by an engineer. This article describes a lesson in which students design, build, and critique cereal boxes. The lesson…

  14. Cardboard Boxes: Learning Concepts Galore!

    ERIC Educational Resources Information Center

    Warner, Laverne; Wilmoth, Linda

    2007-01-01

    Mrs. Keenan, a preschool teacher, observed her 3-year-old granddaughter Riley pull, tug, and stack piles of holiday boxes on the floor. She remembered that her child care director had suggested using boxes as a curriculum theme, but she hadn't given much thought about the idea until now. She said to herself, "I wonder if my children would be as…

  15. Dead cell counts during serum cultivation are underestimated by the fluorescent live/dead assay.

    PubMed

    Zhou, Shengda; Cui, Zhanfeng; Urban, Jill

    2011-05-01

    The live/dead fluorescent assay provides a quick method for assessing the proportion of live and dead cells in cell culture systems or tissues and is widely used. Dead cells are detected by the fluorescence produced when propidium iodide (PI) binds to DNA; PI and similar molecules are excluded from live cells but can penetrate dead cells because of their loss of membrane integrity. Here we investigated the effect of serum in the culture medium on the reliability of the method. We assessed viability of chondrocytes with/without serum using both a live/dead assay kit and also trypan blue staining. We found that after 2 days of culture, the DNA-binding dye PI could no longer detect dead cells if serum was present but they were readily detected in serum-free medium or if an inhibitor to DNase I was added to the serum-containing medium. Dead cells could be detected by trypan blue staining in all cultures. Hence dead cells are no longer detected as the DNase I present in serum degrades their DNA. DNA-binding dyes may thus not give a reliable estimate of the number of dead cells in systems that have been cultured in the presence of serum for several days.

  16. The taxonomic status of "Halobacterium marismortui" from the Dead Sea: a comparison with Halobacterium vallismortis

    NASA Technical Reports Server (NTRS)

    Oren, A.; Lau, P. P.; Fox, G. E.

    1988-01-01

    A Halobacterium strain, isolated by Ginzburg et al. from the Dead Sea in the late 1960's, often referred to as "Halobacterium marismortui" or "Halobacterium of the Dead Sea" (deposited in the American Type Culture Collection as ATCC 43049) was compared with Halobacterium (Haloarcula) vallismortis ATCC 29715. The strains appeared to be very closely related, as shown by the near identity of their 5S and 16S ribosomal RNA's, and a large number of other common properties. Distinct differences exist, however, in cell morphology, and in their potency to utilize different sugars and other compounds.

  17. Possible Role of MADS AFFECTING FLOWERING 3 and B-BOX DOMAIN PROTEIN 19 in Flowering Time Regulation of Arabidopsis Mutants with Defects in Nonsense-Mediated mRNA Decay

    PubMed Central

    Nasim, Zeeshan; Fahim, Muhammad; Ahn, Ji Hoon

    2017-01-01

    Eukaryotic cells use nonsense-mediated mRNA decay (NMD) to clear aberrant mRNAs from the cell, thus preventing the accumulation of truncated proteins. In Arabidopsis, two UP-Frameshift (UPF) proteins, UPF1 and UPF3, play a critical role in NMD. Although deficiency of UPF1 and UPF3 leads to various developmental defects, little is known about the mechanism underlying the regulation of flowering time by NMD. Here, we showed that the upf1-5 and upf3-1 mutants had a late-flowering phenotype under long-day conditions and the upf1-5 upf3-1 double mutants had an additive effect in delaying flowering time. RNA sequencing of the upf mutants revealed that UPF3 exerted a stronger effect than UPF1 in the UPF-mediated regulation of flowering time. Among genes known to regulate flowering time, FLOWERING LOCUS C (FLC) mRNA levels increased (up to 8-fold) in upf mutants, as confirmed by qPCR. The upf1-5, upf3-1, and upf1-5 upf3-1 mutants responded to vernalization, suggesting a role of FLC in delayed flowering of upf mutants. Consistent with the high FLC transcript levels and delayed flowering in upf mutants, levels of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) mRNAs were reduced in the upf mutants. However, RNA-seq did not identify an aberrant FLC transcript containing a premature termination codon (PTC), suggesting that FLC is not a direct target in the regulation of flowering time by NMD. Among flowering time regulators that act in an FLC-dependent manner, we found that MAF3, NF-YA2, NF-YA5, and TAF14 showed increased transcript levels in upf mutants. We also found that BBX19 and ATC, which act in an FLC-independent manner, showed increased transcript levels in upf mutants. An aberrant transcript containing a PTC was identified from MAF3 and BBX19 and the levels of the aberrant transcripts increased in upf mutants. Taking these results together, we propose that the late-flowering phenotype of upf mutants is mediated by at least two different

  18. Possible Role of MADS AFFECTING FLOWERING 3 and B-BOX DOMAIN PROTEIN 19 in Flowering Time Regulation of Arabidopsis Mutants with Defects in Nonsense-Mediated mRNA Decay.

    PubMed

    Nasim, Zeeshan; Fahim, Muhammad; Ahn, Ji Hoon

    2017-01-01

    Eukaryotic cells use nonsense-mediated mRNA decay (NMD) to clear aberrant mRNAs from the cell, thus preventing the accumulation of truncated proteins. In Arabidopsis, two UP-Frameshift (UPF) proteins, UPF1 and UPF3, play a critical role in NMD. Although deficiency of UPF1 and UPF3 leads to various developmental defects, little is known about the mechanism underlying the regulation of flowering time by NMD. Here, we showed that the upf1-5 and upf3-1 mutants had a late-flowering phenotype under long-day conditions and the upf1-5 upf3-1 double mutants had an additive effect in delaying flowering time. RNA sequencing of the upf mutants revealed that UPF3 exerted a stronger effect than UPF1 in the UPF-mediated regulation of flowering time. Among genes known to regulate flowering time, FLOWERING LOCUS C (FLC) mRNA levels increased (up to 8-fold) in upf mutants, as confirmed by qPCR. The upf1-5, upf3-1, and upf1-5 upf3-1 mutants responded to vernalization, suggesting a role of FLC in delayed flowering of upf mutants. Consistent with the high FLC transcript levels and delayed flowering in upf mutants, levels of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) mRNAs were reduced in the upf mutants. However, RNA-seq did not identify an aberrant FLC transcript containing a premature termination codon (PTC), suggesting that FLC is not a direct target in the regulation of flowering time by NMD. Among flowering time regulators that act in an FLC-dependent manner, we found that MAF3, NF-YA2, NF-YA5, and TAF14 showed increased transcript levels in upf mutants. We also found that BBX19 and ATC, which act in an FLC-independent manner, showed increased transcript levels in upf mutants. An aberrant transcript containing a PTC was identified from MAF3 and BBX19 and the levels of the aberrant transcripts increased in upf mutants. Taking these results together, we propose that the late-flowering phenotype of upf mutants is mediated by at least two different

  19. Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts.

    PubMed Central

    Mahbubani, M H; Bej, A K; Perlin, M; Schaefer, F W; Jakubowski, W; Atlas, R M

    1991-01-01

    A method was developed for the detection of Giardia cysts by using the polymerase chain reaction (PCR) and the giardin gene as the target. DNA amplification by PCR, using giardin DNA as the target, resulted in detection of both live and dead cysts. When giardin mRNA was used as the target, the ability to amplify cDNA by PCR depended on the mode of killing. Cysts killed by freezing were not detected by PCR when giardin mRNA was the target. Cysts killed by heating or exposure to monochloramine, however, gave positive detection signals for both DNA and giardin mRNA targets. The amount of giardin mRNA and total RNA was significantly increased in live cysts following the induction of excystation. Cysts killed by freezing, heating, or exposure to monochloramine did not show a change in RNA content. The detection of the giardin gene by PCR permits a sensitive and specific diagnosis for Giardia spp. Discrimination between live and dead cysts can be made by measuring the amounts of RNA or PCR-amplified product from the giardin mRNA target before and after the induction of excystation. Images PMID:1785923

  20. BC1 RNA: transcriptional analysis of a neural cell-specific RNA polymerase III transcript.

    PubMed Central

    Martignetti, J A; Brosius, J

    1995-01-01

    Rodent BC1 RNA represents the first example of a neural cell-specific RNA polymerase III (Pol III) transcription product. By developing a rat brain in vitro system capable of supporting Pol III-directed transcription, we showed that the rat BC1 RNA intragenic promoter elements, comprising an A box element and a variant B box element, as well as its upstream region, containing octamer-binding consensus sequences and functional TATA and proximal sequence element sites, are necessary for transcription. The BC1 B box, lacking the invariant A residue found in the consensus B boxes of tRNAs, represents a functionally related and possibly distinct promoter element. The transcriptional activity of the BC1 B box element is greatly increased, in both a BC1 RNA and a chimeric tRNA(Leu) gene construct, when the BC1 5' flanking region is present and is appropriately spaced. Moreover, a tRNA consensus B-box sequence can efficiently replace the BC1 B box only if the BC1 upstream region is removed. These interactions, identified only in a homologous in vitro system, between upstream Pol II and intragenic Pol III promoters suggest a mechanism by which the tissue-specific BC1 RNA gene and possibly other Pol III-transcribed genes can be regulated. PMID:7862155

  1. "Dead End Kids in Dead End Jobs"? Reshaping Debates on Young People in Jobs without Training

    ERIC Educational Resources Information Center

    Quinn, Jocey; Lawy, Robert; Diment, Kim

    2008-01-01

    Young people who are in "jobs without training" (JWT) are commonly seen as "dead end kids in dead end jobs". They have been identified as a problem group who need to be encouraged back into formal education and training. Following the Leitch report and the new policy goal to involve all young people in education and training up…

  2. Recruitment of Arabidopsis RNA Helicase AtRH9 to the Viral Replication Complex by Viral Replicase to Promote Turnip Mosaic Virus Replication

    PubMed Central

    Li, Yinzi; Xiong, Ruyi; Bernards, Mark; Wang, Aiming

    2016-01-01

    Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly dependent on host components to fulfill their life cycle. Recent studies have suggested that DEAD-box RNA helicases play vital roles in many aspects of RNA metabolism. To explore the possible role of the RNA helicases in viral infection, we used the Turnip mosaic virus (TuMV)-Arabidopsis pathosystem. The Arabidopsis genome encodes more than 100 putative RNA helicases (AtRH). Over 41 Arabidopsis T-DNA insertion mutants carrying genetic lesions in the corresponding 26 AtRH genes were screened for their requirement in TuMV infection. TuMV infection assays revealed that virus accumulation significantly decreased in the Arabidopsis mutants of three genes, AtRH9, AtRH26, and PRH75. In the present work, AtRH9 was further characterized. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that AtRH9 interacted with the TuMV NIb protein, the viral RNA-dependent RNA polymerase. Moreover, the subcellular distribution of AtRH9 was altered in the virus-infected cells, and AtRH9 was recruited to the viral replication complex. These results suggest that Arabidopsis AtRH9 is an important component of the TuMV replication complex, possibly recruited via its interaction with NIb. PMID:27456972

  3. Fungal life in the dead sea.

    PubMed

    Oren, Aharon; Gunde-Cimerman, Nina

    2012-01-01

    The waters of the Dead Sea currently contain about 348 g/l salts (2 M Mg(2+), 0.5 M Ca(2+), 1.5 M Na(+), 0.2 M K(+), 6.5 M Cl(-), 0.1 M Br(-)). The pH is about 6.0. After rainy winters the surface waters become diluted, triggering development of microbial blooms. The 1980 and 1992 blooms were dominated by the unicellular green alga Dunaliella and red Archaea. At least 70 species (in 26 genera) of Oomycota (Chromista), Mucoromycotina, Ascomycota, and Basidiomycota (Fungi) were isolated from near-shore localities and offshore stations, including from deep waters. Aspergillus and Eurotium were most often recovered. Aspergillus terreus, A. sydowii, A. versicolor, Eurotium herbariorum, Penicillium westlingii, Cladosporium cladosporioides, C. sphaerospermum, C. ramnotellum, and C. halotolerans probably form the stable core of the community. The species Gymnascella marismortui may be endemic. Mycelia of Dead Sea isolates of A. versicolor and Chaetomium globosum remained viable for up to 8 weeks in Dead Sea water; mycelia of other species survived for many weeks in 50% Dead Sea water. Many isolates showed a very high tolerance to magnesium salts. There is no direct proof that fungi contribute to the heterotrophic activity in the Dead Sea, but fungi may be present at least locally and temporarily, and their enzymatic activities such as amylase, protease, and cellulase may play a role in the lake's ecosystem.

  4. Can the dead be brought into disrepute?

    PubMed

    Masterton, Malin; Hansson, Mats G; Höglund, Anna T; Helgesson, Gert

    2007-01-01

    Queen Christina of Sweden was unconventional in her time, leading to hypotheses on her gender and possible hermaphroditic nature. If genetic analysis can substantiate the latter claim, could this bring the queen into disrepute 300 years after her death? Joan C. Callahan has argued that if a reputation changes, this constitutes a change only in the group of people changing their views and not in the person whose reputation it is. Is this so? This paper analyses what constitutes change and draws out the implications to the reputation of the dead. It is argued that a reputation is a relational property which can go through changes. The change is "real" for the group changing their views on Queen Christina and of a Cambridge kind for the long dead queen herself. Cambridge changes result in new properties being acquired, some of which can be of significance. Although the dead cannot go through any non-relational changes, it is possible for the dead to change properties through Cambridge changes. In this sense changes in reputation do affect the dead, and thus Queen Christina can acquire a new property, in this case possibly a worse reputation.

  5. Comparative genomic analysis of T-box regulatory systems in bacteria

    PubMed Central

    Vitreschak, Alexey G.; Mironov, Andrei A.; Lyubetsky, Vassily A.; Gelfand, Mikhail S.

    2008-01-01

    T-box antitermination is one of the main mechanisms of regulation of genes involved in amino acid metabolism in Gram-positive bacteria. T-box regulatory sites consist of conserved sequence and RNA secondary structure elements. Using a set of known T-box sites, we constructed the common pattern and used it to scan available bacterial genomes. New T-boxes were found in various Gram-positive bacteria, some Gram-negative bacteria (δ-proteobacteria), and some other bacterial groups (Deinococcales/Thermales, Chloroflexi, Dictyoglomi). The majority of T-box-regulated genes encode aminoacyl-tRNA synthetases. Two other groups of T-box-regulated genes are amino acid biosynthetic genes and transporters, as well as genes with unknown function. Analysis of candidate T-box sites resulted in new functional annotations. We assigned the amino acid specificity to a large number of candidate amino acid transporters and a possible function to amino acid biosynthesis genes. We then studied the evolution of the T-boxes. Analysis of the constructed phylogenetic trees demonstrated that in addition to the normal evolution consistent with the evolution of regulated genes, T-boxes may be duplicated, transferred to other genes, and change specificity. We observed several cases of recent T-box regulon expansion following the loss of a previously existing regulatory system, in particular, arginine regulon in Clostridium difficile and methionine regulon in Lactobacillaceae. Finally, we described a new structural class of T-boxes containing duplicated terminator–antiterminator elements and unusual reduced T-boxes regulating initiation of translation in the Actinobacteria. PMID:18359782

  6. Breaking out of Our Boxes.

    ERIC Educational Resources Information Center

    Patterson, William

    2003-01-01

    Argues that educators must "think outside the box" to improve school performance. Suggests several areas for expanded thought, including school size, curriculum coverage, grading practices, use of time, organization of students, time management, and belief statement. (PKP)

  7. Center Spot: Shoe Box Science

    ERIC Educational Resources Information Center

    Hoffman, Jan

    1976-01-01

    This is the second "Center Spot" devoted to Jan Hoffman's "Shoe Box Science," a program that organizes manipulative materials so that children can identify, describe, order, construct, name and distinguish on their own.

  8. The ATP-Dependent RNA Helicase DDX3X Modulates Herpes Simplex Virus 1 Gene Expression.

    PubMed

    Khadivjam, Bita; Stegen, Camille; Hogue-Racine, Marc-Aurèle; El Bilali, Nabil; Döhner, Katinka; Sodeik, Beate; Lippé, Roger

    2017-04-15

    The human protein DDX3X is a DEAD box ATP-dependent RNA helicase that regulates transcription, mRNA maturation, and mRNA export and translation. DDX3X concomitantly modulates the replication of several RNA viruses and promotes innate immunity. We previously showed that herpes simplex virus 1 (HSV-1), a human DNA virus, incorporates DDX3X into its mature particles and that DDX3X is required for optimal HSV-1 infectivity. Here, we show that viral gene expression, replication, and propagation depend on optimal DDX3X protein levels. Surprisingly, DDX3X from incoming viral particles was not required for the early stages of the HSV-1 infection, but, rather, the protein controlled the assembly of new viral particles. This was independent of the previously reported ability of DDX3X to stimulate interferon type I production. Instead, both the lack and overexpression of DDX3X disturbed viral gene transcription and thus subsequent genome replication. This suggests that in addition to its effect on RNA viruses, DDX3X impacts DNA viruses such as HSV-1 by an interferon-independent pathway.IMPORTANCE Viruses interact with a variety of cellular proteins to complete their life cycle. Among them is DDX3X, an RNA helicase that participates in most aspects of RNA biology, including transcription, splicing, nuclear export, and translation. Several RNA viruses and a limited number of DNA viruses are known to manipulate DDX3X for their own benefit. In contrast, DDX3X is also known to promote interferon production to limit viral propagation. Here, we show that DDX3X, which we previously identified in mature HSV-1 virions, stimulates HSV-1 gene expression and, consequently, virion assembly by a process that is independent of its ability to promote the interferon pathway.

  9. Effect of dead material in a calorimeter

    SciTech Connect

    Green, D.

    1995-10-01

    The existence of dead material in any practical calorimeter system is simply a fact of life. The task for the designer, then, is to understand the impact on the Physics in question, and strive to minimize it. The aim of this note is to use the ``Hanging File`` test data, which has fined grained individual readout of about 100 depth segments, to explore impact of dead material on the mean and r.m.s. of the hadronic distribution. The amount and location of the dead material is varied. It important to remember that the Hanging File data was calibrated, EM to HCAL compartment, so as to minimize the electron to pion energy dependence. In practical terms e/pie was made = 1.0 at an incident energy of about 100 GeV. Note that the PB(EM) + FE(HCAL) calorimeter was not a compensating device.

  10. Dead Zone Accretion Flows in Protostellar Disks

    NASA Technical Reports Server (NTRS)

    Turner, Neal; Sano, T.

    2008-01-01

    Planets form inside protostellar disks in a dead zone where the electrical resistivity of the gas is too high for magnetic forces to drive turbulence. We show that much of the dead zone nevertheless is active and flows toward the star while smooth, large-scale magnetic fields transfer the orbital angular momentum radially outward. Stellar X-ray and radionuclide ionization sustain a weak coupling of the dead zone gas to the magnetic fields, despite the rapid recombination of free charges on dust grains. Net radial magnetic fields are generated in the magnetorotational turbulence in the electrically conducting top and bottom surface layers of the disk, and reach the midplane by ohmic diffusion. A toroidal component to the fields is produced near the midplane by the orbital shear. The process is similar to the magnetization of the solar tachocline. The result is a laminar, magnetically driven accretion flow in the region where the planets form.

  11. Microbial and chemical characterization of underwater fresh water springs in the Dead Sea.

    PubMed

    Ionescu, Danny; Siebert, Christian; Polerecky, Lubos; Munwes, Yaniv Y; Lott, Christian; Häusler, Stefan; Bižić-Ionescu, Mina; Quast, Christian; Peplies, Jörg; Glöckner, Frank Oliver; Ramette, Alban; Rödiger, Tino; Dittmar, Thorsten; Oren, Aharon; Geyer, Stefan; Stärk, Hans-Joachim; Sauter, Martin; Licha, Tobias; Laronne, Jonathan B; de Beer, Dirk

    2012-01-01

    Due to its extreme salinity and high Mg concentration the Dead Sea is characterized by a very low density of cells most of which are Archaea. We discovered several underwater fresh to brackish water springs in the Dead Sea harboring dense microbial communities. We provide the first characterization of these communities, discuss their possible origin, hydrochemical environment, energetic resources and the putative biogeochemical pathways they are mediating. Pyrosequencing of the 16S rRNA gene and community fingerprinting methods showed that the spring community originates from the Dead Sea sediments and not from the aquifer. Furthermore, it suggested that there is a dense Archaeal community in the shoreline pore water of the lake. Sequences of bacterial sulfate reducers, nitrifiers iron oxidizers and iron reducers were identified as well. Analysis of white and green biofilms suggested that sulfide oxidation through chemolitotrophy and phototrophy is highly significant. Hyperspectral analysis showed a tight association between abundant green sulfur bacteria and cyanobacteria in the green biofilms. Together, our findings show that the Dead Sea floor harbors diverse microbial communities, part of which is not known from other hypersaline environments. Analysis of the water's chemistry shows evidence of microbial activity along the path and suggests that the springs supply nitrogen, phosphorus and organic matter to the microbial communities in the Dead Sea. The underwater springs are a newly recognized water source for the Dead Sea. Their input of microorganisms and nutrients needs to be considered in the assessment of possible impact of dilution events of the lake surface waters, such as those that will occur in the future due to the intended establishment of the Red Sea-Dead Sea water conduit.

  12. Microbial and Chemical Characterization of Underwater Fresh Water Springs in the Dead Sea

    PubMed Central

    Ionescu, Danny; Siebert, Christian; Polerecky, Lubos; Munwes, Yaniv Y.; Lott, Christian; Häusler, Stefan; Bižić-Ionescu, Mina; Quast, Christian; Peplies, Jörg; Glöckner, Frank Oliver; Ramette, Alban; Rödiger, Tino; Dittmar, Thorsten; Oren, Aharon; Geyer, Stefan; Stärk, Hans-Joachim; Sauter, Martin; Licha, Tobias; Laronne, Jonathan B.; de Beer, Dirk

    2012-01-01

    Due to its extreme salinity and high Mg concentration the Dead Sea is characterized by a very low density of cells most of which are Archaea. We discovered several underwater fresh to brackish water springs in the Dead Sea harboring dense microbial communities. We provide the first characterization of these communities, discuss their possible origin, hydrochemical environment, energetic resources and the putative biogeochemical pathways they are mediating. Pyrosequencing of the 16S rRNA gene and community fingerprinting methods showed that the spring community originates from the Dead Sea sediments and not from the aquifer. Furthermore, it suggested that there is a dense Archaeal community in the shoreline pore water of the lake. Sequences of bacterial sulfate reducers, nitrifiers iron oxidizers and iron reducers were identified as well. Analysis of white and green biofilms suggested that sulfide oxidation through chemolitotrophy and phototrophy is highly significant. Hyperspectral analysis showed a tight association between abundant green sulfur bacteria and cyanobacteria in the green biofilms. Together, our findings show that the Dead Sea floor harbors diverse microbial communities, part of which is not known from other hypersaline environments. Analysis of the water’s chemistry shows evidence of microbial activity along the path and suggests that the springs supply nitrogen, phosphorus and organic matter to the microbial communities in the Dead Sea. The underwater springs are a newly recognized water source for the Dead Sea. Their input of microorganisms and nutrients needs to be considered in the assessment of possible impact of dilution events of the lake surface waters, such as those that will occur in the future due to the intended establishment of the Red Sea−Dead Sea water conduit. PMID:22679498

  13. The lithium vapor box divertor

    NASA Astrophysics Data System (ADS)

    Goldston, R. J.; Myers, R.; Schwartz, J.

    2016-02-01

    It has long been recognized that volumetric dissipation of the plasma heat flux from a fusion power system is preferable to its localized impingement on a material surface. Volumetric dissipation mitigates both the anticipated very high heat flux and intense particle-induced damage due to sputtering. Recent projections to a tokamak demonstration power plant suggest an immense upstream parallel heat flux, of order 20 GW m-2, implying that fully detached operation may be a requirement for the success of fusion power. Building on pioneering work on the use of lithium by Nagayama et al and by Ono et al as well as earlier work on the gas box divertor by Watkins and Rebut, we present here a concept for a lithium vapor box divertor, in which lithium vapor extracts momentum and energy from a fusion-power-plant divertor plasma, using fully volumetric processes. At the high powers and pressures that are projected this requires a high density of lithium vapor, which must be isolated from the main plasma in order to avoid lithium build-up on the chamber walls or in the plasma. Isolation is achieved through a powerful multi-box differential pumping scheme available only for condensable vapors. The preliminary box-wise calculations are encouraging, but much more work is required to demonstrate the practical viability of this scheme, taking into account at least 2D plasma and vapor flows within and between the vapor boxes and out of the vapor boxes to the main plasma.

  14. Dead-time Corrected Disdrometer Data

    DOE Data Explorer

    Bartholomew, Mary Jane

    2008-03-05

    Original and dead-time corrected disdrometer results for observations made at SGP and TWP. The correction is based on the technique discussed in Sheppard and Joe, 1994. In addition, these files contain calculated radar reflectivity factor, mean Doppler velocity and attenuation for every measurement for both the original and dead-time corrected data at the following wavelengths: 0.316, 0.856, 3.2, 5, and 10cm (W,K,X,C,S bands). Pavlos Kollias provided the code to do these calculations.

  15. Dbp9p, a putative ATP-dependent RNA helicase involved in 60S-ribosomal-subunit biogenesis, functionally interacts with Dbp6p.

    PubMed Central

    Daugeron, M C; Kressler, D; Linder, P

    2001-01-01

    Ribosome synthesis is a highly complex process and constitutes a major cellular activity. The biogenesis of this ribonucleoprotein assembly requires a multitude of protein trans-acting factors including several putative ATP-dependent RNA helicases of the DEAD-box and related protein families. Here we show that the previously uncharacterized Saccharomyces cerevisiae open reading frame YLR276C, hereafter named DBP9 (DEAD-box protein 9), encodes an essential nucleolar protein involved in 60S-ribosomal-subunit biogenesis. Genetic depletion of Dbp9p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This terminal phenotype is likely due to the instability of early pre-ribosomal particles, as evidenced by the low steady-state levels and the decreased synthesis of the 27S precursors to mature 25S and 5.8S rRNAs. In agreement with a role of Dbp9p in 60S subunit synthesis, we find that increased Dbp9p dosage efficiently suppresses certain dbp6 alleles and that dbp6/dbp9 double mutants show synthetic lethality. Furthermore, Dbp6p and Dbp9p weakly interact in a yeast two-hybrid assay. Altogether, our findings indicate an intimate functional interaction between Dbp6p and Dbp9p during the process of 60S-ribosomal-subunit assembly. PMID:11565753

  16. 30 CFR 57.12006 - Distribution boxes.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Distribution boxes. 57.12006 Section 57.12006... and Underground § 57.12006 Distribution boxes. Distribution boxes shall be provided with a... deenergized, and the distribution box shall be labeled to show which circuit each device controls....

  17. Plate forming and break down pizza box

    DOEpatents

    Pantisano, Frank; Devine, Scott M.

    1992-01-01

    A standard corrugated paper pizza box is provided with slit cuts cut through the top panel of the pizza box in a shape to form four circular serving plates with a beveled raised edge and cross slit cuts through the bottom panel of the pizza box separating the box into four essentially equal portions for easy disposal.

  18. The HMG-1 box protein family: classification and functional relationships.

    PubMed Central

    Baxevanis, A D; Landsman, D

    1995-01-01

    The abundant and highly-conserved nucleoproteins comprising the high mobility group-1/2 (HMG-1/2) family contains two homologous basic domains of about 75 amino acids. These basic domains, termed HMG-1 boxes, are highly structured and facilitate HMG-DNA interactions. Many proteins that regulate various cellular functions involving DNA binding and whose target DNA sequences share common structural characteristics have been identified as having an HMG-1 box; these proteins include the RNA polymerase I transcription factor UBF, the mammalian testis-determining factor SRY and the mitochondrial transcription factors ABF2 and mtTF1, among others. The sequences of 121 HMG-1 boxes have been compiled and aligned in accordance with thermodynamic results from homology model building (threading) experiments, basing the alignment on structure rather than by using traditional sequence homology methods. The classification of a representative subset of these proteins was then determined using standard least-squares distance methods. The proteins segregate into two groups, the first consisting of HMG-1/2 proteins and the second consisting of proteins containing the HMG-1 box but which are not canonical HMG proteins. The proteins in the second group further segregate based on their function, their ability to bind specific sequences of DNA, or their ability to recognize discrete non-B-DNA structures. The HMG-1 box provides an excellent example of how a specific protein motif, with slight alteration, can be used to recognize DNA in a variety of functional contexts. Images PMID:7784217

  19. Survey of Claimed Encounters with the Dead.

    ERIC Educational Resources Information Center

    Haraldsson, Erlendur

    1989-01-01

    National survey in Iceland revealed 31 percent of respondents reported having perceived presence of dead person. Interviewed 127 persons on nature of experiences, relationship with deceased, conditions of experiences, and characteristics of interviewees and deceased persons. Attempted to test theories of what may elicit such experiences.…

  20. Effect of Dead Algae on Soil Permeability

    SciTech Connect

    Harvey, R.S.

    2003-02-21

    Since existing basins support heavy growths of unicellular green algae which may be killed by temperature variation or by inadvertent pH changes in waste and then deposited on the basin floor, information on the effects of dead algae on soil permeability was needed. This study was designed to show the effects of successive algal kills on the permeability of laboratory soil columns.

  1. Unethical and Deadly Symbiosis in Higher Education

    ERIC Educational Resources Information Center

    Crumbley, D. Larry; Flinn, Ronald; Reichelt, Kenneth J.

    2012-01-01

    As administrators are pressured to increase retention rates in accounting departments, and higher education in general, a deadly symbiosis is occurring. Most students and parents only wish for high grades, so year after year many educators engage in unethical grade inflation and course work deflation. Since administrators use the students to audit…

  2. 46 CFR 171.117 - Dead covers.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Dead covers. 171.117 Section 171.117 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO VESSELS CARRYING PASSENGERS Openings in the Side of a Vessel Below the Bulkhead or Weather Deck §...

  3. 46 CFR 171.117 - Dead covers.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Dead covers. 171.117 Section 171.117 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO VESSELS CARRYING PASSENGERS Openings in the Side of a Vessel Below the Bulkhead or Weather Deck §...

  4. 46 CFR 171.117 - Dead covers.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Dead covers. 171.117 Section 171.117 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO VESSELS CARRYING PASSENGERS Openings in the Side of a Vessel Below the Bulkhead or Weather Deck §...

  5. 46 CFR 171.117 - Dead covers.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Dead covers. 171.117 Section 171.117 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO VESSELS CARRYING PASSENGERS Openings in the Side of a Vessel Below the Bulkhead or Weather Deck §...

  6. Cheatgrass Dead Zones in Northern Nevada

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reports of areas of cheatgrass die-off are becoming more frequent. In 2009, we investigated cheatgrass die-off in north-central Nevada. Dead zones ranged from several to hundreds of acres in size and were largely unvegetated and covered by cheatgrass litter with a distinct gray cast. We collected re...

  7. Dbp7p, a putative ATP-dependent RNA helicase from Saccharomyces cerevisiae, is required for 60S ribosomal subunit assembly.

    PubMed Central

    Daugeron, M C; Linder, P

    1998-01-01

    Putative ATP-dependent RNA helicases are ubiquitous, highly conserved proteins that are found in most organisms and they are implicated in all aspects of cellular RNA metabolism. Here we present the functional characterization of the Dbp7 protein, a putative ATP-dependent RNA helicase of the DEAD-box protein family from Saccharomyces cerevisiae. The complete deletion of the DBP7 ORF causes a severe slow-growth phenotype. In addition, the absence of Dbp7p results in a reduced amount of 60S ribosomal subunits and an accumulation of halfmer polysomes. Subsequent analysis of pre-rRNA processing indicates that this 60S ribosomal subunit deficit is due to a strong decrease in the production of 27S and 7S precursor rRNAs, which leads to reduced levels of the mature 25S and 5.8S rRNAs. Noticeably, the overall decrease of the 27S pre-rRNA species is neither associated with the accumulation of preceding precursors nor with the emergence of abnormal processing intermediates, suggesting that these 27S pre-rRNA species are degraded rapidly in the absence of Dbp7p. Finally, an HA epitope-tagged Dbp7 protein is localized in the nucleolus. We propose that Dbp7p is involved in the assembly of the pre-ribosomal particle during the biogenesis of the 60S ribosomal subunit. PMID:9582098

  8. More dead than dead: perceptions of persons in the persistent vegetative state.

    PubMed

    Gray, Kurt; Knickman, T Anne; Wegner, Daniel M

    2011-11-01

    Patients in persistent vegetative state (PVS) may be biologically alive, but these experiments indicate that people see PVS as a state curiously more dead than dead. Experiment 1 found that PVS patients were perceived to have less mental capacity than the dead. Experiment 2 explained this effect as an outgrowth of afterlife beliefs, and the tendency to focus on the bodies of PVS patients at the expense of their minds. Experiment 3 found that PVS is also perceived as "worse" than death: people deem early death better than being in PVS. These studies suggest that people perceive the minds of PVS patients as less valuable than those of the dead - ironically, this effect is especially robust for those high in religiosity.

  9. The lithium vapor box divertor

    DOE PAGES

    Goldston, R. J.; Myers, R.; Schwartz, J.

    2016-01-13

    It has long been recognized that volumetric dissipation of the plasma heat flux from a fusion power system is preferable to its localized impingement on a material surface. Volumetric dissipation mitigates both the anticipated very high heat flux and intense particle-induced damage due to sputtering. Our recent projections to a tokamak demonstration power plant suggest an immense upstream parallel heat flux, of order 20 GW m-2, implying that fully detached operation may be a requirement for the success of fusion power. Building on pioneering work on the use of lithium by Nagayama et al and by Ono et al asmore » well as earlier work on the gas box divertor by Watkins and Rebut, we present here a concept for a lithium vapor box divertor, in which lithium vapor extracts momentum and energy from a fusion-power-plant divertor plasma, using fully volumetric processes. Furthermore, at the high powers and pressures that are projected this requires a high density of lithium vapor, which must be isolated from the main plasma in order to avoid lithium build-up on the chamber walls or in the plasma. Isolation is achieved through a powerful multi-box differential pumping scheme available only for condensable vapors. The preliminary box-wise calculations are encouraging, but much more work is required in order to demonstrate the practical viability of this scheme, taking into account at least 2D plasma and vapor flows within and between the vapor boxes and out of the vapor boxes to the main plasma.« less

  10. The lithium vapor box divertor

    SciTech Connect

    Goldston, R. J.; Myers, R.; Schwartz, J.

    2016-01-13

    It has long been recognized that volumetric dissipation of the plasma heat flux from a fusion power system is preferable to its localized impingement on a material surface. Volumetric dissipation mitigates both the anticipated very high heat flux and intense particle-induced damage due to sputtering. Our recent projections to a tokamak demonstration power plant suggest an immense upstream parallel heat flux, of order 20 GW m-2, implying that fully detached operation may be a requirement for the success of fusion power. Building on pioneering work on the use of lithium by Nagayama et al and by Ono et al as well as earlier work on the gas box divertor by Watkins and Rebut, we present here a concept for a lithium vapor box divertor, in which lithium vapor extracts momentum and energy from a fusion-power-plant divertor plasma, using fully volumetric processes. Furthermore, at the high powers and pressures that are projected this requires a high density of lithium vapor, which must be isolated from the main plasma in order to avoid lithium build-up on the chamber walls or in the plasma. Isolation is achieved through a powerful multi-box differential pumping scheme available only for condensable vapors. The preliminary box-wise calculations are encouraging, but much more work is required in order to demonstrate the practical viability of this scheme, taking into account at least 2D plasma and vapor flows within and between the vapor boxes and out of the vapor boxes to the main plasma.

  11. Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

    PubMed

    Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

    2016-01-01

    The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

  12. Classical three-box 'paradox'

    NASA Astrophysics Data System (ADS)

    Kirkpatrick, K. A.

    2003-05-01

    A simple classical probabilistic system (a simple card game) classically exemplifies Aharonov and Vaidman's 'three-box 'paradox'' (1991 J. Phys. A: Math. Gen. 24 2315), implying that the three-box example is neither quantal nor a paradox and leaving one with less difficulty to busy the interpreters of quantum mechanics. An ambiguity in the usual expression of the retrodiction formula is shown to have misled Albert et al (1985 Phys. Rev. Lett. 54 5) to a result not, in fact, 'curious'; the discussion illustrates how to avoid this ambiguity.

  13. Chronic infections of West Nile virus detected in California dead birds.

    PubMed

    Reisen, William K; Padgett, Kerry; Fang, Ying; Woods, Leslie; Foss, Leslie; Anderson, Jaynia; Kramer, Vicki

    2013-06-01

    During 2010 and 2011, 933 recently deceased birds, submitted as part of the dead bird surveillance program, tested positive for West Nile virus RNA at necropsy. The relative amount of RNA measured by qRT-PCR cycles ranged from 8.2 to 37.0 cycle threshold (Ct) and formed a bimodal frequency distribution, with maxima at 20 and 36 Ct and minima at 28-30 Ct. On the basis of frequency distributions among different avian species with different responses to infection following experimental inoculation, field serological data indicating survival of infection, and the discovery of persistent RNA in experimentally infected birds, dead birds collected in nature were scored as "recent" or "chronic" infections on the basis of Ct scores. The percentage of birds scored as having chronic infections was highest during late winter/spring, when all birds were after hatching year, and lowest during late summer, when enzootic transmission was typically highest as indicated by mosquito infections. Our data indicated that intervention efforts should not be based on dead birds with chronic infections unless supported by additional surveillance metrics.

  14. Exploring challenges to TTO utilities: valuing states worse than dead.

    PubMed

    Robinson, Angela; Spencer, Anne

    2006-04-01

    The conventional time trade off (TTO) method relies on fundamentally different procedures to assess states better than and worse than dead. Arbitrary transformation mechanisms are then applied to worse than dead scores in order to achieve symmetry with those rated as better than dead. We use a 'life profile' approach along with a ranking procedure in order to show how states rated worse than dead may be assessed in exactly the same manner as better than dead scores. We then explore a common issue associated with states worse than dead that has received some attention recently: maximal endurable time. Our results showed that, although the severe health state was commonly rated as worse than dead, there were relatively few respondents that exhibited MET preferences. We discuss the implications of our findings for the use of the TTO method in deriving values for states that are worse than dead.

  15. Cajal body proteins differentially affect the processing of box C/D scaRNPs.

    PubMed

    Enwerem, Isioma I; Wu, Guowei; Yu, Yi Tao; Hebert, Michael D

    2015-01-01

    Small nuclear ribonucleoproteins (snRNPs), which are required for pre-mRNA splicing, contain extensively modified snRNA. Small Cajal body-specific ribonucleoproteins (scaRNPs) mediate these modifications. It is unknown how the box C/D class of scaRNPs localizes to Cajal Bodies (CBs). The processing of box C/D scaRNA is also unclear. Here, we explore the processing of box C/D scaRNA 2 and 9 by coilin. We also broaden our investigation to include WRAP53 and SMN, which accumulate in CBs, play a role in RNP biogenesis and associate with coilin. These studies demonstrate that the processing of an ectopically expressed scaRNA2 is altered upon the reduction of coilin, WRAP53 or SMN, but the extent and direction of this change varies depending on the protein reduced. We also show that box C/D scaRNP activity is reduced in a cell line derived from coilin knockout mice. Collectively, the findings presented here further implicate coilin as being a direct participant in the formation of box C/D scaRNPs, and demonstrate that WRAP53 and SMN may also play a role, but the activity of these proteins is divergent to coilin.

  16. The final stages of spliceosome maturation require Spp2p that can interact with the DEAH box protein Prp2p and promote step 1 of splicing.

    PubMed Central

    Roy, J; Kim, K; Maddock, J R; Anthony, J G; Woolford, J L

    1995-01-01

    Pre-mRNA processing occurs by assembly of splicing factors on the substrate to form the spliceosome followed by two consecutive RNA cleavage-ligation reactions. The Prp2 protein hydrolyzes ATP and is required for the first reaction (Yean SL, Lin RJ, 1991, Mol Cell Biol 11:5571-5577; Kim SH, Smith J, Claude A, Lin RJ, 1992, EMBO J 11:2319-2326). The Saccharomyces cerevisiae SPP2 gene was previously identified as a high-copy suppressor of temperature-sensitive prp2 mutants (Last RL, Maddock JR, Woolford JL Jr, 1987, Genetics 117:619-631). We have characterized the function of Spp2p in vivo and in vitro. Spp2p is an essential protein required for the first RNA cleavage reaction in vivo. Depletion of Spp2p from yeast cells results in accumulation of unspliced pre-mRNAs. A temperature-sensitive spp2-1 mutant accumulates pre-mRNAs in vivo and is unable to undergo the first splicing reaction in vitro. However, spliceosomal complexes are assembled in extracts prepared from the mutant. We show that Spp2p function is required after spliceosome assembly but prior to the first reaction. Spp2p associates with the spliceosome before the first RNA cleavage reaction and is likely to be released from the spliceosome following ATP hydrolysis by Prp2p. The Prp2 and Spp2 proteins are capable of physically interacting with each other. These results suggest that Spp2p interacts with Prp2p in the spliceosome prior to the first cleavage-ligation reaction. Spp2p is the first protein that has been found to interact with a DEAD/H box splicing factor. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 PMID:7493316

  17. Survival of filamentous fungi in hypersaline Dead Sea water.

    PubMed

    Kis-Papo, T; Oren, A; Wasser, S P; Nevo, E

    2003-02-01

    A variety of filamentous fungi have recently been isolated from the Dead Sea (340 g/L total dissolved salts). To assess the extent to which such fungi can survive for prolonged periods in Dead Sea water, we examined the survival of both spores and mycelia in undiluted Dead Sea water and in Dead Sea water diluted to different degrees with distilled water. Mycelia of Aspergillus versicolor and Chaetomium globosum strains isolated from the Dead Sea remained viable for up to 8 weeks in undiluted Dead Sea water. Four Dead Sea isolates (A. versicolor, Eurotium herbariorum, Gymnascella marismortui, and C. globosum) retained their viability in Dead Sea water diluted to 80% during the 12 weeks of the experiment. Mycelia of all species survived for the full term of the experiment in Dead Sea water diluted to 50% and 10% of its original salinity. Comparison of the survival of Dead Sea species and closely related isolates obtained from other locations showed prolonged viability of the strains obtained from the Dead Sea. Spores of isolates obtained from the terrestrial shore of the Dead Sea generally proved less tolerant to suspension in undiluted Dead Sea water than spores of species isolated from the water column. Spores of the species isolated from the control sites had lost their viability in undiluted Dead Sea water within 12 weeks. However, with the exception of Emericella spores, which showed poor survival, a substantial fraction of the spores of Dead Sea fungal isolates remained viable for that period. The difference in survival rate between spores and mycelia of isolates of the same species points to the existence of adapted halotolerant and/or halophilic fungi in the Dead Sea.

  18. Regular satellite formation and evolution in a dead zone

    NASA Astrophysics Data System (ADS)

    Chen, Cheng; Martin, Rebecca G.

    2017-01-01

    The dead zone in a circumplanetary disk is a non-turbulent region at the disk midplane that is an ideal location for regular satellite formation. The lower viscosity in the dead zone allows small objects to accrete and grow. We model the evolution of a circumplanetary disk with a dead zone for a range of disk and dead zone parameters. We investigate how these affect the formation and subsequent evolution of regular satellites that form in the disk.

  19. 10 CFR 1047.7 - Use of deadly force.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 4 2011-01-01 2011-01-01 false Use of deadly force. 1047.7 Section 1047.7 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) LIMITED ARREST AUTHORITY AND USE OF FORCE BY PROTECTIVE FORCE OFFICERS General Provisions § 1047.7 Use of deadly force. (a) Deadly force means that force which...

  20. 14 CFR 1203b.106 - Use of deadly force.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Use of deadly force. 1203b.106 Section... AUTHORITY AND USE OF FORCE BY NASA SECURITY FORCE PERSONNEL § 1203b.106 Use of deadly force. Deadly force shall be used only in those circumstances where the security force officer reasonably believes...

  1. 14 CFR 1203b.106 - Use of deadly force.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 5 2011-01-01 2010-01-01 true Use of deadly force. 1203b.106 Section 1203b... AUTHORITY AND USE OF FORCE BY NASA SECURITY FORCE PERSONNEL § 1203b.106 Use of deadly force. Deadly force shall be used only in those circumstances where the security force officer reasonably believes...

  2. 10 CFR 1047.7 - Use of deadly force.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Use of deadly force. 1047.7 Section 1047.7 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) LIMITED ARREST AUTHORITY AND USE OF FORCE BY PROTECTIVE FORCE OFFICERS General Provisions § 1047.7 Use of deadly force. (a) Deadly force means that force which...

  3. 46 CFR 190.10-30 - Dead end corridors.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Dead end corridors. 190.10-30 Section 190.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OCEANOGRAPHIC RESEARCH VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 190.10-30 Dead end corridors. (a) Dead end corridors, or the equivalent,...

  4. 46 CFR 72.10-30 - Dead end corridors.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 3 2014-10-01 2014-10-01 false Dead end corridors. 72.10-30 Section 72.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) PASSENGER VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 72.10-30 Dead end corridors. (a) Dead end corridors, or the equivalent, more than...

  5. 46 CFR 190.10-30 - Dead end corridors.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Dead end corridors. 190.10-30 Section 190.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OCEANOGRAPHIC RESEARCH VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 190.10-30 Dead end corridors. (a) Dead end corridors, or the equivalent,...

  6. 46 CFR 92.10-30 - Dead end corridors.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Dead end corridors. 92.10-30 Section 92.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CARGO AND MISCELLANEOUS VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 92.10-30 Dead end corridors. (a) Dead end corridors, or...

  7. 46 CFR 72.10-30 - Dead end corridors.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 3 2013-10-01 2013-10-01 false Dead end corridors. 72.10-30 Section 72.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) PASSENGER VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 72.10-30 Dead end corridors. (a) Dead end corridors, or the equivalent, more than...

  8. 46 CFR 108.161 - Dead end corridors.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Dead end corridors. 108.161 Section 108.161 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS DESIGN AND EQUIPMENT Construction and Arrangement Means of Escape § 108.161 Dead end corridors. No dead end...

  9. 46 CFR 92.10-30 - Dead end corridors.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Dead end corridors. 92.10-30 Section 92.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CARGO AND MISCELLANEOUS VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 92.10-30 Dead end corridors. (a) Dead end corridors, or...

  10. 46 CFR 72.10-30 - Dead end corridors.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 3 2012-10-01 2012-10-01 false Dead end corridors. 72.10-30 Section 72.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) PASSENGER VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 72.10-30 Dead end corridors. (a) Dead end corridors, or the equivalent, more than...

  11. 46 CFR 108.161 - Dead end corridors.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Dead end corridors. 108.161 Section 108.161 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS DESIGN AND EQUIPMENT Construction and Arrangement Means of Escape § 108.161 Dead end corridors. No dead end...

  12. 46 CFR 190.10-30 - Dead end corridors.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Dead end corridors. 190.10-30 Section 190.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OCEANOGRAPHIC RESEARCH VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 190.10-30 Dead end corridors. (a) Dead end corridors, or the equivalent,...

  13. 46 CFR 72.10-30 - Dead end corridors.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 3 2011-10-01 2011-10-01 false Dead end corridors. 72.10-30 Section 72.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) PASSENGER VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 72.10-30 Dead end corridors. (a) Dead end corridors, or the equivalent, more than...

  14. 46 CFR 92.10-30 - Dead end corridors.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Dead end corridors. 92.10-30 Section 92.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CARGO AND MISCELLANEOUS VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 92.10-30 Dead end corridors. (a) Dead end corridors, or...

  15. 46 CFR 108.161 - Dead end corridors.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Dead end corridors. 108.161 Section 108.161 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS DESIGN AND EQUIPMENT Construction and Arrangement Means of Escape § 108.161 Dead end corridors. No dead end...

  16. 46 CFR 92.10-30 - Dead end corridors.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Dead end corridors. 92.10-30 Section 92.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CARGO AND MISCELLANEOUS VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 92.10-30 Dead end corridors. (a) Dead end corridors, or...

  17. 46 CFR 190.10-30 - Dead end corridors.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Dead end corridors. 190.10-30 Section 190.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OCEANOGRAPHIC RESEARCH VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 190.10-30 Dead end corridors. (a) Dead end corridors, or the equivalent,...

  18. Preventing Deadly Conflict: Toward a World without War.

    ERIC Educational Resources Information Center

    Francis, Greg

    Although some people believed that the end of the Cold War would herald a new age of peace, the 1990s have seen more than five million people die in over 35 deadly conflicts. New technologies have made warfare ever more deadly. There is, however, a breadth of options available to prevent or control deadly conflict in the world. This curriculum…

  19. 46 CFR 111.10-7 - Dead ship.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Dead ship. 111.10-7 Section 111.10-7 Shipping COAST... REQUIREMENTS Power Supply § 111.10-7 Dead ship. (a) The generating plant of each self-propelled vessel must provide the electrical services necessary to start the main propulsion plant from a dead ship...

  20. 46 CFR 111.10-7 - Dead ship.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Dead ship. 111.10-7 Section 111.10-7 Shipping COAST... REQUIREMENTS Power Supply § 111.10-7 Dead ship. (a) The generating plant of each self-propelled vessel must provide the electrical services necessary to start the main propulsion plant from a dead ship...

  1. 46 CFR 111.10-7 - Dead ship.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Dead ship. 111.10-7 Section 111.10-7 Shipping COAST... REQUIREMENTS Power Supply § 111.10-7 Dead ship. (a) The generating plant of each self-propelled vessel must provide the electrical services necessary to start the main propulsion plant from a dead ship...

  2. 46 CFR 111.10-7 - Dead ship.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Dead ship. 111.10-7 Section 111.10-7 Shipping COAST... REQUIREMENTS Power Supply § 111.10-7 Dead ship. (a) The generating plant of each self-propelled vessel must provide the electrical services necessary to start the main propulsion plant from a dead ship...

  3. 46 CFR 111.10-7 - Dead ship.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Dead ship. 111.10-7 Section 111.10-7 Shipping COAST... REQUIREMENTS Power Supply § 111.10-7 Dead ship. (a) The generating plant of each self-propelled vessel must provide the electrical services necessary to start the main propulsion plant from a dead ship...

  4. 46 CFR 190.10-30 - Dead end corridors.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Dead end corridors. 190.10-30 Section 190.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OCEANOGRAPHIC RESEARCH VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 190.10-30 Dead end corridors. (a) Dead end corridors, or the equivalent,...

  5. 46 CFR 72.10-30 - Dead end corridors.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 3 2010-10-01 2010-10-01 false Dead end corridors. 72.10-30 Section 72.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) PASSENGER VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 72.10-30 Dead end corridors. (a) Dead end corridors, or the equivalent, more than...

  6. 46 CFR 108.161 - Dead end corridors.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Dead end corridors. 108.161 Section 108.161 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS DESIGN AND EQUIPMENT Construction and Arrangement Means of Escape § 108.161 Dead end corridors. No dead end...

  7. 46 CFR 92.10-30 - Dead end corridors.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Dead end corridors. 92.10-30 Section 92.10-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CARGO AND MISCELLANEOUS VESSELS CONSTRUCTION AND ARRANGEMENT Means of Escape § 92.10-30 Dead end corridors. (a) Dead end corridors, or...

  8. 9 CFR 314.8 - Dead animal carcasses.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Dead animal carcasses. 314.8 Section 314.8 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... Dead animal carcasses. (a) With the exception of dead livestock which have died en route and...

  9. 9 CFR 314.8 - Dead animal carcasses.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Dead animal carcasses. 314.8 Section 314.8 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... Dead animal carcasses. (a) With the exception of dead livestock which have died en route and...

  10. 9 CFR 314.8 - Dead animal carcasses.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Dead animal carcasses. 314.8 Section 314.8 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... Dead animal carcasses. (a) With the exception of dead livestock which have died en route and...

  11. 9 CFR 314.8 - Dead animal carcasses.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Dead animal carcasses. 314.8 Section 314.8 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... Dead animal carcasses. (a) With the exception of dead livestock which have died en route and...

  12. 9 CFR 314.8 - Dead animal carcasses.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Dead animal carcasses. 314.8 Section 314.8 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... Dead animal carcasses. (a) With the exception of dead livestock which have died en route and...

  13. 46 CFR 108.161 - Dead end corridors.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Dead end corridors. 108.161 Section 108.161 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS DESIGN AND EQUIPMENT Construction and Arrangement Means of Escape § 108.161 Dead end corridors. No dead end...

  14. Dead Sea Minerals loaded polymeric nanoparticles.

    PubMed

    Dessy, Alberto; Kubowicz, Stephan; Alderighi, Michele; Bartoli, Cristina; Piras, Anna Maria; Schmid, Ruth; Chiellini, Federica

    2011-10-15

    Therapeutic properties of Dead Sea Water (DSW) in the treatment of skin diseases such as atopic dermatitis, psoriasis and photo aging UV damaged skin have been well established. DSW is in fact rich in minerals such as calcium, magnesium, sodium, potassium, zinc and strontium which are known to exploit anti-inflammatory effects and to promote skin barrier recovery. In order to develop a Dead Sea Minerals (DSM) based drug delivery system for topical therapy of skin diseases, polymeric nanoparticles based on Poly (maleic anhydride-alt-butyl vinyl ether) 5% grafted with monomethoxy poly(ethyleneglycol) 2000 MW (PEG) and 95% grafted with 2-methoxyethanol (VAM41-PEG) loaded with DSM were prepared by means of a combined miniemulsion/solvent evaporation process. The resulting nanoparticles were characterized in terms of dimension, morphology, biocompatibility, salt content and release. Cytocompatible spherical nanoparticles possessing an average diameter of about 300 nm, a time controlled drug release profile and a high formulation yield were obtained.

  15. Potential Evaporite Biomarkers from the Dead Sea

    NASA Technical Reports Server (NTRS)

    Morris, Penny A.; Wentworth, Susan J.; Thomas-Keprta, Kathie; Allen, Carlton C.; McKay, David S.

    2001-01-01

    The Dead Sea is located on the northern branch of the African-Levant Rift systems. The rift system, according to one model, was formed by a series of strike slip faults, initially forming approximately two million years ago. The Dead Sea is an evaporite basin that receives freshwater from springs and from the Jordan River. The Dead Sea is different from other evaporite basins, such as the Great Salt Lake, in that it possesses high concentrations of magnesium and has an average pH of 6.1. The dominant cation in the Great Salt Lake is sodium, and the pH is 7.7. Calcium concentrations are also higher in the Dead Sea than in the Great Salt Lake. Both basins are similar in that the dominant anion is chlorine and the salinity levels are approximately 20 %. Other common cations that have been identified from the waters of the Dead Sea and the Great Salt Lake include sodium and potassium. A variety of Archea, Bacteria, and a single genus of a green algal, Dunaliella, has been described from the Dead Sea. Earlier studies concentrated on microbial identification and analysis of their unique physiology that allows them to survive in this type of extreme environment. Potential microbial fossilization processes, microbial fossils, and the metallic ions associated with fossilization have not been studied thoroughly. The present study is restricted to identifying probable microbial morphologies and associated metallic ions. XRD (X Ray Diffraction) analysis indicates the presence of halite, quartz, and orthoclase feldspar. In addition to these minerals, other workers have reported potassium chloride, magnesium bromide, magnesium chloride, calcium chloride, and calcium sulfate. Halite, calcium sulfate, and orthoclase were examined in this report for the presence of microbes, microbially induced deposits or microbial alteration. Neither the gypsum nor the orthoclase surfaces possesses any obvious indications of microbial life or fossilization. The sand-sized orthoclase particles are

  16. Zero dead volume tube to surface seal

    DOEpatents

    Benett, William J.; Folta, James A.

    2000-01-01

    A method and apparatus for connecting a tube to a surface that creates a dead volume seal. The apparatus is composed of three components, a body, a ferrule, and a threaded fitting. The ferrule is compressed onto a tube and a seal is formed between the tube and a device retained in the body by threading the fitting into the body which provides pressure that seals the face of the ferrule to a mating surface on the device. This seal can be used at elevated temperatures depending on the materials used. While the invention has been developed for use with micro-machined silicon wafers used in Capillary Gas Chromatograph (GC), it can be utilized anywhere for making a gas or fluid face seal to the surface of a device that has near zero dead volume.

  17. Expo-Box Contact Us

    EPA Pesticide Factsheets

    EPA-Expo-Box is a toolbox for exposure assessors. Its purpose is to provide a compendium of exposure assessment and risk characterization tools that will present comprehensive step-by-step guidance and links to relevant exposure assessment data bases, mode

  18. Black Boxes in Workplace Mathematics

    ERIC Educational Resources Information Center

    Williams, Julian; Wake, Geoff

    2007-01-01

    We ground Cultural-Historical Activity Theory (CHAT) in studies of workplace practices from a mathematical point of view. We draw on multiple case study visits by college students and teacher-researchers to workplaces. By asking questions that "open boxes", we "outsiders and boundary-crossers" sought to expose contradictions between College and…

  19. NETL's JIC in a box

    ScienceCinema

    David Anna

    2016-07-12

    The National Energy Technology Laboratory developed the idea of a portable joint information center AKA JIC in-a-box. This video discribes some of the equipment in the portable JIC as well as some of the methodology that NETL developed as a result of this portable JIC concept.

  20. The Bird Box Survey Project

    ERIC Educational Resources Information Center

    Willis, Patrick

    2014-01-01

    When high school students are asked what's the best part of science class, many will say it's the field trips. Students enjoy engaging in authentic, community-based science outside the classroom. To capitalize on this, Patrick Willis created the Bird Box Survey Project for his introductory field biology class. The project takes students…

  1. NETL's JIC in a box

    SciTech Connect

    David Anna

    2009-06-03

    The National Energy Technology Laboratory developed the idea of a portable joint information center AKA JIC in-a-box. This video discribes some of the equipment in the portable JIC as well as some of the methodology that NETL developed as a result of this portable JIC concept.

  2. EPA-Expo-Box Terminology

    EPA Pesticide Factsheets

    EPA-Expo-Box is a toolbox for exposure assessors. Its purpose is to provide a compendium of exposure assessment and risk characterization tools that will present comprehensive step-by-step guidance and links to relevant exposure assessment data bases,

  3. The Cereal Box Problem Revisited.

    ERIC Educational Resources Information Center

    Wilkins, Jesse L. M.

    1999-01-01

    Investigates the cereal box problem using both an experimental and theoretical framework, and Monte Carlo methods. Using empirical data, students can discover patterns and relationships that help them understand the origin of the theoretical solution to the problem. Contains 17 references. (Author/ASK)

  4. On the Dirichlet's Box Principle

    ERIC Educational Resources Information Center

    Poon, Kin-Keung; Shiu, Wai-Chee

    2008-01-01

    In this note, we will focus on several applications on the Dirichlet's box principle in Discrete Mathematics lesson and number theory lesson. In addition, the main result is an innovative game on a triangular board developed by the authors. The game has been used in teaching and learning mathematics in Discrete Mathematics and some high schools in…

  5. Immune Reconstitution Inflammatory Syndrome: Opening Pandora's Box

    PubMed Central

    2017-01-01

    One of the purposes of antiretroviral therapy (ART) is to restore the immune system. However, it can sometimes lead to an aberrant inflammatory response and paradoxical clinical worsening known as the immune reconstitution inflammatory syndrome (IRIS). We describe a 23-year-old male, HIV1 infected with a rapid progression phenotype, who started ART with TCD4+ of 53 cells/mm3 (3,3%) and HIV RNA = 890000 copies/mL (6 log). Four weeks later he was admitted to the intensive care unit with severe sepsis. The diagnostic pathway identified progressive multifocal leukoencephalopathy, digestive Kaposi sarcoma, and P. aeruginosa bacteraemia. Five weeks after starting ART, TCD4+ cell count was 259 cells/mm3 (15%) and HIV RNA = 3500 copies/mL (4 log). He developed respiratory failure and progressed to septic shock and death. Those complications might justify the outcome but its autopsy opened Pandora's box: cerebral and cardiac toxoplasmosis was identified, as well as hemophagocytic syndrome, systemic candidiasis, and Mycobacterium avium complex infection. IRIS remains a concern and eventually a barrier to ART. Male gender, young age, low TCD4 cell count, and high viral load are risk factors. The high prevalence of subclinical opportunistic diseases highlights the need for new strategies to reduce IRIS incidence. PMID:28163944

  6. De Plein Fouet: Is Strategy Dead?

    DTIC Science & Technology

    2015-06-01

    10 | Air & Space Power Journal De Plein Fouet: Is Strategy Dead? Robert Cardillo Richard Szafranski Disclaimer: The views and opinions expressed or...implied in the Journal are those of the authors and should not be construed as carry- ing the official sanction of the Department of Defense, Air ...Force, Air Education and Training Command, Air University, or other agencies or departments of the US government. This article may be reproduced in

  7. Bullets with Names: The Deadly Dilemma

    DTIC Science & Technology

    1992-06-01

    108 1. Ideology and National Power ....................... 109 2 . The Force of Example ............................. 111 C ...experience. ix L . THE DEADLY DILEMMA Begin on December 22, 1974, Seymour Hersh wrote a series of articles for the New- XYkI imes which profoundly...plots were not a part of the Committee’s statutory mandate, committee chairman Frank Church established a special Subcommittee on Assassination in

  8. Nasal high flow reduces dead space.

    PubMed

    Möller, Winfried; Feng, Sheng; Domanski, Ulrike; Franke, Karl-Josef; Celik, Gülnaz; Bartenstein, Peter; Becker, Sven; Meyer, Gabriele; Schmid, Otmar; Eickelberg, Oliver; Tatkov, Stanislav; Nilius, Georg

    2017-01-01

    Recent studies show that nasal high flow (NHF) therapy can support ventilation in patients with acute or chronic respiratory disorders. Clearance of dead space has been suggested as being the key mechanism of respiratory support with NHF therapy. The hypothesis of this study was that NHF in a dose-dependent manner can clear dead space of the upper airways from expired air and decrease rebreathing. The randomized crossover study involved 10 volunteers using scintigraphy with (81m)Krypton ((81m)Kr) gas during a breath-holding maneuver with closed mouth and in 3 nasally breathing tracheotomized patients by volumetric capnography and oximetry through sampling CO2 and O2 in the trachea and measuring the inspired volume with inductance plethysmography following NHF rates of 15, 30, and 45 l/min. The scintigraphy revealed a decrease in (81m)Kr gas clearance half-time with an increase of NHF in the nasal cavities [Pearson's correlation coefficient cc = -0.55, P < 0.01], the pharynx (cc = -0.41, P < 0.01), and the trachea (cc = -0.51, P < 0.01). Clearance rates in nasal cavities derived from time constants and MRI-measured volumes were 40.6 ± 12.3 (SD), 52.5 ± 17.7, and 72.9 ± 21.3 ml/s during NHF (15, 30, and 45 l/min, respectively). Measurement of inspired gases in the trachea showed an NHF-dependent decrease of inspired CO2 that correlated with an increase of inspired O2 (cc = -0.77, P < 0.05). NHF clears the upper airways of expired air, which reduces dead space by a decrease of rebreathing making ventilation more efficient. The dead space clearance is flow and time dependent, and it may extend below the soft palate.

  9. Improving measurement of Chesapeake Bay's dead zone

    NASA Astrophysics Data System (ADS)

    Schultz, Colin

    2013-09-01

    In the 1930s, researchers first noticed that the Chesapeake Bay had a dead zone, an expanse of water with drastically reduced concentrations of oxygen. In the 1980s, hypoxia—low-oxygen conditions—gave way in some places to anoxia—a near-total depletion of dissolved oxygen. A lack of oxygen makes the water inhospitable for many marine organisms, and the Chesapeake Bay is the focus of major ecosystem rehabilitation efforts.

  10. Pre-stem cell formation by non-platelet RNA-containing particle fusion

    PubMed Central

    Kong, Wuyi; Nuo, Mu; Zhu, Xiao Ping; Han, Xiu Juan; Luo, Lihua; Wang, Xian

    2013-01-01

    1. We found a group of non-platelet RNA-containing particles (NPRCP) in human umbilical cord blood. To understand the origin, characterization and differentiation of NPRCP, we examined cord blood-isolated NPRCP in vitro. 2. The NPRCP range in size from < 1 to 5 μm, have a thin bilayer membrane and various morphological features, contain short RNA and microRNA and express octamer-binding transcription factor 4 (OCT4), sex-determining region Y 2 (SOX2) and DEAD box polypeptide 4 (DDX4). On coculture with nucleated cells from umbilical cord blood, NPRCP fuse to small, active, non-nucleated cells called ‘particle fusion-derived non-nucleated cells’ (PFDNC). The PFDNC are approximately 8 μm in diameter and are characterized by their twisting movement in culture plates. They can easily move into and out of nucleated cells and finally differentiate into mesenchymal-like cells. In addition, the larger non-nucleated cellular structures that are derived from the aggregation and fusion of multiple NPRCP can further differentiate into large stem cells that also release OCT4- and SOX2-positive non-nucleated small cells. 3. Our data provide strong evidence that NPRCP can fuse into PFDNC, which further differentiate into mesenchymal-like cells. Multiple NPRCP also fuse into other types of large stem cells. We believe that stem cells are derived from NPRCP fusion. There is considerable potential for the use of NPRCP in clinical therapy. PMID:23611023

  11. A facial mask comprising Dead Sea mud.

    PubMed

    Abu-Jdayil, Basim; Mohameed, Hazim A

    2006-01-01

    Many investigators have proved that Dead Sea salt and mud are useful in treating skin disorders and skin diseases. Therefore, the black mud has been extensively used as a base for the preparation of soaps, creams, and unguents for skin care. This study concerns a facial mask made mainly of Dead Sea mud. The effects of temperature and shearing conditions on the rheological behavior of the facial mask were investigated. The mud facial mask exhibited a shear thinning behavior with a yield stress. It was found that the apparent viscosity of the mask has a strong dependence on the shear rate as well as on the temperature. The facial mask exhibited a maximum yield stress and very shear thinning behavior at 40 degrees C, which is attributed to the gelatinization of the polysaccharide used to stabilize the mud particles. On the other hand, the mud mask exhibited a time-independent behavior at low temperatures and shear rates and changed to a thixotropic behavior upon increasing both the temperature and the shear rate. The shear thinning and thixotropic behaviors have a significant importance in the ability of the facial mask to spread on the skin: the Dead Sea mud mask can break down for easy spreading, and the applied film can gain viscosity instantaneously to resist running. Moreover, particle sedimentation, which in this case would negatively affect consumer acceptance of the product, occurs slowly due to high viscosity at rest conditions.

  12. Surviving deadness in the analytic experience.

    PubMed

    Koritar, Endre

    2014-12-01

    The transference/countertransference (third space) analysis is considered to be central in the therapeutic effectiveness of the analytic process. Less emphasis has been placed on the actual experiences of analyst and analysand in the conflictual reenactment of third space experience and its resolution. This paper recounts the shared experience of a patient who was silent throughout most of the analysis, and my reaction, in fantasy and enactment, to this disturbing experience-both for him and for myself. I argue that it is the affective re-experiencing of past repressed trauma in the analytic space that has a therapeutic impact, leading to growth in the patient and also the therapist. I contrast Freud's emphasis on insight, making the unconscious conscious, with Ferenczi's suggestion that the therapeutic impact lies in the repetition of past traumatic experience in the analysis but with the possibility of a different outcome with a more benign object, leading to symbolic representation of repressed trauma. Re-experiencing and symbolization, in the third space, of past traumatic experience can be an exit point from the endless repetition of trauma in internal and external object relations, leading to a new beginning in the patient's life. Immersed in the experience of deadness in the analysis, which had become a dead womb, the struggle to remain alive and thinking led to a rupture out of the dead womb, like the Caesura of birth, into aliveness and the ability to mentalize what had previously remained unmentalized.

  13. Cellular microRNAs up-regulate transcription via interaction with promoter TATA-box motifs.

    PubMed

    Zhang, Yijun; Fan, Miaomiao; Zhang, Xue; Huang, Feng; Wu, Kang; Zhang, Junsong; Liu, Jun; Huang, Zhuoqiong; Luo, Haihua; Tao, Liang; Zhang, Hui

    2014-12-01

    The TATA box represents one of the most prevalent core promoters where the pre-initiation complexes (PICs) for gene transcription are assembled. This assembly is crucial for transcription initiation and well regulated. Here we show that some cellular microRNAs (miRNAs) are associated with RNA polymerase II (Pol II) and TATA box-binding protein (TBP) in human peripheral blood mononuclear cells (PBMCs). Among them, let-7i sequence specifically binds to the TATA-box motif of interleukin-2 (IL-2) gene and elevates IL-2 mRNA and protein production in CD4(+) T-lymphocytes in vitro and in vivo. Through direct interaction with the TATA-box motif, let-7i facilitates the PIC assembly and transcription initiation of IL-2 promoter. Several other cellular miRNAs, such as mir-138, mir-92a or mir-181d, also enhance the promoter activities via binding to the TATA-box motifs of insulin, calcitonin or c-myc, respectively. In agreement with the finding that an HIV-1-encoded miRNA could enhance viral replication through targeting the viral promoter TATA-box motif, our data demonstrate that the interaction with core transcription machinery is a novel mechanism for miRNAs to regulate gene expression.

  14. Zebrafish P54 RNA helicases are cytoplasmic granule residents that are required for development and stress resilience

    PubMed Central

    Zampedri, Cecilia; Tinoco-Cuellar, Maryana; Carrillo-Rosas, Samantha; Diaz-Tellez, Abigail; Ramos-Balderas, Jose Luis; Pelegri, Francisco

    2016-01-01

    ABSTRACT Stress granules are cytoplasmic foci that directly respond to the protein synthesis status of the cell. Various environmental insults, such as oxidative stress or extreme heat, block protein synthesis; consequently, mRNA will stall in translation, and stress granules will immediately form and become enriched with mRNAs. P54 DEAD box RNA helicases are components of RNA granules such as P-bodies and stress granules. We studied the expression, in cytoplasmic foci, of both zebrafish P54 RNA helicases (P54a and P54b) during development and found that they are expressed in cytoplasmic granules under both normal conditions and stress conditions. In zebrafish embryos exposed to heat shock, some proportion of P54a and P54b helicases move to larger granules that exhibit the properties of genuine stress granules. Knockdown of P54a and/or P54b in zebrafish embryos produces developmental abnormalities restricted to the posterior trunk; further, these embryos do not form stress granules, and their survival upon exposure to heat-shock conditions is compromised. Our observations fit the model that cells lacking stress granules have no resilience or ability to recover once the stress has ended, indicating that stress granules play an essential role in the way organisms adapt to a changing environment. PMID:27489304

  15. 9 CFR 82.6 - Interstate movement of dead birds and dead poultry from a quarantined area.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... dead poultry from a quarantined area. 82.6 Section 82.6 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS EXOTIC NEWCASTLE DIS- EASE (END) AND CHLAMYDIOSIS Exotic Newcastle Disease (END) § 82.6 Interstate movement of dead birds and dead poultry from a quarantined area. (a) Except...

  16. 9 CFR 82.6 - Interstate movement of dead birds and dead poultry from a quarantined area.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... dead poultry from a quarantined area. 82.6 Section 82.6 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS NEWCASTLE DISEASE AND CHLAMYDIOSIS Newcastle Disease § 82.6 Interstate movement of dead birds and dead poultry from a quarantined area. (a) Except as provided in paragraph (b)...

  17. 9 CFR 82.6 - Interstate movement of dead birds and dead poultry from a quarantined area.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... dead poultry from a quarantined area. 82.6 Section 82.6 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS EXOTIC NEWCASTLE DIS- EASE (END) AND CHLAMYDIOSIS Exotic Newcastle Disease (END) § 82.6 Interstate movement of dead birds and dead poultry from a quarantined area. (a) Except...

  18. 9 CFR 82.6 - Interstate movement of dead birds and dead poultry from a quarantined area.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... dead poultry from a quarantined area. 82.6 Section 82.6 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS EXOTIC NEWCASTLE DISEASE (END) AND CHLAMYDI-OSIS Exotic Newcastle Disease (END) § 82.6 Interstate movement of dead birds and dead poultry from a quarantined area. (a) Except...

  19. 9 CFR 82.6 - Interstate movement of dead birds and dead poultry from a quarantined area.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... dead poultry from a quarantined area. 82.6 Section 82.6 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS EXOTIC NEWCASTLE DISEASE (END) AND CHLAMYDI-OSIS Exotic Newcastle Disease (END) § 82.6 Interstate movement of dead birds and dead poultry from a quarantined area. (a) Except...

  20. Vertical Mixing in the Dead Sea

    NASA Astrophysics Data System (ADS)

    Gertman, Isaac; Ozer, Tal; Katsenelson, Boris; Lensky, Nadav

    2015-04-01

    For hundreds of years, the Dead Sea was characterized by a stable haline stratification, supported by runoff. The penetration of the winter convection was limited to an upper mixed layer (UML) of about 30-50 m. Below the UML, a stable halocline prevented the mixing. As a result of the runoff reduction, the UML salinity increased and the gravitational stability diminished. During the winter of 1978-1979, the sea water overturned, ending the long-term stable hydrological regime. Since 1979, the haline stratification structure reoccurred twice after extremely rainy winters, in 1980-82 and 1992-1995. In other years, the sea was entirely mixed by winter thermal convection ( which occurs from November to March ) and had a seasonal pycnocline beneath the UML during summer. Profiles of temperature and quasi-salinity (density anomaly from 1000 kg/m3 for the chosen reference temperature of 32° C) during the last 19 years, show the formation of summer ``overturning halocline'' beneath the UML, and the thermocline that supports the stable stratification. Another warm and saline layer is formed also during the summer period near the bottom. This layer spreads from the southern part of the sea, where end-brine is discharged to the sea from the Israeli and Jordanian salt plants' evaporation ponds. The end-brine has extremely high salinity (˜ 350 g/kg) and, in spite of the high temperatures ( ˜ 45° C), high density (1350 kg/m^3), it therefore spreads as a gravitational current in the Dead Sea deep basin. Estimation of the density ratio (Rρ) for the Dead Sea water (where measurements of water salinity is quite difficult) was done using quasi-salinity (σ32) and potential temperature (θ): Rρ= [α(partialθ/partial z)]/[β(partial σ32/partial z)], where α and β are temperature expansion and quasi-salinity contraction coefficients respectively. The values of α and β for the Dead Sea water were defined from water samples collected during 2008. The Rρ values confirm that

  1. Nasal high flow reduces dead space

    PubMed Central

    Feng, Sheng; Domanski, Ulrike; Franke, Karl-Josef; Celik, Gülnaz; Bartenstein, Peter; Becker, Sven; Meyer, Gabriele; Schmid, Otmar; Eickelberg, Oliver; Tatkov, Stanislav; Nilius, Georg

    2017-01-01

    Recent studies show that nasal high flow (NHF) therapy can support ventilation in patients with acute or chronic respiratory disorders. Clearance of dead space has been suggested as being the key mechanism of respiratory support with NHF therapy. The hypothesis of this study was that NHF in a dose-dependent manner can clear dead space of the upper airways from expired air and decrease rebreathing. The randomized crossover study involved 10 volunteers using scintigraphy with 81mKrypton (81mKr) gas during a breath-holding maneuver with closed mouth and in 3 nasally breathing tracheotomized patients by volumetric capnography and oximetry through sampling CO2 and O2 in the trachea and measuring the inspired volume with inductance plethysmography following NHF rates of 15, 30, and 45 l/min. The scintigraphy revealed a decrease in 81mKr gas clearance half-time with an increase of NHF in the nasal cavities [Pearson’s correlation coefficient cc = −0.55, P < 0.01], the pharynx (cc = −0.41, P < 0.01), and the trachea (cc = −0.51, P < 0.01). Clearance rates in nasal cavities derived from time constants and MRI-measured volumes were 40.6 ± 12.3 (SD), 52.5 ± 17.7, and 72.9 ± 21.3 ml/s during NHF (15, 30, and 45 l/min, respectively). Measurement of inspired gases in the trachea showed an NHF-dependent decrease of inspired CO2 that correlated with an increase of inspired O2 (cc = −0.77, P < 0.05). NHF clears the upper airways of expired air, which reduces dead space by a decrease of rebreathing making ventilation more efficient. The dead space clearance is flow and time dependent, and it may extend below the soft palate. NEW & NOTEWORTHY Clearance of expired air in upper airways by nasal high flow (NHF) can be extended below the soft palate and de facto causes a reduction of dead space. Using scintigraphy, the authors found a relationship between NHF, time, and clearance. Direct measurement of CO2 and O2 in the trachea confirmed a reduction of rebreathing, providing

  2. Affinity capture and identification of host cell factors associated with hepatitis C virus (+) strand subgenomic RNA.

    PubMed

    Upadhyay, Alok; Dixit, Updesh; Manvar, Dinesh; Chaturvedi, Nootan; Pandey, Virendra N

    2013-06-01

    Hepatitis C virus (HCV) infection leading to chronic hepatitis is a major factor in the causation of liver cirrhosis, hepatocellular carcinoma, and liver failure. This process may involve the interplay of various host cell factors, as well as the interaction of these factors with viral RNA and proteins. We report a novel strategy using a sequence-specific biotinylated peptide nucleic acid (PNA)-neamine conjugate targeted to HCV RNA for the in situ capture of subgenomic HCV (+) RNA, along with cellular and viral factors associated with it in MH14 host cells. Using this affinity capture system in conjunction with LC/MS/MS, we have identified 83 cellular factors and three viral proteins (NS5B, NS5A, and NS3-4a protease-helicase) associated with the viral genome. The capture was highly specific. These proteins were not scored with cured MH14 cells devoid of HCV replicons because of the absence of the target sequence in cells for the PNA-neamine probe and also because, unlike oligomeric DNA, cellular proteins have no affinity for PNA. The identified cellular factors belong to different functional groups, including signaling, oncogenic, chaperonin, transcriptional regulators, and RNA helicases as well as DEAD box proteins, ribosomal proteins, translational regulators/factors, and metabolic enzymes, that represent a diverse set of cellular factors associated with the HCV RNA genome. Small interfering RNA-mediated silencing of a diverse class of selected proteins in an HCV replicon cell line either enhanced or inhibited HCV replication/translation, suggesting that these cellular factors have regulatory roles in HCV replication.

  3. Diverse Evolutionary Trajectories for Small RNA Biogenesis Genes in the Oomycete Genus Phytophthora

    PubMed Central

    Bollmann, Stephanie R.; Fang, Yufeng; Press, Caroline M.; Tyler, Brett M.; Grünwald, Niklaus J.

    2016-01-01

    Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. The genomes of several oomycetes including Phytophthora species such as the soybean pathogen P. sojae, have been sequenced, allowing evolutionary analysis of small RNA-processing enzymes. This study examined the evolutionary origins of the oomycete small RNA-related genes Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) through broad phylogenetic analyses of the key domains. Two Dicer gene homologs, DCL1 and DCL2, and one RDR homolog were cloned and analyzed from P. sojae. Gene expression analysis revealed only minor changes in transcript levels among different life stages. Oomycete DCL1 homologs clustered with animal and plant Dicer homologs in evolutionary trees, whereas oomycete DCL2 homologs clustered basally to the tree along with Drosha homologs. Phylogenetic analysis of the RDR homologs confirmed a previous study that suggested the last common eukaryote ancestor possessed three RDR homologs, which were selectively retained or lost in later lineages. Our analysis clarifies the position of some Unikont and Chromalveolate RDR lineages within the tree, including oomycete homologs. Finally, we analyzed alterations in the domain structure of oomycete Dicer and RDR homologs, specifically focusing on the proposed domain transfer of the DEAD-box helicase domain from Dicer to RDR. Implications of the oomycete domain structure are discussed, and possible roles of the two oomycete Dicer homologs are proposed. PMID:27014308

  4. Dbp3p, a putative RNA helicase in Saccharomyces cerevisiae, is required for efficient pre-rRNA processing predominantly at site A3.

    PubMed Central

    Weaver, P L; Sun, C; Chang, T H

    1997-01-01

    In Saccharomyces cerevisiae, ribosomal biogenesis takes place primarily in the nucleolus, in which a single 35S precursor rRNA (pre-rRNA) is first transcribed and sequentially processed into 25S, 5.8S, and 18S mature rRNAs, leading to the formation of the 40S and 60S ribosomal subunits. Although many components involved in this process have been identified, our understanding of this important cellular process remains limited. Here we report that one of the evolutionarily conserved DEAD-box protein genes in yeast, DBP3, is required for optimal ribosomal biogenesis. DBP3 encodes a putative RNA helicase, Dbp3p, of 523 amino acids in length, which bears a highly charged amino terminus consisting of 10 tandem lysine-lysine-X repeats ([KKX] repeats). Disruption of DBP3 is not lethal but yields a slow-growth phenotype. This genetic depletion of Dbp3p results in a deficiency of 60S ribosomal subunits and a delayed synthesis of the mature 25S rRNA, which is caused by a prominent kinetic delay in pre-rRNA processing at site A3 and to a lesser extent at sites A2 and A0. These data suggest that Dbp3p may directly or indirectly facilitate RNase MRP cleavage at site A3. The direct involvement of Dbp3p in ribosomal biogenesis is supported by the finding that Dbp3p is localized predominantly in the nucleolus. In addition, we show that the [KKX] repeats are dispensable for Dbp3p's function in ribosomal biogenesis but are required for its proper localization. The [KKX] repeats thus represent a novel signaling motif for nuclear localization and/or retention. PMID:9032262

  5. Glove box for water pit applications

    DOEpatents

    Mills, William C.; Rabe, Richard A.

    2005-01-18

    A glove box assembly that includes a glove box enclosure attached to a longitudinally extending hollow tube having an entranceway, wherein the portion of the tube is in a liquid environment. An elevator member is provided for raising an object that is introduced into the hollow tube from the liquid environment to a gas environment inside the glove box enclosure while maintaining total containment.

  6. The Guide to the Ecology Box.

    ERIC Educational Resources Information Center

    Ontario Inst. for Studies in Education, Toronto.

    Cooperating with the Canadian Commission for UNESCO, the Ontario Institute for Studies in Education has prepared boxes of experimental curriculum materials on the subject of ecology. This guide summarizes the design and contents of the boxes and provides instructions for those using the boxes--principals, teachers, parents, librarians, and…

  7. The Heuristic Interpretation of Box Plots

    ERIC Educational Resources Information Center

    Lem, Stephanie; Onghena, Patrick; Verschaffel, Lieven; Van Dooren, Wim

    2013-01-01

    Box plots are frequently used, but are often misinterpreted by students. Especially the area of the box in box plots is often misinterpreted as representing number or proportion of observations, while it actually represents their density. In a first study, reaction time evidence was used to test whether heuristic reasoning underlies this…

  8. Illumination box and camera system

    DOEpatents

    Haas, Jeffrey S.; Kelly, Fredrick R.; Bushman, John F.; Wiefel, Michael H.; Jensen, Wayne A.; Klunder, Gregory L.

    2002-01-01

    A hand portable, field-deployable thin-layer chromatography (TLC) unit and a hand portable, battery-operated unit for development, illumination, and data acquisition of the TLC plates contain many miniaturized features that permit a large number of samples to be processed efficiently. The TLC unit includes a solvent tank, a holder for TLC plates, and a variety of tool chambers for storing TLC plates, solvent, and pipettes. After processing in the TLC unit, a TLC plate is positioned in a collapsible illumination box, where the box and a CCD camera are optically aligned for optimal pixel resolution of the CCD images of the TLC plate. The TLC system includes an improved development chamber for chemical development of TLC plates that prevents solvent overflow.

  9. Comet 'Bites the Dust' Around Dead Star

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Infrared Spectrometer Graph

    This artist's concept illustrates a comet being torn to shreds around a dead star, or white dwarf, called G29-38. NASA's Spitzer Space Telescope observed a cloud of dust around this white dwarf that may have been generated from this type of comet disruption. The findings suggest that a host of other comet survivors may still orbit in this long-dead solar system.

    The white dwarf G29-38 began life as a star that was about three times as massive as our sun. Its death involved the same steps that the sun will ultimately undergo billions of years from now. According to theory, the G29-38 star became brighter and brighter as it aged, until it bloated up into a dying star called a red giant. This red giant was large enough to engulf and evaporate any terrestrial planets like Earth that happened to be in its way. Later, the red giant shed its outer atmosphere, leaving behind a shrunken skeleton of star, called a white dwarf. If the star did host a planetary system, outer planets akin to Jupiter and Neptune and a remote ring of icy comets would remain.

    The Spitzer observations provide observational evidence for this orbiting outpost of comet survivors. Astronomers speculate that one such comet was knocked into the inner regions of G29-38, possibly by an outer planet. As the comet approached very close to the white dwarf, it may have been torn apart by the star's tidal forces. Eventually, all that would be left of the comet is a disk of dust.

    This illustration shows a comet in the process of being pulverized: part of it still exists as a chain of small clumps, while the rest has already spread out into a dusty disk. Comet Shoemaker-Levy 9 broke apart in a similar fashion when it plunged into Jupiter in 1994. Evidence for Comets Found in Dead Star's Dust The graph of data, or spectrum, from NASA's Spitzer Space Telescope indicates that a dead star, or white dwarf, called G29

  10. The five deadly sins of science publishing

    PubMed Central

    Tracz, Vitek

    2015-01-01

    Science cannot progress without scientists reporting their findings. And yet researchers have given control of this central pillar of the scientific process to science publishers, who are in the business of serving the interests of their journals; these are not always the same as the interests of science. This editorial describes the problems with the process of preparing and publishing research findings, and with judging their veracity and significance, and then explains how we at Faculty of 1000 are starting to tackle the ‘deadly sins’ of science publishing. PMID:26097694

  11. NC2 mobilizes TBP on core promoter TATA boxes.

    PubMed

    Schluesche, Peter; Stelzer, Gertraud; Piaia, Elisa; Lamb, Don C; Meisterernst, Michael

    2007-12-01

    The general transcription factors (GTFs) of eukaryotic RNA polymerase II, in a process facilitated by regulatory and accessory factors, target promoters through synergistic interactions with core elements. The specific binding of the TATA box-binding protein (TBP) to the TATA box has led to the assumption that GTFs recognize promoters directly, producing a preinitiation complex at a defined position. Using biochemical analysis as well as biophysical single-pair Förster resonance energy transfer, we now provide evidence that negative cofactor-2 (NC2) induces dynamic conformational changes in the TBP-DNA complex that allow it to escape and return to TATA-binding mode. This can lead to movement of TBP along the DNA away from TATA.

  12. RNA Helicase DDX5 Inhibits Reprogramming to Pluripotency by miRNA-Based Repression of RYBP and its PRC1-Dependent and -Independent Functions.

    PubMed

    Li, Huanhuan; Lai, Ping; Jia, Jinping; Song, Yawei; Xia, Qing; Huang, Kaimeng; He, Na; Ping, Wangfang; Chen, Jiayu; Yang, Zhongzhou; Li, Jiao; Yao, Mingze; Dong, Xiaotao; Zhao, Jicheng; Hou, Chunhui; Esteban, Miguel A; Gao, Shaorong; Pei, Duanqing; Hutchins, Andrew P; Yao, Hongjie

    2017-04-06

    RNA-binding proteins (RBPs), in addition to their functions in cellular homeostasis, play important roles in lineage specification and maintaining cellular identity. Despite their diverse and essential functions, which touch on nearly all aspects of RNA metabolism, the roles of RBPs in somatic cell reprogramming are poorly understood. Here we show that the DEAD-box RBP DDX5 inhibits reprogramming by repressing the expression and function of the non-canonical polycomb complex 1 (PRC1) subunit RYBP. Disrupting Ddx5 expression improves the efficiency of iPSC generation and impedes processing of miR-125b, leading to Rybp upregulation and suppression of lineage-specific genes via RYBP-dependent ubiquitination of H2AK119. Furthermore, RYBP is required for PRC1-independent recruitment of OCT4 to the promoter of Kdm2b, a histone demethylase gene that promotes reprogramming by reactivating endogenous pluripotency genes. Together, these results reveal important functions of DDX5 in regulating reprogramming and highlight the importance of a Ddx5-miR125b-Rybp axis in controlling cell fate.

  13. Invariant box-parameterization of neutrino oscillations

    SciTech Connect

    Weiler, Thomas J.; Wagner, DJ

    1998-10-19

    The model-independent 'box' parameterization of neutrino oscillations is examined. The invariant boxes are the classical amplitudes of the individual oscillating terms. Being observables, the boxes are independent of the choice of parameterization of the mixing matrix. Emphasis is placed on the relations among the box parameters due to mixing-matrix unitarity, and on the reduction of the number of boxes to the minimum basis set. Using the box algebra, we show that CP-violation may be inferred from measurements of neutrino flavor mixing even when the oscillatory factors have averaged. General analyses of neutrino oscillations among n{>=}3 flavors can readily determine the boxes, which can then be manipulated to yield magnitudes of mixing matrix elements.

  14. Asteroid 'Bites the Dust' Around Dead Star

    NASA Technical Reports Server (NTRS)

    2009-01-01

    NASA's Spitzer Space Telescope set its infrared eyes upon the dusty remains of shredded asteroids around several dead stars. This artist's concept illustrates one such dead star, or 'white dwarf,' surrounded by the bits and pieces of a disintegrating asteroid. These observations help astronomers better understand what rocky planets are made of around other stars.

    Asteroids are leftover scraps of planetary material. They form early on in a star's history when planets are forming out of collisions between rocky bodies. When a star like our sun dies, shrinking down to a skeleton of its former self called a white dwarf, its asteroids get jostled about. If one of these asteroids gets too close to the white dwarf, the white dwarf's gravity will chew the asteroid up, leaving a cloud of dust.

    Spitzer's infrared detectors can see these dusty clouds and their various constituents. So far, the telescope has identified silicate minerals in the clouds polluting eight white dwarfs. Because silicates are common in our Earth's crust, the results suggest that planets similar to ours might be common around other stars.

  15. [Reflexes in brain-dead patients].

    PubMed

    Ulvik, A; Salvesen, R; Nielsen, E W

    1998-05-20

    We report on a patient who suffered an acute, extensive intracerebral haemorrhage, leading to symptoms of cerebral herniation within a few hours. The clinical diagnosis of brain death was made based on a neurological examination, and an apnoea test eight hours after the haemorrhage. A few hours later the diagnosis was changed, as several reflexes reappeared. After six days mechanical ventilation was withdrawn, as the brain damage was considered so serious as to render further therapy futile. It was considered unethical to sustain therapy for a possible organ donation at a later date. A review of relevant the literature, however, shows that brain-dead patients may exhibit such varying degrees of autonomic and spinal reflexes as to cause confusion, thus delaying the physician in making a diagnosis. Often, an opportunity for organ donation is lost. Based on this review, we believe that our patient was indeed brain dead when the first diagnosis was made, and that a cerebral angiography should have been performed. Because organ donation is an important issue, the diagnosis of brain death must be definitive.

  16. RNA Interference

    MedlinePlus

    ... NIGMS Home > Science Education > RNA Interference Fact Sheet RNA Interference Fact Sheet Tagline (Optional) Middle/Main Content Area What is RNA interference? RNA interference (RNAi) is a natural process ...

  17. Research on the recently dead: an historical and ethical examination.

    PubMed

    Tomasini, F

    2008-01-01

    INTRODUCTION AND SOURCES OF DATA: This paper briefly outlines the history of research on the recently dead, before critically exploring 1. In what sense can we harm the dead? 2. What are the justificatory arguments for employing family or parental consent for medical research on dead relatives? AREAS OF AGREEMENT, CONTROVERSY, GROWING POINTS OF INTEREST AND AREAS TIMELY FOR DEVELOPING RESEARCH: The controversy surrounding harm in relation to research on dead bodies largely depends on how dead people are perceived. That is, 1. As Cadavers. 2. As Ante-mortem persons. 3. By Significant others. Controversy over whether we need to have consent from significant others (bereaved relatives) depends on the weight we give to the bereaved and their experience of the dead. Understanding this is timely in developing research and is relevant to the issue of consent around organ donation for transplants.

  18. BOX DNA: a novel regulatory element related to embryonal carcinoma cell differentiation.

    PubMed Central

    Kihara-Negishi, F; Tsujita, R; Negishi, Y; Ariga, H

    1993-01-01

    BOX DNA was previously isolated from the DNA sequence inserted in the enhancer B domain of mutant polyomavirus (fPyF9) DNA. We also reported that BOX DNA functioned negatively on DNA replication and transcription of another polyomavirus mutant (PyhrN2) in F9-28 cells, a subclone of mouse F9 embryonal carcinoma (EC) cells expressing the polyomavirus large T antigen. In this study, we demonstrate that BOX DNA enhances transcription from the thymidine kinase (TK) promoter in various EC cells. One or three copies of BOX DNA, linked to the bacterial chloramphenicol acetyltransferase gene under the control of the herpes simplex virus TK promoter, activated promoter activity in F9, P19, and ECA2 cells. Band shift assays using BOX DNA as a probe revealed that specific binding proteins were present in all EC cells examined; the patterns of BOX DNA-protein complexes were the same among them. A mutation introduced within BOX DNA abolished enhancer activity as well as the formation of specific DNA-protein complexes. In non-EC cells, including L and BALB/3T3 cells, the enhancer activity of BOX DNA on the TK promoter was not observed, although binding proteins specific to the sequence exist. In band shift assays, the patterns of the DNA-protein complexes of either L or BALB/3T3 cells were different from those of EC cells. Furthermore, the enhancer activity of BOX DNA decreased upon differentiation induction in all EC cells examined, of different origins and distinct differentiation ability. In parallel with the loss of enhancer activity, the binding proteins specific for BOX DNA decreased in these cells. Moreover, we cloned a genomic DNA of F9, termed BOXF1, containing BOX DNA sequence approximately 400 bp upstream from the RNA start site of the gene. BOXF1, containing a TATA-like motif and the binding elements for Sp1 and Oct in addition to BOX DNA, possessed promoter activity deduced by a BOXF1-chloramphenicol acetyltransferase construct. Deletion analyses of the construct

  19. Haloactinomyces albus gen. nov., sp. nov., isolated from Dead Sea.

    PubMed

    Lai, Hangxian; Jiang, Yingying; Chen, Xiu; Li, Qinyuan; Jiang, Chenglin; Jiang, Yi; Wei, Xiaomin

    2017-01-10

    A novel halophilic, filamentous actinomycete strain, designated AFM 10258T, was isolated from a sediment sample collected from Dead Sea of Israel. The isolate grew with 10-35% NaCl and did not grow without NaCl. The isolate formed white aerial mycelium and long spore chains, and two spores were separated by sterile mycelium. The spores were non-motile, spherical and rugose-surfaced. The isolate contained meso-diaminopimelic acid as the diagnostic diamino acid and galactose and arabinose as the major whole-cell sugars. The polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and three unknown phospholipids. Major fatty acids were anteiso-C17:0, iso-C16:0 and iso-C15:0. MK-9(H4) was the predominant menaquinone and the DNA G+C content was 62.8 mol%. 16S rRNA gene sequence analysis indicated that strain AFM 10258T shared low sequence similarity with the closely related representatives of the families Pseudonocardiaceae (below 94.47%) and Actinopolysporaceae (below 93.76%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain AFM 10258T formed a robust clade with members of the family Actinopolysporaceae. On the basis of analysis of phenotypic, chemical and molecular characteristics, strain AFM 10258T is considered to represent a novel species of a new genus , for which the name Haloactinomyces albus gen. nov., sp. nov., is proposed. The type strain is AFM 10258T (=DSM 45977T = CGMCC 4.7115T).

  20. Analysis of the Isolated SecA DEAD Motor Suggests a Mechanism for Chemical-Mechanical Coupling

    SciTech Connect

    Nithianantham, Stanley; Shilton, Brian H

    2011-09-28

    The preprotein cross-linking domain and C-terminal domains of Escherichia coli SecA were removed to create a minimal DEAD motor, SecA-DM. SecA-DM hydrolyzes ATP and has the same affinity for ADP as full-length SecA. The crystal structure of SecA-DM in complex with ADP was solved and shows the DEAD motor in a closed conformation. Comparison with the structure of the E. coli DEAD motor in an open conformation (Protein Data Bank ID 2FSI) indicates main-chain conformational changes in two critical sequences corresponding to Motif III and Motif V of the DEAD helicase family. The structures that the Motif III and Motif V sequences adopt in the DEAD motor open conformation are incompatible with the closed conformation. Therefore, when the DEAD motor makes the transition from open to closed, Motif III and Motif V are forced to change their conformations, which likely functions to regulate passage through the transition state for ATP hydrolysis. The transition state for ATP hydrolysis for the SecA DEAD motor was modeled based on the conformation of the Vasa helicase in complex with adenylyl imidodiphosphate and RNA (Protein Data Bank ID 2DB3). A mechanism for chemical-mechanical coupling emerges, where passage through the transition state for ATP hydrolysis is hindered by the conformational changes required in Motif III and Motif V, and may be promoted by binding interactions with the preprotein substrate and/or other translocase domains and subunits.

  1. Analysis of the Isolated SecA DEAD Motor Suggests a Mechanism for Chemical-Mechanical Coupling

    SciTech Connect

    Nithianantham, Stanley; Shilton, Brian H

    2010-09-20

    The preprotein cross-linking domain and C-terminal domains of Escherichia coli SecA were removed to create a minimal DEAD motor, SecA-DM. SecA-DM hydrolyzes ATP and has the same affinity for ADP as full-length SecA. The crystal structure of SecA-DM in complex with ADP was solved and shows the DEAD motor in a closed conformation. Comparison with the structure of the E. coli DEAD motor in an open conformation (Protein Data Bank ID 2FSI) indicates main-chain conformational changes in two critical sequences corresponding to Motif III and Motif V of the DEAD helicase family. The structures that the Motif III and Motif V sequences adopt in the DEAD motor open conformation are incompatible with the closed conformation. Therefore, when the DEAD motor makes the transition from open to closed, Motif III and Motif V are forced to change their conformations, which likely functions to regulate passage through the transition state for ATP hydrolysis. The transition state for ATP hydrolysis for the SecA DEAD motor was modeled based on the conformation of the Vasa helicase in complex with adenylyl imidodiphosphate and RNA (Protein Data Bank ID 2DB3). A mechanism for chemical-mechanical coupling emerges, where passage through the transition state for ATP hydrolysis is hindered by the conformational changes required in Motif III and Motif V, and may be promoted by binding interactions with the preprotein substrate and/or other translocase domains and subunits.

  2. Respect for the dead and the ethics of anatomy.

    PubMed

    Wilkinson, T M

    2014-04-01

    Dead bodies are not persons but nor are they just things. What, ethically speaking, do the living owe the dead when it comes to using their bodies in anatomy? The article begins with the general question of respect for the dead. It distinguishes between why we should respect the dead, how we should respect them, and the weight to be given to respect. It sets out an account of the reason to respect the dead based on their interests. The article then turns to how the dead should be respected and the importance of doing so. Specifically, it considers three ethical issues in anatomy: the role of the family, the use of unclaimed bodies, and the public display of bodies donated for that purpose. This article claims that what it is to respect the dead is substantially determined by their wishes. Nonetheless the article argues that respect is consistent with allowing the family to veto anatomical use even when the deceased has consented because respect for the dead does not require following all their possible wishes. Respect is also consistent with using unclaimed bodies to which no one--deceased or family--has consented because the interests of the dead do not directly require consent and the interests of the family are unlikely to be relevant. Finally, the article does not see anything disrespectful in the public display of the bodies of those who have consented.

  3. The ecosystem service value of living versus dead biogenic reef

    NASA Astrophysics Data System (ADS)

    Sheehan, E. V.; Bridger, D.; Attrill, M. J.

    2015-03-01

    Mixed maerl beds (corralline red algae) comprise dead thalli with varying amounts of live maerl fragments, but previously it was not known whether the presence of the live maerl increases the ecosystem service 'habitat provision' of the dead maerl for the associated epibenthos. A 'flying array' towed sled with high definition video was used to film transects of the epibenthos in dead maerl and mixed maerl beds in two locations to the north and south of the English Channel (Falmouth and Jersey). Mixed maerl beds supported greater number of taxa and abundance than dead beds in Falmouth, while in Jersey, mixed and dead beds supported similar number of taxa and dead beds had a greater abundance of epifauna. Scallops tended to be more abundant on mixed beds than dead beds. Tube worms were more abundant on mixed beds in Falmouth and dead beds in Jersey. An increasing percentage occurrence of live maerl thalli correlated with increasing number of taxa in Falmouth but not Jersey. It was concluded that while live thalli can increase the functional role of dead maerl beds for the epibenthos, this is dependent on location and response variable. As a result of this work, maerl habitat in SE Jersey has been protected from towed demersal fishing gear.

  4. Dead-Time Modifications to Fast Fourier Transform Power Spectra

    NASA Astrophysics Data System (ADS)

    Zhang, W.; Jahoda, K.; Swank, J. H.; Morgan, E. H.; Giles, A. B.

    1995-08-01

    Time series analysis in X-ray astronomy is complicated by the effects of detector dead time which can cause significant departures of power spectra from those expected from simple Poisson statistics. In this paper we study the effects of both the paralyzable and nonparalyzable dead times on the underlying time series which obeys Poisson counting statistics. We present analytical formulae for the autocorrelation function, auto-covariances, and power spectra, for both kinds of dead time. These formulae, taken as a whole, offer a general qualitative and quantitative framework to further understand the effects of dead time in an X-ray detection and data processing system.

  5. Turbulence, Transport, and Waves in Ohmic Dead Zones

    NASA Astrophysics Data System (ADS)

    Gole, Daniel; Simon, Jacob B.; Lubow, Stephen H.; Armitage, Philip J.

    2016-07-01

    We use local numerical simulations to study a vertically stratified accretion disk with a resistive mid-plane that damps magnetohydrodynamic (MHD) turbulence. This is an idealized model for the dead zones that may be present at some radii in protoplanetary and dwarf novae disks. We vary the relative thickness of the dead and active zones to quantify how forced fluid motions in the dead zone change. We find that the residual Reynolds stress near the mid-plane decreases with increasing dead zone thickness, becoming negligible in cases where the active to dead mass ratio is less than a few percent. This implies that purely Ohmic dead zones would be vulnerable to episodic accretion outbursts via the mechanism of Martin & Lubow. We show that even thick dead zones support a large amount of kinetic energy, but this energy is largely in fluid motions that are inefficient at angular momentum transport. Confirming results from Oishi & Mac Low, the perturbed velocity field in the dead zone is dominated by an oscillatory, vertically extended circulation pattern with a low frequency compared to the orbital frequency. This disturbance has the properties predicted for the lowest order r mode in a hydrodynamic disk. We suggest that in a global disk similar excitations would lead to propagating waves, whose properties would vary with the thickness of the dead zone and the nature of the perturbations (isothermal or adiabatic). Flows with similar amplitudes would buckle settled particle layers and could reduce the efficiency of pebble accretion.

  6. Deformity in the "Boxing Boys".

    PubMed

    Ferrence, Susan; Bendersky, Gordon

    2005-01-01

    The late Bronze Age wall painting the Boxing Boys (c. 17th-16th century BCE) was excavated in the ancient town of Akrotiri on the Greek island of Thera. This article considers a medical interpretation for the spinal-pelvic anomaly in the anatomy of one of the boys. The artist has depicted a combination of structural anatomical adjustments diagnostic of spondylolisthesis, a forward slippage of one of the lumbar vertebrae. The accurate portrayal of the surface appearance of this condition suggests that the artist painted directly from a live subject. Thus, the Boxing Boys mural may be the earliest visual record of a sports-induced injury. Although the meaning of the wall paintings is unclear, the wild goats (agrimia) on the adjoining walls simulate swayback as a reflection of the boy's torso deformity and share other features with the boxers, adding to the unifying characteristics of the room. The abnormal morphology appears to be the earliest achievement of transforming disease into aesthetic charm on a monumental scale.

  7. Lighting up a Dead Star's Layers

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This image from NASA's Spitzer Space Telescope shows the scattered remains of an exploded star named Cassiopeia A. Spitzer's infrared detectors 'picked' through these remains and found that much of the star's original layering had been preserved.

    In this false-color image, the faint, blue glow surrounding the dead star is material that was energized by a shock wave, called the forward shock, which was created when the star blew up. The forward shock is now located at the outer edge of the blue glow. Stars are also seen in blue. Green, yellow and red primarily represent material that was ejected in the explosion and heated by a slower shock wave, called the reverse shock wave.

    The picture was taken by Spitzer's infrared array camera and is a composite of 3.6-micron light (blue); 4.5-micron light (green); and 8.0-micron light (red).

  8. An in silico analysis of T-box regulated genes and T-box evolution in prokaryotes, with emphasis on prediction of substrate specificity of transporters

    PubMed Central

    Wels, Michiel; Kormelink, Tom Groot; Kleerebezem, Michiel; Siezen, Roland J; Francke, Christof

    2008-01-01

    Background T-box anti-termination is an elegant and sensitive mechanism by which many bacteria maintain constant levels of amino acid-charged tRNAs. The amino acid specificity of the regulatory element is related to a so-called specifier codon and can in principle be used to guide the functional annotation of the genes controlled via the T-box anti-termination mechanism. Results Hidden Markov Models were defined to search the T-box regulatory element and were applied to all completed prokaryotic genomes. The vast majority of the genes found downstream of the retrieved elements encoded functionalities related to transport and synthesis of amino acids and the charging of tRNA. This is completely in line with findings reported in literature and with the proposed biological role of the regulatory element. For several species, the functional annotation of a large number of genes encoding proteins involved in amino acid transport could be improved significantly on basis of the amino acid specificity of the identified T-boxes. In addition, these annotations could be extrapolated to a larger number of orthologous systems in other species. Analysis of T-box distribution confirmed that the element is restricted predominantly to species of the phylum Firmicutes. Furthermore, it appeared that the distribution was highly species specific and that in the case of amino acid transport some boxes seemed to "pop-up" only recently. Conclusion We have demonstrated that the identification of the molecular specificity of a regulatory element can be of great help in solving notoriously difficult annotation issues, e.g. by defining the substrate specificity of genes encoding amino acid transporters on basis of the amino acid specificity of the regulatory T-box. Furthermore, our analysis of the species-dependency of the occurrence of specific T-boxes indicated that these regulatory elements propagate in a semi-independent way from the genes that they control. PMID:18625071

  9. Haloplanus natans gen. nov., sp. nov., an extremely halophilic, gas-vacuolate archaeon isolated from Dead Sea-Red Sea water mixtures in experimental outdoor ponds.

    PubMed

    Bardavid, Rahel Elevi; Mana, Lily; Oren, Aharon

    2007-04-01

    To study biological phenomena in the Dead Sea and to simulate the effects of mixing Dead Sea water with Red Sea water, experimental mesocosms were operated at the Dead Sea Works at Sedom, Israel. Dense communities of red halophilic archaea developed in mesocosms filled with 80 % Dead Sea water and 20 % Red Sea water after enrichment with phosphate. The most common type of colonies isolated from these brines belonged to the genus Halorubrum. A few white-pinkish opaque colonies contained pleomorphic flat cells with gas vesicles. Three strains isolated from the latter colonies were characterized in depth. Their 16S rRNA gene sequences showed only 91 % similarity to the closest cultured relative (Haloferax mediterranei), indicating that the new strains represent a novel species of a new genus. The name Haloplanus natans gen. nov., sp. nov. is proposed for this novel organism. The type strain of Haloplanus natans is RE-101(T) (=DSM 17983(T)=JCM 14081(T)).

  10. Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression

    PubMed Central

    Sharma, Manju; Dearth, Andrea; Smith, Benjamin; Braun, Robert E.

    2015-01-01

    The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2 -/- ;Ybx3 -/- double mutants using a previously reported Ybx2 -/- model and a newly generated global Ybx3 -/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets. PMID:26646932

  11. Adenovirus and mycoplasma infection in an ornate box turtle (Terrapene ornata ornata) in Hungary.

    PubMed

    Farkas, Szilvia L; Gál, János

    2009-07-02

    A female, adult ornate box turtle (Terrapene ornata ornata) with fatty liver was submitted for virologic examination in Hungary. Signs of an adenovirus infection including degeneration of the liver cells, enlarged nuclei and intranuclear inclusion bodies were detected by light microscopic examination. The presence of an adenovirus was later confirmed by obtaining partial sequence data from the adenoviral DNA-dependent DNA-polymerase. Phylogenetic analyses revealed that this novel chelonian adenovirus was distinct from previously described reptilian adenoviruses, not belonging to any of the recognized genera of the family Adenoviridae. As a part of the routine diagnostic procedure for chelonians the detection of herpes-, rana- and iridoviruses together with Mycoplasma spp. was attempted. Amplicons were generated by a general mycoplasma polymerase chain reaction (PCR) targeting the 16S/23S ribosomal RNA (rRNA) intergenic spacer region, as well as, a specific Mycoplasma agassizii PCR targeting the 16S rRNA gene. Based on the analyses of partial sequences of the 16S rRNA gene, the Mycoplasma sp. of the ornate box turtle seemed to be identical with the recently described eastern box turtle (Terrapene carolina carolina) Mycoplasma sp. This is the first report of a novel chelonian adenovirus and a mycoplasma infection in an ornate box turtle (T. ornata ornata) in Europe.

  12. Interchangeable breech lock for glove boxes

    DOEpatents

    Lemonds, David Preston

    2015-11-24

    A breech lock for a glove box is provided that may be used to transfer one or more items into the glove box. The breech lock can be interchangeably installed in place of a plug, glove, or other device in a port or opening of a glove box. Features are provided to aid the removal of items from the breech lock by a gloved operator. The breech lock can be reused or, if needed, can be replaced with a plug, glove, or other device at the port or opening of the glove box.

  13. Repackaging SRS Black Box TRU Waste

    SciTech Connect

    Swale, D. J.; Stone, K.A.; Milner, T. N.

    2006-01-09

    Historically, large items of TRU Waste, which were too large to be packaged in drums for disposal have been packaged in various sizes of custom made plywood boxes at the Savannah River Site (SRS), for many years. These boxes were subsequently packaged into large steel ''Black Boxes'' for storage at SRS, pending availability of Characterization and Certification capability, to facilitate disposal of larger items of TRU Waste. There are approximately 107 Black Boxes in inventory at SRS, each measuring some 18' x 12' x 7', and weighing up to 45,000 lbs. These Black Boxes have been stored since the early 1980s. The project to repackage this waste into Standard Large Boxes (SLBs), Standard Waste Boxes (SWB) and Ten Drum Overpacks (TDOP), for subsequent characterization and WIPP disposal, commenced in FY04. To date, 10 Black Boxes have been repackaged, resulting in 40 SLB-2's, and 37 B25 overpack boxes, these B25's will be overpacked in SLB-2's prior to shipping to WIPP. This paper will describe experience to date from this project.

  14. Global Mapping of Human RNA-RNA Interactions.

    PubMed

    Sharma, Eesha; Sterne-Weiler, Tim; O'Hanlon, Dave; Blencowe, Benjamin J

    2016-05-19

    The majority of the human genome is transcribed into non-coding (nc)RNAs that lack known biological functions or else are only partially characterized. Numerous characterized ncRNAs function via base pairing with target RNA sequences to direct their biological activities, which include critical roles in RNA processing, modification, turnover, and translation. To define roles for ncRNAs, we have developed a method enabling the global-scale mapping of RNA-RNA duplexes crosslinked in vivo, "LIGation of interacting RNA followed by high-throughput sequencing" (LIGR-seq). Applying this method in human cells reveals a remarkable landscape of RNA-RNA interactions involving all major classes of ncRNA and mRNA. LIGR-seq data reveal unexpected interactions between small nucleolar (sno)RNAs and mRNAs, including those involving the orphan C/D box snoRNA, SNORD83B, that control steady-state levels of its target mRNAs. LIGR-seq thus represents a powerful approach for illuminating the functions of the myriad of uncharacterized RNAs that act via base-pairing interactions.

  15. Speaking to the dead: images of the dead in contemporary art.

    PubMed

    O'Neill, Mary

    2011-05-01

    In this article I explore works by three artists in which we can see images that relate to bereavement. In the work of the first two, Araya Rasdjarmrearnsook and Andres Serrano, we can see photographic images (still and moving) of human corpses, which have been criticized as morbid and unhealthy. However I argue that it is not in fact images of death or the dead that are problematic but those images which present or evoke evidence of the emotions associated with death, and create a situation where we imagine the circumstances of our own deaths or the death of those we love. Images of the dead are acceptable as long as they do not cause pain to the living, as in a video game fantasy or a fiction, or are seen as other and distant. In the second group of works, by Gustgav Metzger, The Absent Dead: The Surrogate Body, the body is not present either because the death has taken place at a distance, either in time or geographically, or both, and a new site must be created. In this section, I discuss Metzger's auto-destructive art and argue that these works, through their ephemerality, embody a form of 'meaning making' and a possibility of the benefits of grief as described by Parkes.

  16. Zombie Vortices: The Dead Zones of Protoplanetary Disks are Not Dead

    NASA Astrophysics Data System (ADS)

    Jiang, Chung-Hsiang; Marcus, Philip; Pei, Suyang; Barranco, Joe; Hassanzadeh, Pedram; Lecoanet, Daniel

    2014-11-01

    Numerical simulations, using both the anelastic and fully compressible equations of motion, show that the ``dead zones'' of protoplanetary disks (PPDs) around forming stars are unstable and filled with vortex-dominated turbulence with Mach and Rossby numbers of order 0.2 - 0.3. The dead zones are regions in which the temperature is too cool for the gas to ionize and be destabilized by instabilities associated with the magnetic field. The ``dead zones'' were thought, by most authors, to be stable to all purely-hydrodynamic instabilities because the flow has an angular momentum that increases with increasing radius in a PPD and is therefore stable by Rayleigh's theorem. However, that theorem in not applicable to stratified flows, such as those in a PPD. We summarize our simulations with emphasis on the finite-amplitude trigger of the instability and show that when the trigger is Kolmogorov noise, the Mach number of the noise that is needed to create instability is proportional to Re - 1 / 2 , where Re is the Reynolds number of the initial noise.

  17. Raccoon Parasite Not as Deadly to Humans as Thought

    MedlinePlus

    ... gov/news/fullstory_164280.html Raccoon Parasite Not as Deadly to Humans as Thought Researchers find confirmed cases of raccoon roundworm ... 24, 2017 FRIDAY, March 24, 2017 (HealthDay News) -- A raccoon parasite that can be deadly in humans ...

  18. Recipe for Hypoxia: Playing the Dead Zone Game

    ERIC Educational Resources Information Center

    Kastler, Jessica A.

    2009-01-01

    Dead zones--areas experiencing low levels of dissolved oxygen--are growing in shallow ocean waters around the world. Research has shown that dead zones form as a result of a specific type of pollution, called nutrient enrichment or eutrophication, and are found in almost every coastal zone where humans have large populations. Concepts related to…

  19. Day of the Dead: A Mexican-American Celebration.

    ERIC Educational Resources Information Center

    Hoyt-Goldsmith, Diane

    This children's book describes how a Mexican-American family celebrates the traditional Mexican holiday, Day of the Dead (Dia de Muertos). The book centers on 10-year-old twins, Ximena and Azucena, who live in Sacramento, California, with their two brothers, older sister, and parents. The Day of the Dead takes place on the first and second day of…

  20. 37 CFR 1.422 - When the inventor is dead.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2012-07-01 2012-07-01 false When the inventor is dead. 1.422 Section 1.422 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE... File An International Application § 1.422 When the inventor is dead. In case of the death of...

  1. 37 CFR 1.422 - When the inventor is dead.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2011-07-01 2011-07-01 false When the inventor is dead. 1.422 Section 1.422 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE... File An International Application § 1.422 When the inventor is dead. In case of the death of...

  2. 37 CFR 1.42 - When the inventor is dead.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2011-07-01 2011-07-01 false When the inventor is dead. 1.42 Section 1.42 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE... for A Patent § 1.42 When the inventor is dead. In case of the death of the inventor, the...

  3. 37 CFR 1.422 - When the inventor is dead.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false When the inventor is dead. 1.422 Section 1.422 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE... File An International Application § 1.422 When the inventor is dead. In case of the death of...

  4. 37 CFR 1.42 - When the inventor is dead.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false When the inventor is dead. 1.42 Section 1.42 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE... for A Patent § 1.42 When the inventor is dead. In case of the death of the inventor, the...

  5. 37 CFR 1.42 - When the inventor is dead.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2012-07-01 2012-07-01 false When the inventor is dead. 1.42 Section 1.42 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE... for A Patent § 1.42 When the inventor is dead. In case of the death of the inventor, the...

  6. Zero-dead-time operation of interleaved atomic clocks.

    PubMed

    Biedermann, G W; Takase, K; Wu, X; Deslauriers, L; Roy, S; Kasevich, M A

    2013-10-25

    We demonstrate a zero-dead-time operation of atomic clocks. This clock reduces sensitivity to local oscillator noise, integrating as nearly 1/τ whereas a clock with dead time integrates as 1/τ(1/2) under identical conditions. We contend that a similar scheme may be applied to improve the stability of optical clocks.

  7. 10 CFR 1047.7 - Use of deadly force.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... explosives). (3) Nuclear weapons or nuclear explosive devices. When deadly force reasonably appears to be necessary to prevent the theft, sabotage, or unauthorized control of a nuclear weapon or nuclear explosive device. (4) Special nuclear material. When deadly force reasonably appears to be necessary to prevent...

  8. 10 CFR 1047.7 - Use of deadly force.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... explosives). (3) Nuclear weapons or nuclear explosive devices. When deadly force reasonably appears to be necessary to prevent the theft, sabotage, or unauthorized control of a nuclear weapon or nuclear explosive device. (4) Special nuclear material. When deadly force reasonably appears to be necessary to prevent...

  9. 10 CFR 1047.7 - Use of deadly force.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... explosives). (3) Nuclear weapons or nuclear explosive devices. When deadly force reasonably appears to be necessary to prevent the theft, sabotage, or unauthorized control of a nuclear weapon or nuclear explosive device. (4) Special nuclear material. When deadly force reasonably appears to be necessary to prevent...

  10. Insights into Metalloregulation by M-box Riboswitch RNAs via Structural Analysis of Manganese-Bound Complexes

    SciTech Connect

    Ramesh, Arati; Wakeman, Catherine A.; Winkler, Wade C.

    2011-12-09

    The M-box riboswitch couples intracellular magnesium levels to expression of bacterial metal transport genes. Structural analyses on other riboswitch RNA classes, which typically respond to a small organic metabolite, have revealed that ligand recognition occurs through a combination of base-stacking, electrostatic, and hydrogen-bonding interactions. In contrast, the M-box RNA triggers a change in gene expression upon association with an undefined population of metals, rather than responding to only a single ligand. Prior biophysical experimentation suggested that divalent ions associate with the M-box RNA to promote a compacted tertiary conformation, resulting in sequestration of a short sequence tract otherwise required for downstream gene expression. Electrostatic shielding from loosely associated metals is undoubtedly an important influence during this metal-mediated compaction pathway. However, it is also likely that a subset of divalent ions specifically occupies cation binding sites and promotes proper positioning of functional groups for tertiary structure stabilization. To better elucidate the role of these metal binding sites, we resolved a manganese-chelated M-box RNA complex to 1.86 {angstrom} by X-ray crystallography. These data support the presence of at least eight well-ordered cation binding pockets, including several sites that had been predicted by biochemical studies but were not observed in prior structural analysis. Overall, these data support the presence of three metal-binding cores within the M-box RNA that facilitate a network of long-range interactions within the metal-bound, compacted conformation.

  11. Dead-time compensation for a logarithmic display rate meter

    DOEpatents

    Larson, John A.; Krueger, Frederick P.

    1988-09-20

    An improved circuit is provided for application to a radiation survey meter that uses a detector that is subject to dead time. The circuit compensates for dead time over a wide range of count rates by producing a dead-time pulse for each detected event, a live-time pulse that spans the interval between dead-time pulses, and circuits that average the value of these pulses over time. The logarithm of each of these values is obtained and the logarithms are subtracted to provide a signal that is proportional to a count rate that is corrected for the effects of dead time. The circuit produces a meter indication and is also capable of producing an audible indication of detected events.

  12. Dead-time compensation for a logarithmic display rate meter

    DOEpatents

    Larson, J.A.; Krueger, F.P.

    1987-10-05

    An improved circuit is provided for application to a radiation survey meter that uses a detector that is subject to dead time. The circuit compensates for dead time over a wide range of count rates by producing a dead-time pulse for each detected event, a live-time pulse that spans the interval between dead-time pulses, and circuits that average the value of these pulses over time. The logarithm of each of these values is obtained and the logarithms are subtracted to provide a signal that is proportional to a count rate that is corrected for the effects of dead time. The circuit produces a meter indication and is also capable of producing an audible indication of detected events. 5 figs.

  13. Boxing and chronic brain damage.

    PubMed

    Stiller, J W; Weinberger, D R

    1985-06-01

    A chronic, and at times, progressive neurologic syndrome associated with boxing has been recognized for some time by boxing fans and people involved with the sport. Since Martland's first description of the syndrome in 1929, there has been only one randomly selected study of ex-boxers, that of Roberts, which showed a 17 per cent prevalence of this syndrome among boxers who fought between 1929 and 1955. This syndrome can be progressive but often is not. Furthermore, the extent of occupational exposure is probably a significant risk factor. Because of this, it would be expected that the prevalance of the syndrome in the modern boxer, as well as the amateur, would be significantly less than during the first half of the century, and, indeed, several studies appear to support this. Recent studies provide evidence that brain damage does exist in modern boxers and suggests that "subclinical" brain damage is likely to be more prevalent than obvious clinical dysfunction. There is clearly a discrepancy between subclinical evidence of neurologic involvement (for example, an abnormal CT scan) and signs of clinical neurologic dysfunction (for example, clinical exam and neuropsychological testing). The latter tend to show less frequent and consistent evidence of brain damage in boxers than does the CT scan. Although it is tempting to assume that an abnormal CT scan presages the development of neurologic dysfunction, it is not clear that this is the case. The prevalence of the syndrome, risk for progression to functional deficit, warning signs, and the natural history cannot be defined at this time. The only way to better define these parameters would be a controlled prospective study, which has yet to be undertaken.

  14. Modeling the circulation of the Dead Sea

    NASA Astrophysics Data System (ADS)

    Brenner, Steve; Lensky, Nadav; Gertman, Isaac

    2015-04-01

    The Dead Sea is a hypersaline, terminal lake located at the lowest point on the land surface of the Earth. Its current level is more than 429 m below MSL, and due to a negative water balance (mainly anthropogenic), the lake level has been dropping at an average rate of more than 1 m/yr for more than 30 years. The mean salinity has also been steadily increasing and today is close to 280 psu. The region of the Dead Sea is a unique landscape that has important historical, cultural, and economic value and therefore such an extreme change of the lake has significant environmental and economic consequences. In recent years there has been a notable increase in observing and monitoring of the lake through continuous measurements from several fixed buoys as well as during quasi-regular cruises. In order to complement the measurements and improve our understanding of the dynamics of this unique lake a three dimensional circulation model is being developed. Previous modeling efforts were limited mainly to a one dimensional column model which was coupled to a comprehensive physio-chemical model and used for long term multi-decadal simulations. In this study the focus is on understanding the dynamical processes that control the lake-wide circulation on time scales ranging from days to seasons. The first step was to replace the equation of state with an equation appropriate for the hypersaline conditions, in addition to some minor tuning of the turbulence closure scheme. Results will be presented from preliminary simulations of the wind driven circulation in various seasons. A case study of a recent unusual winter flooding event, during which the lake level rose by more than 20 cm over a two month period, will also be presented. The model successfully simulated the observed transition from holomictic to meromictic conditions and epilimnion dilution during this event, as well as the restoration of holomictic conditions when the level started to drop again. The relationship

  15. 36 CFR 1192.33 - Fare box.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Fare box. 1192.33 Section 1192.33 Parks, Forests, and Public Property ARCHITECTURAL AND TRANSPORTATION BARRIERS COMPLIANCE BOARD... Systems § 1192.33 Fare box. Where provided, the farebox shall be located as far forward as practicable...

  16. 49 CFR 38.33 - Fare box.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Fare box. 38.33 Section 38.33 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES Buses, Vans and Systems § 38.33 Fare box. Where provided, the...

  17. 49 CFR 38.33 - Fare box.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 1 2013-10-01 2013-10-01 false Fare box. 38.33 Section 38.33 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES Buses, Vans and Systems § 38.33 Fare box. Where provided, the...

  18. 49 CFR 38.33 - Fare box.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 1 2014-10-01 2014-10-01 false Fare box. 38.33 Section 38.33 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES Buses, Vans and Systems § 38.33 Fare box. Where provided, the...

  19. BLS: Box-fitting Least Squares

    NASA Astrophysics Data System (ADS)

    Kovács, G.; Zucker, S.; Mazeh, T.

    2016-07-01

    BLS (Box-fitting Least Squares) is a box-fitting algorithm that analyzes stellar photometric time series to search for periodic transits of extrasolar planets. It searches for signals characterized by a periodic alternation between two discrete levels, with much less time spent at the lower level.

  20. Box Plots in the Australian Curriculum

    ERIC Educational Resources Information Center

    Watson, Jane M.

    2012-01-01

    This article compares the definition of "box plot" as used in the "Australian Curriculum: Mathematics" with other definitions used in the education community; describes the difficulties students experience when dealing with box plots; and discusses the elaboration that is necessary to enable teachers to develop the knowledge…

  1. PINE Discovery Box, 101 Stimulating Ideas.

    ERIC Educational Resources Information Center

    Busch, Phyllis S.

    This manual is intended for use with the PINE (Projects in Imaginative Nature Education) discovery box in elementary school conservation education. The box contains 21 natural specimens which can serve as the starting point for simple student investigations. Specimens and activities are keyed for grade level. For each item, background information…

  2. Cereal Box Design: An Interdisciplinary Graphics Activity

    ERIC Educational Resources Information Center

    Fitzgerald, Mike; Tsosie, Teri

    2004-01-01

    This article describes cereal box design, an interdisciplinary graphics activity. The cereal box design activity is intriguing both for its simplicity and the resourcefulness that it can generate in young people. It lends itself to a variety of curriculums. It covers both consumerism and Design for the Environment (DfE) concepts broadly and in…

  3. Dynamics of TBP binding to the TATA box

    NASA Astrophysics Data System (ADS)

    Schluesche, Peter; Heiss, Gregor; Meisterernst, Michael; Lamb, Don C.

    2008-02-01

    Gene expression is highly controlled and regulated in living cells. One of the first steps in gene transcription is recognition of the promoter site by the TATA box Binding Protein (TBP). TBP recruits other transcriptions factors and eventually the RNA polymerase II to transcribe the DNA in mRNA. We developed a single pair Förster Resonance Energy Transfer (spFRET) assay to investigate the mechanism of gene regulation. Here, we apply this assay to investigate the initial binding process of TBP to the adenovirus major late (AdML) promoter site. From the spFRET measurements, we were able to identify two conformations of the TBP-DNA complex that correspond to TBP bound in the correct and the opposite orientation. Increased incubation times or the presence of the transcription factor TFIIA improved the alignment of TBP on the promoter site. Binding of TBP to the TATA box shows a rich dynamics with abrupt transitions between multiple FRET states. A frame-wise histogram analysis revealed the presence of at least six discrete states, showing that TBP binding is more complicated than previously thought. Hence, the spFRET assay is very sensitive to the conformation of the TBP-DNA complex and is very promising tool for investigating the pathway of TBP binding in detail.

  4. 46 CFR 111.81-3 - Cables entering boxes.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Cables entering boxes. 111.81-3 Section 111.81-3...-GENERAL REQUIREMENTS Outlet Boxes and Junction Boxes § 111.81-3 Cables entering boxes. Each cable entering a box or fitting must be protected from abrasion and must meet the following: (a) Each...

  5. RNA nanoparticle as a vector for targeted siRNA delivery into glioblastoma mouse model.

    PubMed

    Lee, Tae Jin; Haque, Farzin; Shu, Dan; Yoo, Ji Young; Li, Hui; Yokel, Robert A; Horbinski, Craig; Kim, Tae Hyong; Kim, Sung-Hak; Kwon, Chang-Hyuk; Nakano, Ichiro; Kaur, Balveen; Guo, Peixuan; Croce, Carlo M

    2015-06-20

    Systemic siRNA administration to target and treat glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP) based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA for gene silencing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. This study provides possible application of pRNA-3WJ RNP for specific delivery of therapeutics such as siRNA, microRNA and/or chemotherapeutic drugs into glioblastoma cells without inflicting collateral damage to healthy tissues.

  6. RNA nanoparticle as a vector for targeted siRNA delivery into glioblastoma mouse model

    PubMed Central

    Lee, Tae Jin; Haque, Farzin; Shu, Dan; Yoo, Ji Young; Li, Hui; Yokel, Robert A.; Horbinski, Craig; Kim, Tae Hyong; Kim, Sung-Hak; Kwon, Chang-Hyuk; Nakano, Ichiro; Kaur, Balveen; Guo, Peixuan; Croce, Carlo M.

    2015-01-01

    Systemic siRNA administration to target and treat glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP) based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA for gene silencing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. This study provides possible application of pRNA-3WJ RNP for specific delivery of therapeutics such as siRNA, microRNA and/or chemotherapeutic drugs into glioblastoma cells without inflicting collateral damage to healthy tissues. PMID:25885522

  7. North American box turtles: A natural history

    USGS Publications Warehouse

    Dodd, C. Kenneth

    2002-01-01

    Once a familiar backyard visitor in many parts of the United States and Mexico, the box turtle is losing the battle against extinction. In North American Box Turtles, C. Kenneth Dodd, Jr., has written the first book-length natural history of the twelve species and subspecies of this endangered animal. This volume includes comprehensive information on the species’ evolution, behavior, courtship and reproduction, habitat use, diet, population structure, systematics, and disease. Special features include color photos of all species, subspecies, and their habitats; a simple identification guide to both living and fossil species; and a summary of information on fossil Terrapene and Native uses of box turtles. End-of-chapter sections highlight future research directions, including the need for long-term monitoring and observation of box turtles within their natural habitat and conservation applications. A glossary and a bibliography of literature on box turtles accompany the text.

  8. Dead regions and noisiness of pure tones.

    PubMed

    Huss, Martina; Moore, Brian C J

    2005-10-01

    Some hearing-impaired subjects report pure tones as sounding highly distorted and noise-like. We assessed whether such reports indicate that the tone frequency falls inside a dead region (DR). Nine hearing-impaired and four normally hearing subjects rated pure tones on a scale from 1 to 7, where 1 indicates clear tone and 7 indicates noise. A white noise was presented as a reference for a sound that should be rated as 7. Stimuli covered the whole audible range of frequencies and levels. The noisiness ratings were, on average, higher for hearing-impaired subjects than for normally hearing subjects. For the former, the ratings were not markedly different for tones with frequencies just outside or inside a DR. However, ratings always exceeded 3 for tones falling more than 1.5 octaves inside a DR. The results indicate that judgement of a tone as sounding noise-like does not reliably indicate that the tone frequency falls in a DR. Both normally hearing and hearing-impaired subjects rated 0.125 kHz and 12 kHz tones as somewhat noise-like, independently of the existence of a DR.

  9. Optical properties of the Dead Sea

    NASA Astrophysics Data System (ADS)

    Boss, Emmanuel; Gildor, Hezi; Slade, Wayne; Sokoletsky, Leonid; Oren, Aharon; Loftin, James

    2013-04-01

    The Dead Sea, located in the rift valley between Jordan and Israel, is a hypersaline lake, resulting in unique biogeochemistry and optical properties. In the spring of 2004 we conducted two days of physical and optical measurements in the lake. Because of the significant effect of dissolved salts on the optical properties of water, our analysis required a novel processing approach to obtain dissolved and total inherent optical properties from the measurements. In addition, we show that the lake's salinity can be estimated from measurements of hyper-spectral absorption or attenuation spectra in the red and infrared parts of the spectrum, using published values of specific absorption of dissolved NaCl, despite the fact that the lake's salt chemistry is complex. In situ observations demonstrated that the lake has a two-layer structure with a warm and more turbid layer at the top 20-30 m and a clearer colder layer below. Both the particulate and dissolved absorption are well approximated by exponentially decreasing functions with the spectral slope of the particulate absorption about half that of the dissolved fraction and consistent with other aquatic environments. Both have relatively low and similar magnitudes in the blue (O(0.15 m-1)). Mean particle size was observed to increase with depth, consistent with precipitating salt crystals (observed in past campaigns) shown here to play a major role in the lake's optical properties.

  10. Dead Time of Single Photon Avalanche Diodes

    NASA Astrophysics Data System (ADS)

    Neri, L.; Tudisco, S.; Musumeci, F.; Scordino, A.; Fallica, G.; Mazzillo, M.; Zimbone, M.

    2011-06-01

    Single Photon Avalanche Diode (SPAD) is the new generation of Geiger-Muller counter device developed in semiconductor technology [S. Privitera et al. Sensors Journal, vol 8 Iss. 8 (2008) 4636; S. Tudisco et al. IEEE Sensors Journal vol 8 ISS 7-8 (2008) 1324; S. Cova et al. Applied Optics 35 (1996) 1956]. Physical dead time model and noise production process has been analyzed and their corrections have been performed [S.H. Lee, R.P. Gardner, M. Jae, Nucl. Instr. and Meth. in Phys. Res. B 263 (2007) 46]. We have been able to extract the real amount of incident photon rate up to 10 7cps using a device with 0.97μs total deadtime. We also developed the equation of the noise count rate vs incoming photon rate, supported by Montecarlo simulation and experimental data. We marked the difference between dark rate and noise count rate, and introduced the noise rate inside the hybrid deadtime equation used for SPAD device.

  11. Boxing injuries: neurologic, radiologic, and neuropsychologic evaluation.

    PubMed

    Ross, R J; Casson, I R; Siegel, O; Cole, M

    1987-01-01

    Boxing is an endeavor that may have to be re-evaluated in the coming years as to whether it should be designated as a sport. It is the only "sport" in which victory is determined by the amount of physical damage done to the opponent. We have presented the largest number of professional and amateur boxers (58) evaluated by various modern diagnostic modalities and have unequivocally demonstrated the deleterious effects of boxing upon the brain. There have been few, if any, meaningful actions taken by the promoters of boxing to correct the conditions under which boxers are subjected to physical abuse. Recommendations regarding the creation of a National Board of Boxing to supervise this "sport" have not been heeded. Suggested safeguards for the boxer, including mandatory medical and boxing history records (passports), use of headgear and approved safe boxing gloves, avoiding blows to the head, improved boxing ring floors, mandatory neurologic examinations, and more competent physicians at ringsides making medical decisions, have essentially not been implemented. The suggestions that mandatory computed tomograms at various stages in a boxer's career be used to determine possible changes of atrophy have not been followed, even when the CT scans have been made available at no cost to the boxers. The effective use of neuropsychologic evaluation, even when offered at no cost, has also been denied. The established medical injuries due to boxing and the lack of any sustained and significant efforts on the part of organized boxing create an atmosphere that is conducive to following the call for the consideration of a ban of boxing.

  12. Dead space mask eliminates central apnea at altitude.

    PubMed

    Patz, David S; Patz, Michael D; Hackett, Peter H

    2013-06-01

    Travelers to high altitude may have disturbed sleep due to periodic breathing with frequent central apneas. We tested whether a mask with added dead space could reduce the central apneas of altitude. 16 subjects were recruited, age 18-35, residing at 4600 ft (1400 m). They each slept one night with full polysomnographic monitoring, including end tidal CO2, in a normobaric hypoxia tent simulating 12,000 ft. (3658 m) altitude. Those who had a central apnea index (CAI) >20/h returned for a night in the tent for dead space titration, during which they slept with increasing amounts of dead space, aiming for a CAI <5/h or <10% of baseline. Then each subject slept another night with the titrated amount of dead space. Of the 16 subjects, 5 had a central apnea index >20/h mean 49.1, range 21.4-131.5/hr. In each of the 5, the dead space mask reduced the CAI by at least 88% to a mean of 3.1, range 0.9-7.1/h, (p=0.04). Hypopnea index was unchanged. Three subjects required 500 cc of dead space or less. One subject required 860 cc, and one required 2.1 L. Morning symptoms and arousal index were not significantly affected by the dead space mask. Dead space did not appear to increase the CO2 reserve. At 12,000 ft., central apneas can be effectively reduced with a dead space mask, but clinical utility will require further evaluation.

  13. Uncertainty evaluation of dead zone of diagnostic ultrasound equipment

    NASA Astrophysics Data System (ADS)

    Souza, R. M.; Alvarenga, A. V.; Braz, D. S.; Petrella, L. I.; Costa-Felix, R. P. B.

    2016-07-01

    This paper presents a model for evaluating measurement uncertainty of a feature used in the assessment of ultrasound images: dead zone. The dead zone was measured by two technicians of the INMETRO's Laboratory of Ultrasound using a phantom and following the standard IEC/TS 61390. The uncertainty model was proposed based on the Guide to the Expression of Uncertainty in Measurement. For the tested equipment, results indicate a dead zone of 1.01 mm, and based on the proposed model, the expanded uncertainty was 0.17 mm. The proposed uncertainty model contributes as a novel way for metrological evaluation of diagnostic imaging by ultrasound.

  14. Genome-wide identification and expression analysis of SBP-box gene family in Sorghum bicolor L.

    PubMed

    Jianzhong, Chang; Fengxia, Yan; Linyi, Qiao; Jun, Zheng; Fuyao, Zhang; Qingshan, Liu

    2016-06-20

    SQUAMOSA PROMOTER BINDING PROTEIN-box (SBP-box) family genes encoding plant-specific transcription factors are involved in many aspects of crop genetic improvement such as yield, plant-type and stress-resistance. The SBP-box gene family have important practical applications. In this study, 18 SBP-box genes were identified from the reference genome of sorghum (Sorghum bicolor L.) using bioinformatics. These genes distributed on nine chromosomes while eight of them located in the segmental duplication region. Phylogenetic reconstruction resulted in six subfamilies of SBP-box genes, among which SbSBP12, SbSBP3 and SbSBP15 are orthologous to ZmLG1, ZmTGA1 and ZmUB2/3 in corn, respectively. RNA-seq data analysis indicated that SbSBP-box genes show the highest expression level in primordial inflorescences. Moreover, SbSBP9 and SbSBP17 exhibited a tissue specific expression in primordial inflorescences. The expression levels of SbSBP5, SbSBP8 and SbSBP18 were increased in response to exogenous ABA and PEG,indicating that SbSBP-box genes are involved in the defense response against abiotic stresses in sorghum. This research provides references for cloning important genes in SbSBP-box gene family. Genes identified in this study could be considered as candidate genes for genetic improvement of sorghum.

  15. 6. VIEW OF SPILLWAY TIMBERS AND WATER CONTROL BOX, SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. VIEW OF SPILLWAY TIMBERS AND WATER CONTROL BOX, SHOWING WATER CONTROL BOX WITH LOWERED LAKE LEVEL - Three Bears Lake & Dams, Water Control Box, North of Marias Pass, East Glacier Park, Glacier County, MT

  16. Groundwater-Lake Interaction in the Dead Sea Area

    NASA Astrophysics Data System (ADS)

    Kiro, Y.; Weinstein, Y.; Starinsky, A.; Yechieli, Y.

    2011-12-01

    The Dead Sea hypersaline water system is unique in terms of its unusual geochemical composition, rapid lake level changes and water composition of the brines discharging along its shoreline. The Dead Sea can be used as a natural lab for studying groundwater-seawater interaction and saline water hydrological circulation along the aquifer-sea boundary. It provides an opportunity to follow the geochemical processes along a flow path from the lake into the aquifer and back into the lake. The lake level has been dropping since the 1960's due to human interference in its water budget, reaching a rate of 1 m/yr in recent years. Saline water circulation in coastal aquifers may be a major process that governs trace element mass balances in coastal areas. This study uses radium isotopes in order to quantify the lake water circulation in the Dead Sea aquifer. There are four naturally-occurring radium isotopes, with half-lives ranging from 3.7 days to 1600 years which are chain products of uranium and thorium isotopes. Radium isotopes are usually enriched in saline groundwater and therefore are good candidates for estimating seawater or hypersaline lake water circulation in the aquifer. Compared to most natural water bodies, the Dead Sea is extremely enriched in radium and barium, where both 226Ra and 228Ra activities and Ba concentration (145, 1-2 dpm/L and 5 mg/L, respectively) are 2-3 orders of magnitude higher than in ocean water, whereas the salinity of the Dead Sea is only 10 times higher. Circulated Dead Sea water in the aquifer contains decreased concentrations of 226Ra (60 dpm/L), Ba (1.5 mg/L), Sr (300 relative to 340 mg/L in the Dead Sea) and Sulfate (250 relative to 392 mg/L). We suggest that the low 226Ra and Ba concentrations are due to precipitation of barite and celestine from the supersaturated Dead Sea water on entering the aquifer. 228Ra and the shorter-lived 224Ra and 223Ra, which have much lower activities in the Dead Sea (up to 1.8, 3 and 0.8 dpm

  17. The ocular complications of boxing.

    PubMed

    Giovinazzo, V J; Yannuzzi, L A; Sorenson, J A; Delrowe, D J; Cambell, E A

    1987-06-01

    In cooperation with the New York State Athletic Commission, 74 boxers applying for a new or yearly renewal license were sequentially referred over a 2-year period for a complete dilated ocular examination at the Sports Vision Institute of the Manhattan Eye, Ear and Throat Hospital. At least one ocular injury was found in 66% of boxers. Vision-threatening injuries, defined as significant damage to the angle, lens, macula, or peripheral retina occurred in 58% of boxers. Nineteen percent of boxers had angle abnormalities. Nineteen percent of boxers had pathologic cataracts, over 70% of these were posterior subcapsular. Six boxers had macular lesions. A total of 24% of boxers had retinal tears. Standardized photographs were used to distinguish pathologic cataracts from congenital opacities and pathologic retinal tears from atrophic holes. Attempts were made to identify risk factors in boxing that might be predictive for ocular injury. Variables included age, weight division, left- or right-handedness, total number of losses, and total number of bouts. Significant correlations were found between the total number of bouts and the total number of losses, and the presence of retinal tears. College varsity athletes were selected as controls. Significant differences were found between boxers and controls for the total number of injuries, total vision-threatening injuries, and the number of retinal tears. A series of recommendations are proposed to aide in the early detection and prevention of serious ocular injuries.

  18. Hydro-Balanced Stuffing Box field test

    SciTech Connect

    Giangiacomo, L.A.

    1999-05-28

    The Hydro-Balanced Stuffing Box is a seal assembly for polished rod pumping installations commonly used in oil and gas pumping well installations to contain produced well fluids. The improved stuffing box was developed and patented by Harold H. Palmour of The Palmour Group of Livingston, TX. The stuffing box is designed to reduce the incidence of seal leakage and to utilize an environmentally safe fluid, so that if there is any leakage, environmental damage is reduced or eliminated. The unit was tested on two wells at the Rocky Mountain Oilfield Testing Center. During the test period, the performance of the stuffing box was measured by monitoring the pressure on the tubing and the inner chamber with a Barton Two-pen recorder. The amount of safe fluid consumed, fluid leakage at the top of the stuffing box, pressure supplied from the nitrogen bottle, ambient temperature, and polish rod temperature was recorded. The stuffing box is capable of providing a better seal between well fluids an d the environment than conventional stuffing boxes. It allows the polished rod to operate cooler and with lubrication, extending the life of the packing elements, and reducing the amount of attention required to prevent leakage.

  19. Are cyanobacterial blooms trophic dead ends?

    PubMed

    Perga, Marie-Elodie; Domaizon, Isabelle; Guillard, Jean; Hamelet, Valérie; Anneville, Orlane

    2013-06-01

    Cyanobacterial blooms induce significant costs that are expected to increase in the near future. Cyanobacterial resistance to zooplankton grazing is one factor thought to promote bloom events. Yet, numerous studies on zooplankton ability to graze upon cyanobacteria have been producing contradictory results and such a puzzle might arise from the lack of direct observations in situ. Our objective was to track, using fatty acid (FA) and fatty acid stable isotope analyses (FA-SIA), the fate of cyanobacterial organic matter in the food web of a lake subjected to summer blooms of Planktothrix rubescens. A metalimnetic bloom of P. rubescens occurred in Lake Bourget (France) during the study period (May-November 2009). The bloom was especially rich in α-linolenic acid, 18:3(n-3), but none of the considered zooplankton taxa exhibited spiking content in this particular FA. FA-SIA revealed, however, that over a quarter of 18:3(n-3) in small zooplankton (<500 μm) was provided by P. rubescens while large cladocerans (>500 μm) did not benefit from it. P. rubescens 18:3(n-3) could be tracked up to perch (Perca fluviatilis) young of the year (YOY) to which it contributed to ~15 % of total 18:3(n-3). Although transferred with a much lower efficiency than micro-algal organic matter, the P. rubescens bloom supported a significant share of the pelagic secondary production and did not constitute, sensu stricto, a 'trophic dead end'. The cyanobacterial bloom also provided perch YOY with components of high nutritional values at a season when these are critical for their recruitment. This cyanobacterial bloom might thus be regarded as a significant dietary bonus for juvenile fish.

  20. Efficiency of box-traps and leg-hold traps with several bait types for capturing small carnivores (mammalia) in a disturbed area of southeastern Brazil.

    PubMed

    Michalski, Fernanda; Crawshaw, Peter G; Oliveira, Tadeu G de; Fabián, Marta E

    2007-03-01

    Capturing small carnivores is often necessary for obtaining key ecological data. We compared the efficiency of box and leg-hold traps, using live and dead bait, to capture six carnivore species (Herpailurus yagouaroundi (E. Geoffroyi, 1803), Leopardus tigrinus (Schreber, 1775), Nasua nasua (Linnaeus, 1766), Cerdocyon thous (Linnaeus, 1766), Eira barbara (Linnaeus, 1758), and Galictis cuja (Molina, 1782)). The use of leg-hold traps significantly increased the capture rate of carnivores (5.77%) and non-target species (non-carnivores, 11.54%). Dead bait significantly attracted more non-carnivores than carnivores and live bait was more efficient for capturing carnivores (2.56%) than non-carnivores (0.77%). Both box and leg-hold traps caused some minor injuries (swelling and claw loss). We provide recommendations for the ethical use of these trap and bait types.